Screen for affecting amyloidogenic cleavage by BACE1

Dissertation

zur Erlangung des akademischen Grades

eines Doktors der Naturwissenschaften (Dr. rer. nat.)

an der Universität Konstanz

Mathematisch-Naturwissenschaftliche Sektion

Fachbereich Biologie

vorgelegt von

Stephan Penzkofer

Konstanz, Juli 2011

Tag der mündlichen Prüfung: 24.10.2011

1. Referent: Professor Dr. Marcel Leist

2. Referent: Professor Dr. Daniel Dietrich Summary: The Amyloid β peptide (Aβ) is suspected to be a causal agent for Alzheimer’s disease (AD). Therefore a screen for kinases downregulating the initial step of its production, the cleavage of the Amyloid Precursor (APP) by Beta-site of APP Cleaving 1 (BACE1), was conducted in this study. Briefly, HEK293 cells were colipofected with one of in total 1357 siRNAs against 60% of the human kinome and either an APP construct with only the β-cleavage site left or normally cleavable APP as control. Remaining β-cleavage was for logistic reasons firstly measured with an activity-test for secreted alkaline phosphatase (SEAP) fused to both types of APP and subjected to Aβ-ELISA when interesting. Before the screen, the APP-constructs were characterized in the cell types HEK293 and CGCs with regards to cleavage, especially by BACE1. The screen resulted in 38 hits of which one, Testis Specific Serine 3, was confirmed once more. In a second, bioinformatic project, an initially suspected APLP-like pseudogenic-like sequence in C3orf52 was refuted. Further, analysis of C3orf52 expression data hints on a role in myeloid leukemia. Lastly, the phylogenetic relationship of the APP family paralogs was examined, also in comparison to neighboring gene families, and found in the topology (APLP1)(APLP2/APP).

Zusammenfassung: Das Amyloid β-Peptid (Aβ) steht im Verdacht, ursächlich für die Alzheimer- Demenz (AD) zu sein. Daher wurden in dieser Studie Kinasen gesucht, die den ersten Schritt seiner Entstehung, Schnitt des Amyloid Precursor durch Beta-site of APP Cleaving Enzyme 1 (BACE1), herabregulieren. Dafür wurden HEK293 colipofiziert mit einer von insgesamt 1357 siRNAs gegen 60% des menschlichen Kinoms und entweder einem ausschliesslich β-spaltbarem APP- Konstrukt oder normal spaltbarem APP als Kontrolle. Aus logistischen Gründen wurde die übriggebliebene β-Spaltung zunächst mit einem Aktivitätstest für sekretierbare alkaline Phosphatase (SEAP), das an beide APPs fusioniert war, gemessen und falls interessant noch mit Aβ-ELISA. Vor dem Screen wurden die APP-Konstrukte in den Zelltypen HEK293 und CGC mit Hinblick auf Spaltung, v. a. durch BACE1, charakterisiert. Der Screen resultierte in 38 Hits, von denen einer, Testis Specific Serine Kinase 3, nochmals bestätigt wurde. In einem zweiten, bioinformatischen Projekt wurde eine zunächst vermeintlich APLP-ähnliche pseudogenartige Sequenz in C3orf52 widerlegt. Weiter deutet die Analyse von C3orf52-Genexpressionsdaten auf eine Rolle in myeloider Leukämie hin. Zuletzt wurden die phylogenetischen Beziehungen der APP-Familie-Paraloge untersucht, auch im Vergleich mit benachbarten Genfamilien, und in der Topologie (APLP1)(APLP2/APP) gefunden.

Abbreviations aa amino acids LINE1 Long Interspersed Nuclear Element1 Aβ Amyloid β peptide LOAD Late-Onset AD AβK16V Aβ with K to V exchange, not mbp mega base pairs cleavable by α-secretases MCA Middle Cerebral Artery ACh Acetyl-Choline MCI Mild Cognitive Impairment ACoA Anterior Communicating Artery MRI Magnetic Resonance Imaging AD Alzheimer's Disease my, mya million years, - ago AICD APP Intracellular Domain µm 10-6 m Alu primate specific repeat class NbMc Nucleus basalis of Meynert complex ANNE APLP-Near Notes Extant NCBI National Center for Biotechnology APLP1 APP-Like Protein 1 Information APLP2 APP-Like Protein 2 NCT nicastrin APP Amyloid Precursor Protein nm 10-9 m APPswe APP with Swedish double mutation NSAID Non-Steroidal Anti-Inflammatory Drug AQD Amino-Quinazoline-Derivative O-GlcNAcylation O-linked-β-N-acetyl- ATCC American Tissue type Culture glucosaminylation Collection PBS Phosphate Buffered Saline AU Absorption Units PFK BACE1 Beta-site of APP Cleaving Enzyme 1 pg picogram BBB Blood Barrier pNPP para-Nitro-Phenyl-Phosphate bp base pairs PS1 preseniline 1 C3orf52 chr 3 open reading frame 52 rev reverse CAA Cerebral Amyloid Angiopathy SD Standard Deviation CArG CC-AT-rich(6)-GG box SEAP Secreted Alkaline Phosphatase CGC Cerebellar Granule Cell siRNA small interfering RNA ChAT choline acetyl SNP Single Polymorphism chr TFA Tierforschungsanlage CSNK1D isoform D tko triple knockout ct cycle of threshold TMD Transmembrane Domain CTF C-Terminal Fragment TTMP TPA-induced Transmembrane Protein dko double knockout us upstream DMSO dimethylsulphoxide WHO World Health Organization DS Down's Syndrome 3'UTR 3' Untranslated Region ds downstream Embry.s.c. Embryonic stem cells ENSEMBL joint project between EMBL - EBI Bestiarium and Wellcome Trust Sanger Institute EOAD Early-Onset AD Ac Anolis carolinensis green anole ER Endoplasmic Reticulum Bt Bos taurus cattle e-value expected value Ce Caenorhabditis elegans worm fwd forward Cf Canis familiaris dog gbp giga base pairs Cm Callorhinchus milii elephant shark GFP Green Fluorescent Protein Dm Drosophila melanogaster fruitfly HBP Hexosamine Biosynthesis Pathway Dr Danio rerio zebrafish HEK293 Human Embryonic Kidney293 cell line Ec Equus caballus horse HIV Human Immunodeficiency Virus Ga Gasterosteus aculeatus stickleback ICD-10 International Classification of Diseases Gg Gallus gallus chicken 10th revision Hs Homo sapiens human inh inhibitor Mm Mus musculus mouse IPAD Iso-Phthal-Amide-Derivative Oa Ornithorhynchus anatinus platypus IQ Intelligence Quotient Oc Oryctolagus cuniculus rabbit kbp kilo base pairs Ol Oryzias latipes medaka kd knockdown Pt Pan troglodytes chimpanzee ko knockout St Silurana tropicalis clawed frog Tn Tetraodon nigroviridis Tr Takifugu rubripes pufferfish

Some parts of this thesis have already been published:

Poster: Identification of BACE1- and APP-regulating kinases Stephan Penzkofer*, Christiane Volbracht&, Karina Fog&, Kenneth Vielsted& and Marcel Leist*! *:Doerenkamp-Zbinden Chair for alternative in vitro methods, University of Konstanz, Konstanz, Germany; !:corresponding author &:H. Lundbeck A/S, Valby, Denmark Presented at PENS Summer School: “Novel molecular strategies to treat neurodegenerative diseases”, Ofir, Portugal, 7. July 2007

Christiane Volbracht, Stephan Penzkofer, David Mansson, Kenneth Vielsted Christensen, Karina Fog, Stefan Schildknecht, Marcel Leist, Jacob Nielsen, Measurement of cellular β-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems, Analytical Biochemistry 387 (2009) 208-220.

Curriculum vitae of Stephan Penzkofer

06.06.1978 born in Munich, Germany 09/1984 – 06/1998 attendance of schools in Munich and Erding 07/1998 – 04/1999 compulsory military service 05/1999 – 09/1999 newspaper catering and car rental at the Munich Airport 10/1999 – 09/2000 studies in civil engineering at the Technical University of Munich

10/2000 – 09/2002 basic study period in biology at the University of Regensburg, Germany, with intermediate examinations in physics (oral), chemistry (oral) and biology (written; best result of the semester) 10/2002 – 03/2004 laboratory research courses in genetics, organic chemistry, biophysics and biochemistry followed by diploma examinations in organic chemistry, biochemistry and biophysics 04/2004 – 04/2005 diploma thesis with the Institute of Biophysics and physical Biochemistry at the University of Regensburg: “Characterisation of the PDZ2-PIP-Interaction with NMR-titration studies”

10/2005 – 05/2006 research attachment in cell culture, proteomics with mass spectrometry, capillary electrophoresis and flow cytometry with the Institute of Bioengineering and Nanotechnology, Biopolis, Singapore

09/2006 – 09/2011 doctoral thesis with the Doerenkamp-Zbinden-Chair of alternative in vitro methods at the University of Konstanz, Germany: “Screen for kinases affecting amyloidogenic cleavage by BACE1” eidesstattliche Erklärung:

"Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quelle gekennzeichnet."

Table of contents

1 Alzheimer’s disease (AD) and screening for BACE1-affecting kinases 1 1.1 History of AD research and AD in Down’s syndrome 1 I 1.2 Plaques and Aβ 4 N 1.3 APP and the discovery of its cleavage by proteases in HEK293 6 T 1.4 The families of APP and BACE1 11 R 1.5 Diabetes type 2 and the families of APP and BACE1 12 O 1.6 Atrophy in AD and the cholinergic hypothesis 13 D 1.7 Tau modifications and BACE1 transcriptional regulation 17 U 1.8 Influence of kinases on Aβ-generation 23 C 1.9 Difficulties with direct inhibition of BACE1 30 T 1.10 Aims of the study 33 I 2 Evolution of the APP family and characteristics of processed 34 O 2.1 Evolution of the APP family 34 N 2.2 Characteristics of processed pseudogenes 37 3 Screen preparation and execution 42 M 3.1 Cells 42 A 3.2 Molecular material 45 T 3.2.1 APP-constructs 45 E 3.2.2 Inhibitors and other compounds 48 R 3.2.3 Antibodies 49 I 3.2.4 Primers 50 A 3.2.5 small interfering RNA (siRNA) 50 L 3.3 Experimental setup 69 S 3.4 Data collection and analysis 71

3.4.1 SEAP activity measurement 72 & 3.4.2 A40 quantification 72 3.4.3 Cell viability measurements 72 M 3.4.4 Western blot analysis 73 E 3.4.5 Immunocytochemistry 73 T 3.4.6 Determination of mRNA knockdown 74 H 3.4.7 Statistics 75 O 3.4.8 Screen data analysis 75 D 3.5 Laboratory equipment 76 S 4 Sequences of C3orf52, orthologs and C3orf52 primers 78 5 Characterization of cells and APP-constructs 80 5.1 Characterization of CGCs, HEK293 and SHSY5Y cells 80 5.2 Optimization of transfection for APP-construct characterization 85 R 5.3 Characterization of the APP-constructs 89 E 6 Kinome screen for BACE1-affecting kinases 114 S 6.1 Optimization of cotransfection with SEAP-APPswe-EpoR and siRNAs 114 U 6.2 Analysis and validation of screen results 123 L 7 Identification of potential APLP-like pseudogenic fragments in C3orf52 137 T 7.1 Examination of potential APLP-like pseudogenic fragments 137 S 7.2 data analysis of C3orf52 141 8 Second attempt to identify a fourth APP family member on chromosome 3 143 9 Examination of the regions containing the APP family 144 10 Additional figures and tables from chapters 7-9 148 11 Interpretation of 185 D 11.1 cell- and APP-construct-characterization 185 I 11.2 the kinome screen for BACE1-affecting kinases 191 S 12 Interpretation of 198 C 12.1 potential APLP-like pseudogenic fragments 198 U 12.2 gene expression data analysis of C3orf52 202 S 13 The search for the 4. APP family member and the examination of the APP family regions206 S I O N

14 References 208 15 Acknowledgements 226

Introduction

1 Alzheimer‟s disease (AD) and screening for BACE1-affecting kinases

1.1 History of AD research and AD in Down‟s syndrome

AD was named in memory of Alois Alzheimer and originally the term was coined by Emil Kraepelin in 1910 to describe the type of presenile dementia which was reported histologically by Alzheimer in 1906 [1]. The age of its onset was earlier than in most cases of dementia and silver staining revealed round non-cellular structures which have earlier been called plaques [2, 3], with an average diameter of 50 µm [4]. Based on clinical (behavioral) symptoms, the disease was earlier identified as distinct from others by Kraepelin in whose laboratory Alzheimer searched on the histological level for (biological) pathologies. These pathologies were postulated by Kraepelin as underlying causes of the major psychiatric disorders. Already in 1904, Alzheimer had published findings of plaques in cases of senile dementia [5, 6] which were corroborated in 1907 by Oskar Fischer in 16 cases [3, 7], but only in the early 1950s, Meta Neumann realized that most senile dementia cases can be classified as AD [8]. Nowadays, they comprise probably around 60% of all dementia patients or 24 Mio worldwide. The WHO labeled this senile dementia in ICD-10 with G30.1: “Alzheimer‟s disease with age of onset 65 or older”, more commonly called late-onset AD (LOAD) as opposed to early-onset AD (EOAD). One of the first EOAD cases after Auguste Deter was Johann Feigl who died at age 57 [9] and was examined by Alzheimer post-mortem in 1910 [10]. In 1992, the brain samples were rediscovered and revaluated and no genetic cause for the disease could be found [11], as in most cases of EOAD. However dementia and mental illness also affected his mother and three siblings, so his dementia might be called familial [9]. Familial AD (FAD) cases require for confirmation two first-degree-relatives diagnosed with AD and account for 7% of all EOAD cases. Around 20% of familial EOAD are passed on in an autosomal-dominant pattern with high penetrance and 25% to 50% of them are caused by mutations in three . They are directly linked to the above-mentioned plaques. Its main constituent, amyloid-β (Aβ), a peptide of mostly 40 or 42 amino acids, was isolated in 1984 by Glenner [12]. Since it was noticed in 1929 that Down‟s syndrome

1 Introduction

(DS) patients develop plaques similar to those of AD patients [13, 14], the plaques of DS patients were also examined and also demonstrated to be made up mainly of Aβ [15, 16]. DS is a chromosomal disorder which is caused by an extra chromosome 21 in all cells (95% of DS patients) or in a fraction of the cells (=mosaicism; 1-2%). Also, familial DS exists (=Robertsonian translocation; 2-3%) and the rare event of duplication of parts or all of chromosome 21 [Wikipedia]. Thus it was supposed, that Aβ is encoded on chromosome 21. Indeed it was identified there in 1987 by the groups around Goldgaber, Tanzi and Kang [17-19] as part of a larger protein, the Aβ Precursor Protein (APP), launching an unprecedented boom in AD research, dealing with each and every facet of APP. The search for genetic causes in EOAD cases resulted in a total of 25 non-silent mutations in APP which have been detected between 1990 and 2009 [from www.alzforum.org, APP Mutations Table]. The best known, the Swedish mutation, is a double mutation found in a Swedish family and leading to higher Aβ production [20- 22], presumably because it leads to more cleavage of APP by a protease, called Beta-site of APP Cleaving Enzyme 1 (BACE1). BACE1 has been identified in 1999 independently by Hussain, Lin, Sinha, Vassar and Yan and their colleagues [23-27]. For the liberation of Aβ a second protease has to cleave the truncated APP, which was called the γ-secretase complex. It consists of the 4 proteins Nicastrin, Aph-1, and the Presenilins-1 and -2 [28, 29] for which 92 and 11 mutations have been linked to AD, respectively [from www.alzforum.org, Presenilin-1 or Presenilin-2 Mutations Table]. The ratio of Aβ40 to Aβ42 is regulated by the γ-secretase complex, and many mutations shift the ratio in favor of Aβ42 [30] which is more prone to aggregation. These and additional facts were the basis for a nowadays widely accepted theory on the cause of AD: the Amyloid Cascade Hypothesis by Selkoe and Hardy in 1991 [31, 32]. Additional facts were the finding that the major genetic risk factor for LOAD, apolipoprotein E ε allel 4 (ApoEε4) [33], leads to a strong buildup of amyloid and the knowledge of amyloid-induced diseases in other organs like kidney which can be successfully treated by prevention of the responsible amyloid protein. Also, it was observed that Aβ oligomers can be toxic to cells and that brain inflammatory cells, the microglia, are activated by plaques. According to the hypothesis, Aβ42 accumulates and oligomerizes in limbic and association cortices and exerts subtle effects on synapses as oligomers. These form fibrils and fibers and then deposit as not matured, diffuse plaques, thereby activating microglia and astrocytes, the key

2 Introduction players of inflammatory response in brain. Neurons in the vicinity can thus be directly injured by oxidative stress or through altered ionic homeostasis. Kinase and phosphatase activities could be changed and lead to a common hallmark of many types of dementia, neurofibrillary tangles. Finally, the widespread neuronal dysfunction and neuronal loss is clinically perceived as AD-typical dementia with memory loss and death occurs in average nine years after disease onset. However, the Amyloid Cascade Hypothesis was questioned in the past years, partly because diffuse plaques as mentioned above are also found in non-demented elderly, although not as many as in AD patients. Interestingly, Alois Alzheimer himself interpreted plaques as signs of brain aging and did not accept Oskar Fischer‟s suggestion that they might correlate with severity of dementia and memory loss [34]. AD in DS patients is one of the strongest arguments for the hypothesis as Aβ being causative for dementia. On a closer look, plaques in DS patients are found as early as 12 years and in 16% from 20-29 years and 80% from 30-39 years [35], whereas estimates for dementia range from 6%-75% [36]. A more recent study determined the prevalence of dementia with 13% and a mean age of onset of 55 years [37], an earlier one with 15%-45% and 52 years [38]. Reasons for difficulties with determination of dementia prevalence in DS patients can have its origins in the overall physical changes coming along with trisomy 21, e.g. hypothyroidism in 40% which leads to cognitive decline when untreated [39], mitral valve prolapse in 50% which increases the risk for cerebrovascular accident [39] and a lifestyle with less physical activity leading to high obesity rates [39]. resistance, also called diabetes type 2, which is linked to obesity in DS patients affects 10% of patients [40, 41]. Also, the liver isozyme of (PFKL) resides on chromosome 21 [42]. However, regarding the facts, that at most half of DS patients with extensive plaque generation develop AD, the existence of differences like a higher prevalence of epilepsy and the different overall DS constitution, one might think of not totally identical etiologies of dementia in DS and sporadic AD patients. For further unraveling the role of Aβ for the development of AD in DS patients it would be of great interest to find DS patients with mosaicism, such that neurons would be spared of trisomy 21 and hence APP not overexpressed, and see whether dementia will still be found in the usual prevalence or not.

3 Introduction

At least it could be possible to find these DS patients by testing their skin cells for trisomy 21, since skin cells like neurons are derived from the ectodermic lineage. They could be of DS phenotype due to mesoderm/endodermic trisomy 21 and with normal IQ, as an inverse relationship of the fraction of skin cells with trisomy 21 and measured IQ demonstrated [43]. Closest to the condition of mosaic DS with only two copies of APP (also in neurons) was a 78 year old woman with partial trisomy 21 in all cells and a phenotype suggestive of DS, although not typical. Her brain was very similar to a DS brain with its weight of 950 g, the round shape and the small cerebellum but lacking extensive amyloid plaques, below 1/mm2, and also atrophy. She was not demented [44], lending support to the Amyloid Cascade Hypothesis. Finally, a direct link exists between AD and DS patients: mothers giving birth to DS children before age 35 have a fivefold higher rate of AD [45]. A DS child mother who developed presenile dementia with mild cerebral atrophy at age 41 showed a connection: at routine karyotyping she was diagnosed with trisomy 21 in 10% of white blood cells, yet had no DS phenotypical features [46]. The lymphocyte trisomy 21 mosaicism affects only 2% of mothers with DS children but occult mosaicism may be more common in brain and germ cells and thus in part explain the association of DS and AD in family histories [47] or also some of the EOAD cases. So, trisomy 21 without DS phenotype leads to a higher risk for AD, thus also strengthening the Amyloid Cascade Hypothesis.

1.2 Plaques and Aβ

Since their discovery, the plaques have been examined for their morphological structure and chemical constitution. Morphologically, mainly two different types of plaques in brain parenchyma are distinguished. The earlier stages are so-called diffuse plaques which are found in non-demented elderly and young DS patients, characterized by diameters of 10-200 µm and blurred boundaries but not containing dystrophic neurites [48]. Also, no microglia or activated astrocytes are found in their vicinity [49]. Diffuse plaques cannot be stained with congo-red or thioflavin-S [50] in contrast to later stages which are called neuritic plaques and typically display a dense core in middle of a corona of degenerating neurites [51] and the final stage of a “burnt-out” plaque with only the core left over [50]. Whereas neuritic plaques are surrounded by microglia and astrocytes, “burnt-out” plaques are not [52]. Neuritic and burnt-out plaques are stainable with iodine, congo-red and thioflavin-S because they consist of amyloid 4 Introduction fibrils [50]. The fibrils are called amyloid since Virchow stained them in 1854 with iodine, which is along with congo-red a classical stain for amylose. The conformation of proteins in amyloid fibrils is a beta-sheet like structure, contributing the “β” in Aβ. The chemical constitution of the proteinaceous main component in the plaques was unknown until Glenner isolated Aβ in 1984 [12]. Before, even the origin of the protein was under discussion – whether of local neuronal production as already postulated by Struwe in 1929 [13] or a blood component like serum amyloid A protein (SAA protein) as suggested by Glenner in 1978 [53]. The discussion was launched by the observation of plaques found in Cerebral Amyloid Angiopathy (CAA)-diseased vessels within 80% of AD patients [54]. In addition, amyloid plaques occur also in other amyloid diseases, amyloidoses, of the blood circulatory system. So, in his final attempt Glenner used plaques from cerebral blood vessels as crude material for isolation; a possible hint on the nature of Aβ could have been the isolation of a low molecular weight protein with around 6 kDa in senile heart amyloid plaques in 1978 [55]. In CAA the amyloid plaques are found at the outer basement membrane in the smooth muscle layer of the lamina media of arteries and veins [54]. Two types of CAA are distinguished, based on the involvement of capillary occlusion (capillary: diameter of 15 µm or less) [54]: Type 2 without and type 1 with capillary occlusion, which is more severe and frequent in AD patients and associated with ApoEε4 [54]. In similarity to diffuse plaques in non-demented elderly, CAA occurs in many cognitively normal individuals, and at the age of 80 and above only 25-40% is free from CAA [54]. CAA-affected vessels in later stages are prone to rupture and a study on CAA and hypertension counted most microbleeds in the vascular territory of the cerebral middle artery [56]. Evidenced linkage to CAA exists for FAD with PS1- and PS2-mutations [57] as well as to the most severe CAA forms [54, 57] for the Aβ mutations Dutch E22Q [58], Flemish A21G and Iowa D23N. These and two other mutations in this critical part of Aβ, Italian E22K and especially Arctic E22G [59], confer Aβ changed fibrillogenic properties [60]. Fiber formation from Aβ42 E22G was even more enhanced with increased ionic strength than from wildtype (wt) Aβ42 [61, 62]. The reasons for stronger aggregative tendencies of Aβ E22K or E22G are supposed to originate in the termination of the usual α-helix from residues 16-23 or its change into a more unstable 310-helix, respectively [60]. As result, the α-helical hydrogen bonds, which are formed intramolecularly by the peptide backbone, can be replaced by

5 Introduction intermolecular β-sheet hydrogen bonds. Similarly, the two extra residues of wt Aβ42 seem to destabilize the α-helix 28-35 and render the whole C-terminus a higher propensity for β-sheet structure in comparison with Aβ40. An additional driving force for β-sheet-structure is exposure of the hydrophobic C-terminal side chains to a hydrous environment following the cleavage of APP. Regarding the sensitivity of Aβ to increased ionic strength, it is tempting to speculate about the effect of blood, which has 22 mm Hg hyperosmotic pressure relative to brain parenchyma [63], leaking out during microbleeds. The kinetics of plaque formation could be in the order of days to weeks, as demonstrated previously in mice [64, 65]. The middle cerebral artery (MCA) is the only one of the three cortical main arteries per hemisphere running the same route directly next to each other antiparallel with its corresponding vein, which is the superficial middle cerebral vein (=Sylvian vein), located along the Sylvian fissure between the frontal and temporal lobe. One might hypothesize that the close proximity of artery and vein could counteract the removal of Aβ in the area, for example when an arterial microbleed elevates the osmotic pressure of the adjacent area including that of the nearby vein and thus reduces the venous osmotic inward pressure which is normally the driving force for fluid being absorbed by veins [63]. So even if Aβ is stable in higher osmolarity, rupture of vessels because of hypertension might lead to its accumulation and start a vicious cycle with Aβ forming plaques leading to more ruptures and so on..

1.3 APP and the discovery of its cleavage by proteases in HEK293

Recognition of Aβ as a cleavage product and probable initiatior of AD called immediately for intense research on the precursor protein APP and the cleaving proteases, with the main intention to find out whether they can be considered as potential drug targets. From the in-depth-examination of APP, basic data piled up, allowing to follow APP‟s course through the cell from its transcription to its degradation. APP is a gene encoded on chromosome 21 and that in 18 exons of which exons 7, 8 and 15 are differentially spliced to result in in seven isoforms [NCBI-GeneID: 351] from 677 to 770 amino acids. In neurons the huge majority of transcripts are spliced to encode for 695 amino acids, whereas in other tissues mostly the 751 and 770 isoforms predominate. Synthesis and N-glycosylation of the type-1-transmembrane protein occurs in the endoplasmatic reticulum (ER) [66]. The signal peptide which is recognized by the signal recognition particle to slow down ribosomal translation and 6 Introduction thus allows for insertion into the membrane is also removed. With the majority being degraded, 20-30% of the still immature APP is trafficked through the Golgi where it is N- and O-glycosylated [66]. During transit on the secretory pathway to the cell surface APP is also sulfated and phosphorylated [66]. The constitutive seems to be restricted to the ectodomain and takes place in a post- Golgi-compartment and at the cell surface [66]. In neurons, APP is also transported anterogradely in vesicles along axons [67] to presynaptic terminals [68]. Once arrived at the cell surface it can be internalized via the two internalization signals NPTY (684- 687 in APP695) and YTSI (653-656 in APP695), with the NPXY believed to mediate interactions with clathrin [69]. Following internalization, APP can be sent retrogradely or transcytotically [70]. The fundamental scheme of APP cleavage was explored to a large degree in the Human Embryonic Kidney 293 cell line (HEK293 [71]), an XX cell line with hypotriploidy, 64 , in 30% of cells [www.atcc.org]. The first cleavage activity to be discovered upon transfection with APP695 was not one leading to Aβ production, but one excluding its production because it occurs within the Aβ- sequence, often before lysine 612 [72]. It results in the release of a large N-terminal moiety of APP into the medium, the soluble 105-125 kDa APPα (sAPPα), therefore called α-cleavage, and leaves behind a membrane bound 10 kDa C-terminal fragment (CTFα). Three proteases with this activity, commonly called α-secretases, have been described. They belong to the family of A Disintegrin And Metalloproteases ADAM9, -10 [73] and -17 (=TACE) [74] and their cleavage sites are located between amino acids 14 and 21 of Aβ with preference for peptide bond cleavage before lysine 16 [72, 75,76]. In addition to this non-amyloid pathway, the amyloid pathway was also demonstrated in HEK293 in 1992 [77]. However, before this cell type could be considered a valid research model system for Aβ generation, the identity of the proteases liberating Aβ in HEK293 and in Alzheimer patients had to be proved. However, at that time the responsible proteases in patients were unknown. For the protease cleaving at the N- terminus of Aβ, several suggestions were made like: The three metalloproteinases thimet [78], MP78 [79] and MP100 [80], two chymotrypsin-like serine proteases [81, 82] and the two aspartate proteases cathepsin G and cathepsin D [83]. The amyloid pathway involves the endosomal-lysosomal system so that cathepsin D with its acidic, lysosomal pH-optimum was tested by Ladror for cleavage of a synthetic Aβ

7 Introduction peptide which worked in the enzymatic test [83] but not when cathepsin D was cotransfected with APP in HEK293, which was published in 1997 [79]. One year earlier it has been found, that APP is processed normally to Aβ in cathepsin D knockout mice [84]. Finally, five groups using three different approaches came at the same time to the same result [23-27]. Hussain, Lin and Yan tried on an aspartate protease which was isolated from mouse kidney and had 47% identity with mouse cathepsin D [85], back then found in brain and reported to an expressed sequence tag (EST) database by SmithKline Beecham [86], where Hussain worked. Sinha‟s and Vassar‟s approaches were screens. Sinha from Elan Pharmaceuticals used a biochemical approach with inhibitors for the different protease classes and affinity-purification with immobilized inhibitor peptide. Vassar from Amgen used a molecular biology approach with a cDNA expression library constructed from HEK293 and transfected, as 8600 pools of 100, into HEK293 expressing APP with the Swedish mutation. The protease found by the five groups was hence renamed to Beta-site of APP Cleaving Enzyme 1 (BACE1) and cleaves APP to set free sAPPβ and the remnant CTFβ. BACE1 as principal β- secretase was demonstrated by abolition of Aβ-production in neuronal cultures from knockout (ko) mice [87] and overexpression of BACE1 in mice increased Aβ [88]. BACE1 is a gene on with 9 exons, which are alternatively spliced to encode four type-1-transmembrane protein isoforms, A with 501 amino acids (aa), B with 476 aa, and the catalytically not active isoforms C with 457 aa [89] and D with 432 aa. Post-translational modifications include cleavage of the signal peptide by furin in the trans-Golgi, at least in HEK293 [90], palmytoylation at three C-terminal cysteines, also in HEK293 [90], and N-glycosylation in the ER at three sites and autocatalytic processing in the medial Golgi to mature BACE1 [91] (Fig. 1).

24-45: autocatalytic propeptide cleaveage membrane enzymatic aspartates (D) N-term. 93:DTGS 289:DSGT C-term. 1-23: signal peptide cleaved by furin 476-501

N-glycosylation sites: 153, 172, 223, 354 sorting signal: L499/L500 C-palmytoylation sites: 478, 482, 485 modulation by phosphorylation of S498

Fig. 1: Posttranslational modifications of BACE1.

8 Introduction

BACE1 also autocatalytically removes its propeptide (Fig. 1); however, the importance of its removal for the activity of BACE1 was questionable in HEK293 [90]. Fully matured BACE1 is subject to phosphorylation at S498 which leads to trafficking to juxtanuclear compartments after retrieval from the cell surface as deferred from to the location of a non-phosphorylatable S498A mutant in peripheral endosomes [92]. Also S498A showed less costaining with APP [92]. When the two proteins colocalize, BACE1 cleaves APP either between methionine 596 and aspartate 597 of APP695 or between tyrosine 606 and glutamate 607. One of the two catalytic aspartates in the BACE1 activates a coordinated water molecule by deprotonating its oxygen which establishes a covalent bond to the keto-/enol-Carboxy-terminal backbone carbon of the methionine or the tyrosine. The tetrahedral oxyanion intermediate is terminated when the scissile amide bond on the new N-terminus gets a proton from the protonated catalytic aspartate to have the backbone bonded to two protons and to regenerate the deprotonated state of the catalytic aspartate [Wikipedia]. Maybe an acidic side chain on the +1 residue, as in aspartate and glutamate, accelerates the cleavage by deprotonating the hydroxyl group in the tetrahedral oxyanion intermediate. The two catalytical aspartate residues at positions 93 and 289 start the conserved motifs DTGS and DSGT. Only the two longer isoforms are proteolytically active. The cleavage produces thus, similarly to α- cleavage, a large N-terminal moiety of approximately the same molecular weight as sAPPα which is called soluble APPβ (sAPPβ) and CTFβ. Concomitantly with the search for the β-secretase, the search for γ-secretase which cleaves at the Aβ-C-terminus was underway. In 1995, cathepsin D was here also under suspicion since it cleaved a synthetic peptide with γ-cleavage sites in an enzymatic test [93] as well as a protease inhibitable with a calpain inhibitor [94]. Also in 1995, PS1 was discovered as a gene linked to very aggressive EOAD [95, 96] with much of the basic PS1 characterization also done in HEK293 [97]. Borchelt suggested in 1996 that FAD linked to PS1 mutations was due to a gain of deleterious PS1 properties because of the absence of nonsense or frame shift mutations in FAD patients [30]. Also in 1996, Citron suggested that the production of Aβ40 and Aβ42 could be conducted by different γ-secretases [98] after obtaining different levels of reduction for the two Aβ species by administration of a γ-secretase inhibitor in HEK293 and other cells.

9 Introduction

By 1998 four calpain inhibitors were demonstrated to act on γ-secretase, thus hinting to a cystein protease. At that time, Wolfe and Citron found an aspartyl protease inhibitor that led to a strong decrease of γ-secretase activity of both Aβ40 and Aβ42 [99, 100]. Also in 1998, it was found that PS1 knockout (ko) led to an extremely strong reduction of Aβ and to an accumulation of CTFs [101, 102]. Two peculiar aspartates in the transmembrane domains (TMDs) 6 and 7 of PS1 were then found to be indispensible for γ-secretase activity as shown with mutation studies in HEK293 [103], thus unifying the two lines of research. The two aspartates are not found in usual aspartyl protease motifs D(S/T)G(S/T), as in BACE1, but in a GXGD motif, at least the one in TMD7. However, data suggested, that PS1 acts not alone but in a high molecular weight complex [104, 105] and indeed binding partners were found (by immunoaffinity isolation), so nicastrin (NCT), preseniline 2 (PEN2) and APH-1 [106]. Regarding the cellular localization, PS1 was found in large amounts in the ER but also in complexes with NCT in endosomes and on the cell surface [105]. Once in the same compartment with either CTFα or CTFβ, γ-secretase cleaves the transmembrane domain approximately in the middle, mostly (90%) after valine 636 and to a minor degree (10%) after isoleucine 638 to release p3 from CTFα or Aβ from CTFβ. PS1 is also an aspartyl protease and maybe it is favorable for the reaction to have at least free electron pairs in the side chain of the +1 residue, as in threonine 639, to facilitate deprotonation of the hydroxyl group in the intermediate tetrahedral oxyanion, even when the topology in the membrane is not perfect then. As result of γ-cleavage Aβ and the larger C-terminal APP Intracellular Domain (AICD) are produced. Fig. 2: Intracellular trafficking of APP. Nascent APP molecules (black bars) mature through the constitutive secretory pathway (1). Once APP reaches the cell surface, it is rapidly internalized (2) and subsequently trafficked through endocytic and recycling compartments back to the cell surface (3) or degraded in the lysosome. Non-amyloidogenic process- sing mainly occurs at the cell surface where α- secretases are present. Amyloidogenic processing involves transit through the endocytic organelles where APP encounters β- and γ- secretases. From [107]. 10 Introduction

1.4 The families of APP and BACE1

Like many other vertebrate proteins, APP and BACE1 belong to families with more than one member. During the cloning of APP other cross-hybridizing cDNAs were obtained which suggested that APP belongs to a family of proteins [18, 108]. Indeed, human paralogs were identified in 1993 and 1997 and named Amyloid Precursor Like Protein 2 (APLP2, [109, 110]) and APLP1 [111], the murine APP paralogs APLP1 [108] and APLP2 [109] in 1992 and 1993, respectively. Triple knockouts (tko) of APP/APLP1/APLP2 in mice are 100% perinatally lethal [112, 113]. APP/APLP2 double ko (dko) mice survive the first month to less than 1% [114] or 26% [115], depending on the strain. APLP1/APLP2 dko mice also die all perinatally [116]. APP/APLP1 dko mice are viable and fertile [116] like all of the single knockout mice [115-118]. Obviously, APLP2 can compensate largely for the APP ko, the APLP1 ko and the APP/APLP1 dko, while it can be substituted by the combined actions of APP and APLP1 in APLP2 ko. Given the higher similarity of APP to APLP2 in sequence and expression pattern throughout the whole body it is likely that APP and not APLP1 compensates the APLP2 ko. The APP/APLP2 dko might thus be lethal because the exclusively neuronally expressed [119, 120] APLP1 cannot sufficiently compensate essential non-neuronal functions of APP/APLP2. In the APLP1/APLP2 dko, APP can probably substitute for APLP2 but not for the role of APLP1 in neurons. BACE1 belongs to a family together with BACE2 which has been found during the search for the chromosomal localization of BACE1 with expressed sequence tags [27, 121]. BACE1 and BACE2 have 51% [27] or 52% [121] identity. BACE1/BACE2 dko mice have a mortality of 60% in the first month, BACE1 ko mice of 43% in the first month and BACE2 ko mice were all healthy [122]. The BACE1 ko suggests a crucial function for BACE1, whereas the difference in mortality of the dko and the BACE1 ko suggests that BACE2 can compensate to a minor degree for the BACE1 ko. Triple knockouts (ko) result in mice to 68% in cortical malformations called dysplasia [112, 113]. APP/APLP2 double ko (dko) mice, of which less than 1% survives the first month [114], have behavioral deficits like ataxia, spinning and head tilting [115, 116].

In the APLP1/APLP2 dko, APP probably substitutes for APLP2 but only poorly for the role of APLP1 in neurons leading to the behavioral deficits described in few survivors.

11 Introduction

The severe cortical dysplasia in the tko on top of perinatal lethality seems to be linked to a missing crucial function which can be afforded by all three proteins even when the mice are not viable. It was suggested that the function is afforded by the C- terminus when it is cleaved off. The requirements for obtaining very similar liberated C-termini are given by the striking conservation and the same fundamental cleavage scheme with one protease cleaving off the N-terminal part close to the membrane and thus rendering it a substrate for γ-secretase which sets free either APP Intracellular Domain (AICD) or the corresponding APLP Intracellular Domain [123]. Their suggested function is corroborated by similar cortical dysplasias in PS1 ko mice having no functional γ-secretase [124] as well as in Fe65/Fe65L dko mice where AICD no longer can bind to these proteins [125]. It would be interesting to test whether the expression of the C-terminal fragments (CTFs), the substrates for γ- secretase, can rescue the dysplasias in tko mice. Also, inducible and neuron-specific ko of the three proteins, as already done for PS1 [126], could give hints, whether the APP-family is important not only for cortical development but also for brain function in the adult vertebrate. Brain development includes neuronal migration and differentiation. The cortical dysplasias described above in ko mice resemble human type 2 lissencephaly [112, 113], also called cobblestone lissencephaly and a symptom of Walker-Warburg syndrome (WWS). Lissencephaly is characterized by the lack of brain folds (gyri) and grooves (sulci), leading to a smooth brain surface, and caused by defective neuronal migration [Wikipedia]. In addition to lissencephaly, APP ko, APLP1 ko, APP/APLP1 dko mice and survivors of APP/APLP2 dko display reduced body weight, which they share with some WWS patients [www.lissencephaly.org.uk]. APP ko leads to reduced grip strength and APP/APLP2 dko to defective formation of neuromuscular junction [127] which is remotely reminiscent of the other main symptom of WWS: congenital muscular dystrophy. WWS was linked to mutations in the O-mannosyl-transferase POMT1 [128]. APP can be O-mannosylated, at least in yeast [129].

1.5 Diabetes type 2 and the families of APP and BACE1

Diabetes type 2 (DT2), which affects 20% of the US population with age 65 and older [Wikipedia] and has been reported as risk factor for AD, leads to lack of glucose in cells due to insulin resistance. Further, 10% of DS patients are diagnosed with DT2 [40, 41]. Insulin resistance of cells entails a high blood sugar level, hyperglycemia, as 12 Introduction characteristic for DT2. Its etiology is not clear, but seems to be linked to toxicity by excess O-linked-β-N-acetylglucosaminylation (O-GlcNAcylation) in pancreatic β-cells [130], maybe caused by continually high glucose or fructose levels. In AD patients on the other hand, blood glucose levels are abnormally low [131]. Hypoglycemia has also been detected in the APP-family ko mice, with up to 66% less blood sugar in the surviving APP/APLP2 dko mice and 31% less in either APP ko or APLP2 ko mice [114]; as of September 2009 no effect on glucose levels was reported for APLP1. In addition to hypoglycemia, a body weight reduction of 20-30% for the APP/APLP2 dko and the APLP2 ko [114, 115] and hyperinsulinemia linked to APLP2 ko [114] was measured. Body weight reduction reached 15% in APP ko mice [117, 118, 132] and 10% in APLP1 ko mice [116]. Not surviving APP/APLP2 mice died of wasting [113], which is defined by 10% or more reduction in weight and caused by “acute malnutrition” [Wikipedia]. BACE1/BACE2 dko mice were 30% reduced in body weight like BACE1 ko and BACE2 ko around 10% [122]. BACE1/BACE2 dko and BACE1 ko mice also died of a wasting syndrome, suggesting an etiogenesis similar as in the APP/APLP2 ko [122].

1.6 Atrophy in AD and the cholinergic hypothesis

Cerebral atrophy is linked to the clinical diagnosis of dementia in AD and also in other types of dementia. Atrophy in general describes a process of reabsorption and breakdown of tissues, with apoptosis involved on the cellular level, and which can be caused by poor nourishment, poor circulation, loss of hormonal supply, disuse/lack of exercise or disease [Wikipedia]. In AD the region exhibiting earliest, in FAD even before onset of symptoms, and strongest atrophy is the medial region of the temporal lobe, followed by the inferior and lateral regions as judged by volume reduction assessed with Magnetic Resonance Imaging (MRI) [133]. The medial temporal lobe comprises the hippocampus and the entorhinal cortex, the sites of the earliest Aβ plaque formation [134]. The hippocampus is involved in the formation of lasting memories, like the basal forebrain. Damage of the basal forebrain can also impair the ability to form lasting memories from recent experiences, a condition known as anterograde amnesia [135, 136]. It can for example be damaged by rupture of an aneurysm [135, 136], which forms usually sporadic by age 50 in 5% of the population in the area of the Anterior Communicating Artery (ACoA) and is usually unrecognized. 13 Introduction

More specifically, the cholinergic projection system provided by the Nucleus basalis of Meynert complex (NbMc) [6] and situated in the basal forebrain, has been implicated in concert with the hippocampus as important for memory and learning [34, 137]. The cholinergic neurons of NbMc are located in the substantia innominata (SI), the Medial Septum (MS) and the Diagonal Band (DB). The SI provides the cortex and in humans partly the hippocampus [138] with acetylcholine while the MS provides 90% of the cholinergic innervation to the hippocampus, at least in rodents [139, 140]. In brains of AD patients, a selective loss of cholinergic neurons was reported [141] and following studies demonstrated that ACh-related neurochemical activities, particularly that of the synthesizing enzyme choline acetyl transferase (ChAT), are mostly decreased in post-mortem AD brains compared with other neurotransmitter systems [141, 142]. The hence resultant cholinergic hypothesis which states that decreasing levels of acetylcholine (ACh) initiate AD is moreover backed by the finding, that blocking cerebral acetylcholine (ACh) receptors with scopolamine induces a several-minute episode of AD-like impairment even in healthy individuals which can be reversed with the cholinesterase inhibitor physostigmine [143]. However, the cholinergic hypothesis is challenged by the finding that, while cholinergic marker enzyme activity was strongly diminished in patients with end-stage dementia, neither the activity of ChAT nor the activity of acetylcholinesterase (AChE) was reduced in patients with mild and moderate AD [144]. Although it can be argued that adaptive changes compensate for ACh deficits in mild dementia, the most straight-forward interpretation of these results is that ACh deficits are characteristic of advanced dementia and contribute hardly to the early phases of cognitive impairment [144]. Another interpretation could be that enzymatic activities in earlier stages of AD are not strong correlates to the amount of ACh available. While in end-stage AD they correlate probably quite well with remaining levels of ACh, with 10-40% of ChAT activity remaining [141, 142] in comparison with 27% [145] to 61% (due to 8 of the 11 AD samples below detection threshold and therefore not included)[146] to 31% [147] of remaining ACh in CSF, this might not be the case in mild and moderate AD because each of the just-mentioned remaining ACh levels was pooled from patients in all different stages of AD, meaning that even if enzymatic activity is not diminished, the ACh level, however, is significantly decreased. Moreover, ACh levels correlated

14 Introduction significantly inversely with cognitive impairment when ACh concentrations were plotted against dementia scale scores [145]. If it holds true, that ACh concentrations are strongly diminished despite the presence of hardly reduced enzymatic ChAT activity, diminished ACh levels could originate in a deficit of either acetyl-Co A (Ac- CoA) or choline (or both), the substrates out of which ChAT synthesizes ACh [148]. The concentration of choline (Ch) in the CSF in the three studies mentioned was very similar for AD patients and controls [145, 147] or slightly elevated [146]. This is at first glance not suggestive of a simple downregulation of ACh production because it is assumed that physiologically the availability of Ch is rate limiting for ACh synthesis [145, 149] which is maybe regulated by Ch transport across the BBB [145, 150]. With Ch concentrations unaltered, it was proposed that rather the availability of Ac- CoA might be decreased due to hypoxia which was suggested [145] to lead to decreased glucose oxidation, decreased pyruvate levels and finally reduced Ac-CoA generation [145, 151, 152]. Hypoxia [153] and also MCA occlusion [154, 155] has been demonstrated to impair ACh synthesis in rats. In humans the SI is supplied by the precommunicating part of the ACA [156] and laterally by the narrow lenticulostriate arteries branching from the MCA which also seem to supply the DB laterally [157]. Posttraumatic infarction in the territory supplied by the lateral lenticulostriate artery can occur even after minor head injury [158]. Hypoxia might indeed contribute to a decreased Ac-CoA generation. A hint on hypoxia occurring in AD might be the very early and very dense Aβ plaque formation which has been reported for the SI and the MS [6, 159] because of demonstrated hypoxia-induced increased BACE1 expression [160, 161]. The plaque formation correlated with neuronal loss in NbMc [6]. In addition to hypoxia, undersupplied regions could suffer from hypoglycemia and it was suspected [6] that the very large neurons of the NbMc are especially vulnerable to a reduction in oxygen and glucose supply. With regards to glucose supply, it was demonstrated that the cholinergic neurons of NbMc are positive for the insulin-dependent glucose transporter 4 (GLUT4) [162]. The generation of Ac-CoA could not only be reduced by hypoxia but also by hypoglycemia or scarcitiy of other molecules precursory to Ac-CoA. Given the role of the cholinergic system in acetylcholine induced arousal and the sleep/wake cycle [163-165], and the cycle‟s coupling to the energy status [164], sensing the energy level via Ac-CoA availability would be reasonable. The brain‟s activity and hence

15 Introduction energy demand would thus be coupled to the available energy in form of Ac-CoA, which is used for the aerobic production of ATP in the Cycle (CAC). Ultimately neuronal damage by energy depletion could be avoided. For such a system to work, the energy status in the sensing neurons would generally have to be more sensitive to changes than that of the neurons which are monitored. At the same time the sensing neuron should possess enough reserves to survive brief low energy episodes like neurons with large cell bodies as the magnocellular cholinergic neurons which are also suspected to be sensitive to hypoxia and hypoglycemia [6]. However, the system is not operating sufficiently well any longer when the energy status of the monitored cells in dependence of the available energy changes considerably relative to that of the sensing neurons or when the sensing neurons have died. Specialized glucose sensing neurons triggering glucagon release rely on ATP-dependent potassium channels [166] and have been postulated in the ventromedial hypothalamus [167-169], the most important regulatory center of the autonomous nervous system which is involved in homeostasis of temperature, blood pressure, osmolarity, regulates feeding and drinking, the circadian rhythm and sleep, largely via control of the pituitary gland [Wikipedia]. It is adjacent to the basal forebrain and richly connected to the DB and MS [Wikipedia]. Reduction in the glucose metabolic rate of the cerebrum is a predominant abnormality even before onset of clinical symptoms or mild cognitive impairment (MCI) [170-174], which leads to a reduction in ATP formation from oxidized glucose and oxygen of 20% in mild LOAD to 35%-50% in advanced LOAD [175]. Also in demented DS patients a 28% reduction of glucose has been measured [176]. The reduced ATP formation is not caused by hypoxia because at least in the beginning the metabolic rate of oxygen is not reduced [175]. The deficit in glucose availability was suggested to be compensated by catabolization amino acids and fatty acids [177]. Indeed, elevated levels of choline in the CSF of AD patients [146] and upregulation of Phospholipase D (PLD) in the AD brain [178] could argue for the use of phosphatidylcholine as Ac-CoA donor. One could further speculate that a prolonged breakdown of membrane lipids leads to a substantial loss of white matter. In patients of anorexia nervosa with an average body mass index (BMI) of 15.6 kg/m2 (1.7 m, 45 kg), white and grey matter loss in association with ventricular enlargement by MRI was measured, which was largely reversible by treatment [179]. In AD patients an association exists between white matter loss and

16 Introduction cortical atrophy, evoked by high blood pressure, and diabetes in the absence of stroke lesions [180]. The brain is not the only organ suffering from atrophy in AD and generally AD patients lose more weight and are thinner than patients of other dementias [181]. About one third of AD patients lose 4% to 7% of weight [181, 182] and one tenth of AD patients experience severe weight loss [182], the reason of which is not yet clear.

1.7 Tau modifications and BACE1 transcriptional regulation

According to the Nobel Prize laureate Eric Kandel, an upregulation of cAMP in the brain is pivotal for AD [183]. A (PKA) could thus be activated and act on substrate proteins like Phospholipase D (PLD) [184] or prephosphorylate the microtubule associated protein Tau [185] for the proline-directed Glycogen Synthase Kinase 3β (GSK3β), another major Tau kinase. Prephosphorylation resulted in an enhanced GSK3β-mediated Tau phosphorylation at 8 sites, among them serines 199, 202 (longest isoform numbering) and threonines 181, 205, 231 [185]. In ground squirrels, hibernation induced reversible Tau hyperphosphorylation, particularly in the entorhinal cortex, hippocampus and isocortex, at 12 sites, among them serines 199, 202 and threonines 181, 205 and 231 (corresponding residues in human Tau) [186]. Hibernation is a behavioral strategy used by several mammalian species to minimize energy expenditure under inhospitable environmental conditions [186], which might be similar to reduced glucose metabolism, as in AD. PKA-mediated prephosphorylation of Tau did not affect phosphorylation of Tau by the also proline-directed Dependent Kinase 5 (CDK5) [185]. Further, CDK5- activating p25 was not required for Tau phosphorylation in hibernating ground squirrels [187]. CDK5 is the only known CDK that displays activity in postmitotic neurons [188] and p25-mediated overactivation results in neurodegeneration [189]. Activity of cdk5 is essential for neurite outgrowth and axon patterning [163, 190-192] and substrates besides Tau [189, 193] are for example neurofilaments [163, 194- 197]. With regards to Aβ pathology it has been shown that inhibition of APP trafficking by Tau does not increase the generation of Aβ [198]. The combined actions of GSK3β and CDK5 resulting in Tau hyperphosphorylation could thus be regarded as an inappropriate molecular atavism in addition to a pathologic deregulation, respectively. Serine and threonine residues can be differently modified by O-linked-β-N- acetylglucosaminylation (O-GlcNAcylation) which is competitive with phosphorylation 17 Introduction but similar because of reversibility and no further modification of the attached glucosamine [199]. O-GlcNAcylation has been reported for Tau [200] and it is decreased on Tau to one quarter in AD [201], which might give way for hyperphosphorylation. Knockdown of O-GlcNAc transferase (OGT) resulted in marked increase of phosphorylation for example on threonine 205 amongst others [201]. Activity of OGT seems to be strongly dependent on availability of the substrate UDP- N-acetylglucosamine (UDP-GlcNAc) [202]. UDP-GlcNAc is produced in the Hexosamine Biosynthesis Pathway (HBP) starting from Fructose-6-Phosphate (Fru- 6-P) (Fig. 3). Approximately 2-5% of total glucose [201, 203] is used as Fru-6-P by the rate limiting enzyme of the HBP [204], Glutamine:Fructose-6-Phosphate Amidotransferase (GFPT) [205] to produce the UDP-GlcNAc-precursor glucosamine- 6-phosphate (GlcN-6-P).

Fig. 3: The hexosamine biosynthetic pathway, and the major O- GlcNAcylated proteins involved in diabetes and AD. The O-GlcNAcylation levels are responsive to flux through HBP. Numbered are as follows: 1. ; 2. glucose-6-phosphate ; 3. GFPT; 4. glucosamine-6-phosphate acetyl -transferase; 5. phosphateacetylglucosamine mutase; 6. UDP-N-acetylglucosamine pyrophosphorylase; 7. or hexokinase. Glc, glucose; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; UDP -GlcNAc, uridine5‟-diphospho- N-acetylglucosamine; OGT, O-GlcNAc transferase. Red arrow indicates rate-limiting enzyme of HBP. Blue arrows and text indicate other biological processes. From [206].

GFPT competes with (PFK) for Fru-6-P, more specifically two isoforms of GFPT (GFPT1 [207] and -2 [208]) compete with homogeneous and heterogeneous tetramers [209, 210] of three isoforms of PFK (PFKL [42], -M, -P for

18 Introduction liver, muscle, platelet). Km values of GFPT1 for Fru-6-P have been determined with 0.2 mM [211], 0.4 mM [212], 1.04 ± 0.09 mM [213], 1.41 mM for Candida albicans GFPT [214] and 7 µM (for purified recombinant human GFPT1) [215]. It is sensitive to product inhibition by GlcN-6-P with a Ki of 6 µM [215] and UDP-GlcNAc with a Ki of 4 µM [215] or 20 µM [211]. GFPT is phosphorylated by cAMP-dependent (PKA). PKA upregulated the activity of GFPT from rat liver 1.7-fold [216], Drosophila melanogaster 1.7-fold [217] and Candida albicans 2-fold [214]. Activity of a glutathione-S-transferase-GFPT1 fusion protein in contrast was completely inhibitable by PKA-dependent phosphorylation of serine 205 [218] which was also suspected as phosphorylation site in the other studies [216]. The authors argued that PKA regulation might be isoform-dependent. Two putative PKA sites, serines 205 and 235, are present in GFPT1, while in GFPT2 only serine 205 is present [213]. Murine GFPT2 was stimulated 2.2-fold by PKA at serine 202 (corresponding to serine 205 in GFPT1) in vitro [219]. GFPT1 was also activated 1.4-fold following phosphorylation at serine 243 by CaMKII and AMPK [213]. On gene expression level, GFPT is strictly tissue-specific regulated in humans [213, 220]. In different rat tissues the activity of GFPT varies up to 200-fold depending on the more or less high expression rates of glycoproteins [217, 221]. Km values of PFK range from 0.1 mM of rabbit erythrocyte PFKL [222] to 0.18 mM of rat heart PFK [223] to 0.45 mM of human erythrocyte PFKL [210]. The affinity for Fru- 6-P increases from PFKP to PFKL to PFKM [224]. PFKL is allosterically activated by Fru-2,6-P2 which is produced by PFK 2. PFK 2 in turn is activated by high levels of Fru-6-P and dephosporylation and deactivated by glucagon and PKA [225]. PFKL expression is coupled to glucose metabolism by hormonal regulation through insulin which induces expression in feeding and glucagon which represses expression in starvation [225]. Also, insulin-induced genes are generally repressed by cAMP and vice versa [226]. The level of GFPT activity correlates with glucose disposal rates in humans and transgenic mice [227]. The glucose level can thus be sensed by the HPB and UDP- GlnNAc can regulate aspects of glucose metabolism [227]. Overexpression of GFPT resulted in transgenic mice in insulin resistance [228]. Sensing of the extracellular glucose level by pancreatic β-cells relies also partly on O-GlcNAcylation as the diabetogenic action of the O-GlcNAcase inhibitor streptozotocin, a UDP-GlcNAc analogue, suggests [130]. Further, OGlcNAc-modification of certain proteins is known

19 Introduction to change in response to T cell activation, insulin signaling, glucose metabolism, and cell cycle progression [229]. Reduced UDP-GlcNAc levels predominantly translate into decreased O-GlcNAc modifications of cytosolic and nuclear proteins [230]. This might have effects on transcription factors which can be O-GlcNAcylated [229], like Sp1 [231], Yin Yang 1 (YY1) [232] and SRF [233]. O-glycosylation of a Sp1-derived peptide blocks known Sp1 protein interactions, like those to the TATA TAF110 [231], whereas YY1-O-GlcNAcylation disrupts binding to the Retinoblastoma protein Rb, possibly liberating it to bind DNA [232]. As of 2003 the role of SRF-glycosylation still needs to be established [233, 234]. Sp1 overexpression enhanced BACE1-mediated cleavage of APP and deletion of a 34 bp Sp1 binding element in the human BACE1 promoter markedly reduced the promoter activity [235]. YY1 has been demonstrated to bind a putative recognition sequence CCATCTCGGCA within the BACE1 promoter at position -572 from the translational start (ATG) and was believed to stimulate BACE1 transcription in activated astrocytes [236]. YY1 and BACE1 are expressed by neurons in rat brain, but only a minor proportion of neurons co-expressed YY1 and BACE1, which lead to the suggestion that YY1 is not required for constitutive neuronal BACE1 expression [236]. A different interpretation could be that YY1 suppresses the expression of BACE1 in neurons. Indeed, YY1 has been demonstrated to repress the expression of a number of proteins including the adeno-associated virus (AAV) P5 promoter [237], the long terminal repeat of Moloney murine leukemia virus [238, 239], the immunoglobulin (Ig) K 3' enhancer [240], the c-fos promoter [241, 242], the human papilloma virus-18 (HPV-18) promoter [243], the N-ras promoter [244], the HPV-16 promoter [245], the histidine decarboxylase gene [246], the human granulocyte-macrophage colony- stimulating factor (HuGMSCF) gene promoter [247], the Dystrophin promoter [248], the skeletal [249] and the smooth muscle [250] α-actin promoter. YY1 is provided with a strong repression domain consisting of four C-terminal GLI-Krüppel type zinc fingers [251]. In the adeno-associated virus (AAV) P5 promoter transcription is repressed by YY1- binding to a site CCAATATGTCA, found at -1242 from TSS [252]. Not only is repression relieved by binding of the adenovirus E1A protein [253] but also YY1 then stimulates transcription. This switch of YY1 function modulates the AAV P5 promoter

20 Introduction activity between 250- and 1,000-fold. In the long terminal repeat of Moloney murine leukemia virus YY1 binds to CGCCATTTT [252]. In the human papilloma virus-18 promotor, YY1 downregulates transcription initiating activity of AP-1 and in the N-ras promoter it downregulates AP-1 or Sp1 activity by binding to 20 bp regions with very high A/T content [243, 244]. The HuGMCSF gene promoter activity is transactivated by an AP1 complex and a Sp1-related complex which is suppressed by a YY1 complex [247]. In the long control region of HPV-16 DNA from cervical carcinomas mutations of YY1-binding sites were identified which increased promoter activity 3–6-fold. The YY1- was positioned upstream of Sp1 [245]. The histidine decarboxylase gene expression is repressed by an YY1/SREBP-1a complex through an upstream Sp1 site [246]. Evidence for a physical interaction between YY1 and Sp1 exists [237] but is not necessary to repress Sp1- and CREB-mediated transcription [251]. In similarity to the switch in YY1 function following binding of E1A, a synergistic enhancement of SP1 and YY1 for transcriptional activity with upstream Sp1 binding site has also been reported [254]. The physical interaction of YY1 and Sp1 involves aa 260-331 (of 414) from YY1 and aa 298-331 (of 331) from Sp1 [237]. In the case of the c-fos promoter [241], the Dystrophin and the α-actin promoters compete with the repressor YY1 and the transcription activator Serum Response Factor (SRF) for a CC-AT-rich(6)-GG (CArG) box. Increased actin polymerization reduced the inhibition of SRF activity by YY1 [255]. Also for the M isozyme of the (MCK) promoter, competition of recombinant SRF and YY1 for the CArG element CCATACAAGG was reported [256]. CArG boxes are the predominant binding sites for SRF as is well established from a large number of biochemical and mutagenesis experiments, since 61% of SRF sites contain CArG boxes [258]. As consensus sequence, CC(A/T)TATA(A/T)GG was calculated [258], although any combination of A and T in the inner six seems to be functional as the description of the CArGome suggests [259]. The occurrence of in in silico identified CArG boxes peaks at 1 kb upstream to the transcription start site (TSS) [259] and verified CArG boxes cluster at around -1250 from TSS [257]. For the study on YY1-binding to the rat BACE1 promoter, the 5‟-flanking region of BACE1 was cloned, beginning at -1541 from start ATG [236]. At position -1828 from ATG, -1401 from TSS in the rat genomic sequence a CArG box CCTTTTTTGG is

21 Introduction located. At position -1732 from ATG, -1275 from TSS [260] in the human genomic sequence a (in [235] and [268] not designated) CArG box CCTTTAATGG is located, 128 bp upstream of the active [235] Sp1 binding element. Binding of YY1 to the CArG box could, as on other promoters, suppress Sp1-mediated BACE1 transcription when it is freed from interaction with Rb by O-GlcNAcylation. Administration of the competitive hexokinase inhibitor 2-deoxy-glucose resulted in Tg2576 mice in more BACE1 on protein level [261]. The effect was attributed to enhanced translation as demonstrated by in vitro experiments with a CMV-promoter-BACE1-construct [261]. The BACE1 promoter also contains a potential AP1 binding site at -1487 from ATG, - 1030 from TSS [235] and an active HIF-1-binding site at -835 from ATG, -378 from TSS [262]. The combination of the hypoxia-responsive Sp1-, AP1- and HIF-1-binding sites is typical for promoters of animal glycolytic enzymes [262, 263]. Active HIF-1 binding sites are found in at least eight glycolytic enzyme genes, among others hexokinase [264], and the key glycolytic regulatory enzymes PFKL and . In the regulation of muscle-specific pyruvate kinase (PKM) and β- [265], Sp1 plays an important role. Also, in PFKP [224] and the human PFKM [266] eight Sp1 binding sites were determined. Hypoxia as sensed by tissues is a more frequent condition than usually anticipated [262] because the normal pO2 of most tissues ranges from 50 mm to 70·mmHg [267], compared with 150 mmHg in air, while less than 40·mmHg

(5% O2) [268] activates the HIF-1 pathway. The activated astrocytes co-expressing BACE1 and YY1 were activated via occlusion of the MCA [236]. Also, regarding the high expression of BACE1 in pancreas [269], HIF-1α and -β, which combine to the transcription factor HIF-1, were upregulated in pancreatic β-cells [270]. Blood glucose levels of DT2 patients are controlled by measuring the fraction of glycated hemoglobin (HbA1c) which, in the T state, has a tenfold higher oxygen affinity accompanied by a slower rate of ligand dissociation than normal hemoglobin (HbA0) [271]. The HbA1c in DT2 patients is at most 10% in comparison to normal values of 5%, however due to the concerted hemoglobin model the difference in glycation could exert a noticeable effect on oxygen supply. PFKP is highly abundant in the brain and, with the allosteric properties, regarded as its key enzyme for the regulation of [224]. PFKL resides in the DS critical region on chromosome 21 [272]. Enhanced PFK activity in erythrocytes of DS patients along with a decrease in Fru-6-P, an increase in Fru-1,6-P2 [273] and a 20-

22 Introduction

40% faster glycolytic rate [274] has been reported. Enhanced glucose metabolism was measured in brains of young DS patients [275] and transgenic mice overexpressing PFKL had a 58% faster initial glucose utilization [276]. One could therefore argue that increased utilization of glucose in brains of DS patients is mediated through increased utilization of Fru-6-P by PFKL – since the other glycolytic enzymes are not encoded on chromosome 21. One could further speculate whether less Fru-6-P is then left for the HPB leading to less O- GlcNAcylation of Tau and YY1, and ultimately to AD pathology. However, in the case of the DS patient with partial trisomy 21 and suggestive of DS phenotype [44], APP was not triploid whereas BACE2 and PFKL were, according to their location with respect to AML1 (RUNX1, [NCBI Map Viewer]). Firstly, this suggests that triploidy for BACE2 does not lead to enhanced Aβ production, in line with previous data ruling out BACE2 as being responsible for AD in DS patients [277]. Secondly, it most probably rules out PFKL overexpression as sufficient cause for AD pathology in DS patients, whereas it could still predispose them.

1.8 Influence of kinases on Aβ-generation

A large body of evidence suggests that Aβ production is regulated by phosphorylation-dependent signaling. ADAM17 for example, one of the α-secretases, was activated upon phosphorylation of threonine (T) 753 by a MEK1/2, upstream of ERK2, which was prevented with the inhibitor (inh) PD98059 [278]. A siRNA-mediated knockdown (kd) or inhibition of the phosphorylating kinase should therefore increase Aβ production. Another prominent protein in AD research, ApoE, was important for the formation of Aβ-plaques in APP-V717F transgenic (tg) mice because plaque number rose dose- dependently in the genotypes ApoE-/- to ApoE+/- to ApoE+/+ [279]. ApoE can be phosphorylated by Casein Kinase (CSNK) 2 in vitro at serine (S) 296 [280]. Tg mice overexpressing mutant forms of APP overproduced Aβ and developed AD-like pathologies in the brain [281]. AP-2 is a transcription factor which bound to the ApoE promoter [282] and might bind to a responsive element in the 5‟ region of BACE1 [235]. The α-isoform was phosphorylated in vitro by PKACa at S239 which stimulated transcriptional activity [282]. Knockdown of PKACa could decrease BACE1-transcription.

23 Introduction

APBA1-(APP-binding, family A, member 1)-overexpression led to an Aβ40 reduction of 20% [283] and overexpression of APBA2 to a reduction of 18-35% Aβ40 and 18- 45% Aβ42 [284]. APP-phosphorylation was abnormal in AD [285] and mature APP was most abundantly phosphorylated on the three residues S655, T654 and T668 (APP695 numbering) [286]. Physiologically it was phosphorylated in neurons on T654 [286] which is found in the YXXI motif important for the endocytic/basolateral sorting of APP in MDCK cells [287, 288]. T668 was physiologically phosphorylated in neurons and most abundant in immature APP [286]. In mice no effect of phosphorylation on Aβ production was measurable [289]. However, a significant upregulation of phosphorylation at T668 was determined in AD brains and in CTFβ and rat cortical neurons transfected with nonphosphorylatable APP-T668A produced less Aβ [290]. T668 was phosphorylated by cdc2 [291], Cdk5 (inh roscovitine) [292], GSK3β [293], JNK (inh SP600125) [294] and SAPK1b/JNK3 [295]. APP can also be phosphorylated at Y682 by Abl non-receptor [296] and Trk [297]. Based on the effect of APP-T668A, it is expected that kd of the phosphorylating kinases results in decreased Aβ production. BACE1 overexpression resulted in a twofold increase of Aβ40 and Aβ42 [26] and S498-phosphorylation by Casein Kinase (CSNK) 1 (inh hymenialdisine) resulted in trafficking to late endosomal compartments [92] and enhanced interaction with GGA1 [298]. CSNK1D (=δ) was the isoform which phosphorylated BACE1„s sorting signal in HEK293 at S498 after full maturation (glycosylation) of BACE1 and a S498A mutation abolished phosphorylation [92]. In comparison with S498A, the phosphorylation mimic S498D and the phosphorylatable wildtype S498 showed more internalization from the cell surface and phosphorylation-dependent retrieval from endocytosed vesicles [92]. CSNK1D was upregulated in AD brain hippocampus 30-fold on protein level [299] and 24-fold on mRNA-level [300]. It colocalized with pathological Tau tangles in AD and DS brains [301]. Overexpression of full length CSNK1E (=ε) or constitutively active CSNK1E-271 resulted in 129% Aβ40 and 121% Aβ42 or 194% Aβ40 and 261% Aβ42 [302]. Inhibition of CSNK1E with D4476 increased CTFβ, thus probably acting on γ-secretase [302]. CSNK1E underwent autophosphorylation at S323, T325, T334, T337, S368, S405, T407, S408 and phosphorylation inhibited the enzymatic activity which increased 8-fold with S/T to A mutations [303]. Kd should result in decreased Aβ production.

24 Introduction

DAPK1, more specifically its SNP rs4878104, had a significant association with LOAD [304]. Its kinase activity was inhibited by phosphorylation at S289 by p90 ribo S6K, downstream of ERK1/2, (inh U0126 [305, 306]) [307] and at S308 by autophosphorylation (inh Ca2+ C6-Ceramide) [308] and activated by phosphorylation at S735 by ERK2 (inh PD98059) [309]. eIF2α-phosphorylation regulated BACE1- mRNA-translation [261] and inhibition of eIF2α-dephosphorylation by 1c with salubrinal (found by [310]) increased BACE1- and Aβ40-levels to 1.5-2-fold. eIF2α was phosphorylated by a number of kinases including double- stranded RNA-activated Kinase (PKR), General Control Nonderepressible 2 kinase (GCN2), Heme-Regulated Inhibitor kinase (HRI), and PKR-like ER Kinase (PERK), which was abundant in pancreatic δ-cells [311], mostly on serine 51 [312]. PERK was identified as eIF2α-phosphorylating kinase in glucose deprivation experiments of HEK293 stably overexpressing a CMV-promoter-BACE1-construct. Transfection of the cells with a dominant-negative PERK or the also pancreatic [313] chaperone P58IPK, which was thought to bind to and thereby inhibit the kinase domain of PERK [314], prevented the increase in BACE1 [261]. The same effect had the administration of tauroursodeoxycholic acid [261] which alleviated ER (Endoplasmic Reticulum) stress and normalized hyperglycemia in obese and diabetic mice [315]. Kd of PERK could reduce BACE1-levels. Fe65-expression in MDCK and H4 neuroglioma led to increased APP on the cell surface and increased Aβ secretion [125, 316], whereas in HEK293 overexpression of Fe65 suppressed slightly the maturation of APP and led to decreased Aβ secretion which was reversed with APP-T668E [317]. FE65 showed a higher affinity to nonphosphoAPP [285, 318]. Phosphorylation at Y547 by c-Abl tyrosine kinase stimulated APP/Fe65-mediated gene transcription, but did not affect APP-Fe65- binding [319]. Fe65 was also phosphorylated at S175 by ERK2 (inh U0126 phorbolester) [320]. GGA1-overexpression or application of a dominant-negative variant reduced BACE1- mediated cleavage as Aβ and CTFβ decreased while anti-GGA1-siRNA increased the secretion of Aβ. The modulation of Aβ generation was probably due to changes in subcellular trafficking of BACE1 or other GGA1-dependent proteins because it was independent of a direct APP-GGA1-interaction and GGA1 expression did not affect the total cellular activity of BACE1 [321]. Phosphorylation of S355 by CSNK2 in vitro led to autoinhibition [322], which suggests that Aβ-production is expected to

25 Introduction decrease upon CSNK2 kd. GGA3-depletion with siRNA increased levels of BACE1 7-fold (H4 neuroglioma cell line with APP751) or 4-fold (H4-swAPP), APP-C99 and Aβ 2-fold (H4-APP751) or 1.5-fold (H4-swAPP) [323]. JIP-1-overexpression promoted the transcription of APP [324] and siRNA-mediated downregulation impaired transport only of APP695 phosphorylated at T668 [318]. JIP-1-phosphorylation at T103 either by JNK-1 [325] or JNK-2 [326] was a prerequisite for activation of the JNK module. c-Jun NH2-terminal kinase (JNK) phosphorylated APP which accumulated in neurites [285]. Based on the effect of APP695-T668A, less Aβ production is expected by JNK-1/2 inhibition or kd. Downregulation of the JNK interacting protein JIP-3 by siRNA also reduced the amount of APP695 phosphorylated at T668 [285]. Further, stress-activated JNK phosphorylated APP only in the cell body while concomitant expression of JIP-3 restored accumulation of phosphorylated APP into neurites. LaminA/C was extensively phosphorylated at T19, S22, S277, S390, S392, T394, S395, S403, S406, S407, T409, S414, S423, S458, S652 [327-331] and its SNP rs505058 was a significant marker for LOAD [332]. LRP (low density lipoprotein receptor-related protein) mediated the endocytosis of APP isoforms containing the KPI domain [333, 334]. The LRP antagonist RAP led to increased cell surface levels of APP with a significant reduction in Aβ synthesis whereas LRP-expression in previously LRP-deficient cells resulted in 3- to 4-fold more Aβ [335]. LRP-phosphorylation at S76 by PKACa led to internalization [336], whereas another study, which also implicated PKCα (inh Ro-32-0432) found that the T16A/S73A/S76A/S79A mutation reduced the endocytic rate [337]. MMP-2 degraded Aβ [338] and was upregulated with 10 µM clioquinol and Cu or Zn which resulted in 90% less Aβ [339]. MMP2 was phosphorylated at S160, T250, S365, T377, T378 [340]. An increased activity of MMP-2 and MMP-3 after clioquinol/Cu/Zn-mediated activation of PI3K and JNK was observed which could be blocked with LY294002 or Wortmannin or SP600125. Clioquinol and Cu also promoted phosphorylation of glycogen synthase kinase 3 (GSK3) which enhanced activation of JNK [339]. Nicastrin-overexpression resulted in a 4-fold increase of Aβ [341] and inhibition of Nicastrin-phosphorylation by ERK1/2 by siRNA or the inhibitors PD98059 and U0126 but not the p38 kinase inhibitor, SB202190, or the JNK inhibitor, SP600125, increased γ-secretase activity by 30% [342].

26 Introduction

PAR-4-overexpression significantly increased and silencing of PAR-4 expression by siRNA significantly decreased β-secretase cleavage of APP [343]. In rat, PAR-4 was phosphorylated at T155, leading to apoptosis [344], and S249 by Akt. Suppression of Akt activation with the PI3K-inhibitor PTEN or LY294002, suppression of Akt expression by siRNA or Akt function by dominant-negative Akt caused apoptosis [345]. Human PAR-4 was phosphorylated at Y226, S228, T229 and S231 [329]. PCK1-SNPs at I94 and G380 were significantly associated with LOAD [346] and in mouse it was phosphorylated at S118 [347]. Pin1 is a prolyl isomerase which catalyzed cis-trans conformational changes in S/T- P-motifs and accelerated the isomerization of T668-phosphorylated APP over 1000- fold [348]. Overexpression reduced Aβ. Pin1 was reduced in AD neurons and its genetic deletion decreased the generation of sAPPα [348]. Pin1-phosphorylation at S16 by PKA enabled Pin1-substrate interactions [349] and at S65 by reduced ubiquitination as tested with kd of Plk1 and the phosphorylation-mimic S65E in HeLaS3 [350]. Inhibition or kd of both PKA and Plk1 should therefore increase Aβ generation. PPARγ-activators or NSAIDS or overexpression of PPARγ, as repressor binding to a PPARγ-responsive element in the BACE1-promoter, reduced BACE1-expression [351]. PPARγ-transcriptional activity was reduced by phosphorylation at S82 by MAPK (inh PD98059, BRL49653) [352] or at S84 or in vitro by ERK2 and JNK [353]. Kd or inhibition of the kinases should decrease Aβ-production if transcriptional and repressive activities rely on similar DNA binding. PSEN1 is the catalytic subunit in γ-secretase complex and was phosphorylated at S310 and S346 by PKACa [354], at S353 and S357 by GSK3β [355], and at T354 by Cdk5 which stabilized PSEN1 [356]. Phosphorylation also reduced binding to N- cadherin and therefore cell surface expression but had no effect on APP processing [357]. PU.1-expression resulted in a 5- to 10-fold enhanced Cathepsin S (CTSS) promoter activity [358] and overexpression of CTSS resulted in 2-fold higher Aβ-levels which was blocked with cysteine protease inhibitor E-64d [359]. PU.1 was phosphorylated by the p38-MAPK-dependent pathway (inh SB203580, anisomycin) at S142 in the SIE motif which was involved in the IL-3 stimulated mcl-1 gene transcription [360] and at S148 by CSNK2-A1 [361] which was required for interaction with the transcription factor ICSBP/IRF8 [362]. Further, LPS induced phosphorylation

27 Introduction potentiates COX-2 gene expression [363]. If phosphorylation of PU.1 also enhances CTSS promoter activity, inhibition or kd of the kinases should lead to decreased Aβ- production. p35-overexpression enhanced the secretion of Aβ, sAPPβ and sAPPα [364]. Phosphorylation of the Cdk5-activator p35 at S8 and T138 by Cdk5 in fetal brain was found in adults and mature neurons at S8 only [365]. S8A/T138A mutation increased cleavage of the N-terminal 98 aa by calpain to p25, whereas S8E/T138E reduced it like phosphorylation, especially of T138 [366]. p35 had a half-life of 20 to 30 min and inhibition of Cdk5 activity by roscovitine or overexpression of a dominant negative mutant of Cdk5 increased the stability of p35 by 2- to 3-fold [367]. A T138A mutant increased the turnover rate 2-fold and a S8A/T138A/S170A/T197A mutant decreased it 3-fold [368]. Rab5-overexpression resulted in a 2-fold increase of CTFβ and a 2.5-fold in Aβ40 and Aβ-42 [368]. Rab5a was phosphorylated after insulin stimulation by ERK1, not ERK2, and Rab5b at S123 by p34cdc2 [369]. SORL1-overexpression in SHSY5Y resulted in 75% Aβ decrease, knock-out in 40% Aβ increase and protein levels were reduced by 80% in AD brain [370]. The intronic SNPs # 8,9,10 and # 22,23,24 were linked to LOAD [371]. It was further demonstrated that SORL1 directed APP to recycling compartments and that with less SORL1 expression APP was sorted into Aβ-generating compartments [371]. The short cytoplasmic part of SORL1 contains motifs for phosphorylation [372]. SUMO-2/3-overexpression reduced Aβ production and dominant-negative mutants increased it [373]. Generally, phosphorylation of proteins affects their SUMOylation [374], however, APP was not directly SUMOylated [375]. Sp1-knockout downregulated BACE1-mRNA by 40% and overexpression upregulated the protein level of BACE1 to 185%, C99 to 212%, Aβ to 216%, which was reversed by mithramycin A down to 50%, 21%, and 56%, respectively [235]. The transcription factor, binding to the Sp1-response element in the BACE1-gene promoter [235], was phosphorylated at T579 by casein kinase II in vitro, which was increased by okadaic acid through inhibition of the liver endogenous PP1. Phosphorylation decreased DNA binding activity [376]. Further Sp1 was phosphorylated at T453 and T739 by ERK1/2, not p38, which enhanced the interaction of Sp1 with the PDGFR-α promoter and switched it from an activator to a repressor of transcription [377]. Also, T453 and T739 phosphorylation by ERK1/2

28 Introduction

(MEK1/2 inhibitor PD98059) enhanced the interaction of Sp1 with the p21 promoter, but this led to increased p21 transcription [378]. T453 and T739 were also phosphorylated by p42/44 MAPK which enhanced the interaction of Sp1 with the VEGF promoter and led to increased VEGF transcription [379]. The Chinese hamster dhfr gene was repressed in the cell-cycle withdrawn G0 phase by Sp1 and hypophosphorylated retinoblastoma protein (Rb) in association with histone deacetylases (HDAC) which could, in serum starved CHOC400 cells, not become derepressed by the HDAC-inhibitor TrichoStatin A (TSA). Phosphorylation of Rb after serum stimulation abolished the Sp1-Rb interaction while the Sp1-HDAC interaction persisted into the S phase [380]. The Rb-related protein p107 also interacted with Sp1 and transient expression of p107 repressed Sp1-dependent transcription [381]. The Luteinizing Hormone Receptor (LHR) gene was silenced via a proximal Sp1 site at its promoter. TSA induced histone acetylation and Sp1-site-dependent gene activation in JAR cells via PI3K/PKC-mediated Sp1 phosphorylation at S641. PKC was shown to associate with Sp1 and the association was enhanced by TSA. The S641-phosphorylation was required for the release of the LHR gene repressor p107. Blockade of PI3K with LY294002, Wortmannin or a dominant-negative p110α catalytic subunit, or the downstream PKC with a myristoylated PKC pseudosubstrate peptide inhibitor or a kinase-deficient mutant, but not by the PKCα,β,γ-inhibitor Gö6976 or the PKCδ-inhibitor Rottlerin, decreased Sp1- phosphorylation, p107 release and LHR-transcription. Kd of either Sp1 or PKC with siRNA had the same effect on LHR transcription [382]. An effect of impaired Sp1- phosphorylation on Aβ-generation cannot be predicted so far. Kinases affecting major players in Aβ-generation are summarized below in Tab. 1.

Major player APP BACE1 α-secretase γ-secretase ApoE Overexpression/ + / - + / - - / + + / - +? / - knockdown effect on Aβ PKACa / + Kinase phosphorylating cdc2 / + CSNK1D / + directly or phosphorylating CDK5 / + CSNK1E / + an intermediate effector GSK3β / + PERK / + (transcription factor, ERK1/2 / - JNK / + PKR / +? MEK1/2 / -? interaction protein) PKACa / +? SAPK1b GCN2 / +? PKA / -? PKACa / +? / demonstrated or expected GSK3β / +? =JNK3 / + HRI / +? Plk1 / -? (?) effect on Aβ CDK5 / +? JNK-1/2 / +? CSNK2 / +? Knockdown by siRNA PKA / -? MAPK / -? should have the opposite Plk1 / -? ERK2 / -? effect JNK / -?

29 Introduction

1.9 Difficulties with direct inhibition of BACE1

Initial results for BACE1 had placed high hopes on it as potential drug target for a disease-modifying therapy. Not only was it demonstrated in ko mice that BACE1 is the sole or at least the main β-secretase for the generation of Aβ [87] but also that its variability in human is quite limited both on the level of single nucleotide polymorphisms (SNPs) and between the different ethnicities. Moreover and maybe more importantly, cleavage at the β-site of APP was regarded as rate-limiting step in Aβ-generation. However, similarly to γ-secretase, drawbacks appeared in the last years in the form of other proteins than APP as substrates for BACE1. Firstly, as already mentioned, APLP2 was cleaved by BACE1 [123] and APLP2 ko mice suffered from hypoglycemia and hyperinsulinemia [114]. Systemic inhibition of BACE1 could prevent the generation of APLP2-cleavage products and result in similar side effects. In 1999 a Golgi-resident α-2,6-sialyltransferase (ST6Gal1) was found to be cleaved between K40/E41 [383] and validated as substrate for BACE1 [384]. Cleavage by BACE1 enhanced the sialylation of soluble glycoproteins [385]. Another sialyltransferase, the polysialyltransferase ST8Sia4, was cleaved by BACE1, however only in vitro and just 10% of ST8Sia4 compared to more than 60% of ST6Gal1 [386]. Interesting about the cleavage of ST6Gal1 is the topology of substrate and protease: ST6Gal1‟s N-terminus is cytosolic and immediately followed by the transmembrane domain and a “stem” which connects to the catalytic lumenal domain [384] whereas BACE1 has a cytosolic C-terminus and is thus antiparallel to ST6Gal1. Therefore, BACE1 probably has to turn around a good part if the same parallel topology as with APP is necessary. An antiparallel cleavage would for BACE1 be tantamount to accommodating a lysine in the glutamate binding site in addition to a point-mirrored amide bond in the active site. The substrate domain can probably not turn around very well because K40/E41 are just 14 aa away from the membrane, with some bulky aa and no glycine-glycine-hinge. In 2003, the transmembranous cell adhesion Protein P-selectin Glycoprotein Ligand- 1 (PSGL-1) was identified as BACE1 substrate which is cleaved between NL and S [387], similarly as in APP with the Swedish double mutation NL↓D instead of the wildtype KM↓D. In 2004, β-subunits of the voltage-gated sodium channels (VGSCβ) were found to be sequentially processed by BACE1 [388] and γ-secretase [388, 389]. A synthetic

30 Introduction peptide with a 39 aa partial sequence of VGSCβ1 was cleaved by BACE1 at L144/E145 as measured by MALDI, 30 aa of VGSCβ2 at L144/Q145, 39 aa of VGSCβ3 at F128/E129 and F153/T154, and 30 aa of VGSCβ4 at L149/Q150 [388]. The auxiliary subunit VGSCβ4 was suggested to have a role as cell adhesion molecule and its overexpression in Neuro2a cells increased neurite extension, which was further increased by BACE1-coexpression, and increased the number of filopodia-like protrusions, which was decreased by BACE1-coexpression [390]. In 2005, LRP was promoted from an interaction protein to a substrate protein for BACE1 [391]. In 2007, the processing of the interleukin-1 receptor 2 (IL-1R2) was characterized because IL-1R2 secretion was observed in AD and the processing involved α-, β- and γ-secretases [392]. Both, BACE1 and also BACE2 cleaved the receptor and the BACE2-cleavage site was F329/Q330 [392]. In 2006, defects were reported for BACE1 ko mice, more specifically, hypomyelination of peripheral nerves and aberrant axonal segregation of small- diameter afferent fibers which are very similar to that in mice with mutations in type 3 Neuregulin 1 (NRG1) or ko of its downstream signaling mediator, the receptor- tyrosine-kinase ErbB2, in Schwann cells [393]. NRG1 was then confirmed as substrate for BACE1 [393] and parallel experiments with BACE1 ko in zebra fish resulted in a movement phenotype, where the fish no longer swam away in response to a shock [394]. On the molecular level, another study found that, in addition to increased full-length NRG1 and decreased cleaved NRG1, phosphorylated Akt was reduced in BACE1 ko mice which led to the suggestion that cleaved NRG1 regulates myelination via phosphorylation of Akt in myelin-forming cells [395]. The cleavage sites and possible side effects of BACE1 inhibition are summarized in Tab. 2 below. BACE1 substrate : cleavage site(s) possible side effect with BACE1 inhibition APLP2 : I655/D656, L659/D660 or K662/E663 hypoglycemia and hyperinsulinemia ST6Gal1 : K40/E41 less sialylation of soluble glycoproteins ST8Sia4 : ? less secretion PSGL-1 : AASNL/SVNYP less secretion VGSCβ1 : L144/E145 less secretion VGSCβ2 : L144/Q145 less secretion VGSCβ3 : F128/Q129 less secretion VGSCβ4 : L149/Q150 more cell adhesion and less neurite extension LRP : 25 kDa before C-terminus altered endocytosis of APP IL-1R2 : BACE2 at F329/Q330 less secretion NRG1 : 51 kDa after N-terminus hypomyelination of peripheral axons

31 Introduction

In 2008, during behavioral studies of BACE1 ko and NRG1+/- mice, rodent analogs of clinical features of schizophrenia were observed, like hypersensitivity to the glutamatergic psychostimulant MK-801, cognitive impairments, deficits in social recognition, deficits in prepulse inhibition and novelty-induced hyperactivity, of which the latter two were responsive to treatment with the atypical antipsychotic drug clozapine [396]. In the study, a 30% decrease in interaction between PSD95 and ErbB4, the likely major mediator of NRG1 functions related to myelination and schizophrenia, was detected [396]. Martin Citron, Christian Haass and other scientists noted that they do not see the role of BACE1 in myelination as a stopper for BACE1 as target, because myelination occurs mostly in development [394] and in humans, myelination is finished prior to the 30ieth year of life. Also, despite the concern that inhibition of γ-secretase might have side effects due to prevented cleavage of other substrates like Notch [397], the benzolactam-derivative LY450139 is now undergoing a phase 3 trial as Semagacestat. The development of drugs to inhibit BACE1 was hence also not stopped and currently several membrane-permeable small molecules with high, nanomolar-affinity are available like an isophthalamide-derivative (IPAD) [398], an aminoquinazoline- derivative (AQD) [399] and an isonicotinamide [400] which are superior to the previous peptidomimetics and statine-like pseudotransition state inhibitors. However, the development of high-affine BACE1-inhibitors, especially of brain-permeable ones, has proved to be extremely difficult [personal communication by Dr. Paolo Paganetti from Novartis, Basel, Switzerland], largely because of the broad active enzymatic cleft of BACE1 [personal communication by Prof. Marcel Leist from the University of Konstanz]. Also, selectivity for BACE1 above other aspartate proteases with similar catalytic sites like Renin, Cathepsin D and BACE2 is challenging to achieve, although the catalytic site of BACE1 is more open and less hydrophobic [398]. For these reasons one might think of a strategy where the problems of direct BACE1- inhibition are circumvented by rather indirectly decreasing BACE1-dependent cleavage of APP. Regarding the influence of signaling pathways on Aβ-production, the identification of kinases, for many of which exist selective inhibitors, might provide an avenue for altering the processing of APP and ultimately for developing an AD- therapy.

32 Introduction

1.10 Aims of the study

In order to elucidate the influence of the kinome on BACE1-dependent APP processing in this project, or rather, the influence of only one defined kinase at a time, siRNA-technology was used. The technology relies on a cellular enzymatic complex which cleaves double stranded RNA, and therefore also usual mRNA with a specific complementary 21 small interfering RNA (siRNA = RNAi) bound to it, and was thus called RNAi(nterference) Induced Silencing Complex (RISC). The targeted and cleaved mRNA is rapidly degraded and can no longer serve as translation template in the ribosome-driven production of proteins, which leads ultimately to knockdown of the protein. In the case of this project the targeted kinase is missing in the cellular signaling machinery which maybe has or has not an effect on Aβ-production and the ultimate aim of the project was to find kinases whose kd leads to an Aβ-decrease as strong as possible. The most straight forward way to assess and judge the effect of kinase knockdown is therefore by measuring the Aβ- production. The large number of individual kinases and siRNAs, however, made a readout desirable which was less costly and time-consuming than the Aβ-ELISA and which was realized at the Danish pharmaceutical company Lundbeck A/S, Valby, Denmark, with APP-constructs containing the reporter enzyme Secreted Alkaline Phosphatase (SEAP). The APP-constructs had to be characterized before the screen together with the cells used for screening with regards to cleavage by the secretases, especially by BACE1.

33 Introduction

2 Evolution of the APP family and characteristics of processed pseudogenes

2.1 Evolution of the APP family

As outlined above, the APP family consists of APP itself and the APP-Like Proteins 1 and 2 (APLP1, APLP2). Knowledge on the relationship of similar genes in one genome, so-called paralogs, and of these to their counterparts (orthologs) in the genomes of other species is useful for mainly two reasons: Firstly, for an initial estimation of functional redundancy among them, which can for example be important in the development of drugs and secondly, in conjunction with knowledge on other gene families, to construct a genome map. Besides insights on evolution, which relies here on back-tracing differences between genomes, similarities in gene order (synteny) can facilitate the detection of genes. In order to scale the relationship of proteins, the degree of conservation on amino acid level is compared, which in the case of the APP family paralogs points to a closer relationship of APP to APLP2 than of both with APLP1. This can be deduced from the amino acid identity of human APLP2 to APP of 52% in comparison to the identity of APLP2 to APLP1 of 43% [109]. Other values but with the same relative outcome are identities of APP to APLP2 of 46% and of APP to APLP1 of 35% [401]. Regarding the higher identity of APLP2 to APLP1 than of APP to APLP1, the better functional substitution of APLP2 in APLP1/APP double-knockout mice as outlined above seems logical. Regarding the comparison of orthologs, human and murine APLP1 possess an identity of 89% [111]. The 11% difference thus corresponds to an anticipated primate- rodent divergence approximately 80 million years ago (mya), with rodents being phylogenetically second closest to primates after tree shrews (Tupaia) [402]. Of rodents, the karyotypes of squirrels and especially those of tree squirrels [403] are very close to that of humans. The differences among the three APP family paralogs are approximately 4-5 times higher which would be tantamount to divergence times of over 400 mya. Since the paralogs are and back then were caught in one genome, the divergences have their origin in two gene duplications instead of speciation. Macroscopically, the genome size rises from from 0.52 giga base pairs (gbp) or 0.59 pg C-value (C-value: mass of haploid DNA in nucleus; [www.genomesize.com]) in the panvertebral ancestor-like

34 Introduction lancelet to 1.2-2.5 pg in lampreys and once more to 3 gbp or 3.5 pg for humans, which, in conjunction with the “one (invertebrate gene) to four (vertebrate genes)”- rule, led Susumu Ohno to suggest that two whole genome duplications (WGDs) took place in early vertebrates [404]. Since its expression 1971, the 2R theory (2 rounds of WGD) was controversial because local duplications could result in a similar genome, however in the recent past more results, also from the group of Axel Meyer, strongly back it [405]. The first two WGDs in vertebrates are thought to have taken place over 500 mya with only a short interim time (Fig. 4).

2. WGD 1. WGD

Fig. 4: The first two WGDs in vertebrates, WGD events of other taxa and major speciation events on geologic time scale. Modified from [412]

35 Introduction

Ohno also suggested that WGDs drove evolution by providing a substrate on which selection could act and thus lead to the evolution of species with more opportunities. For example, it was proposed that early still jawless vertebrates like Haikouella, known as fossils, started to develop the primordial cerebral vesicle of lancelets into diencephalon and hindbrain firstly [406], with the telencephalon, possessed by all present-day vertebrates, added later, and adopted a more active lifestyle than the rather sessile lancelet-ancestor. Another example is the invention of the adaptive immune system of which the beginnings are seen to have evolved at the stage of lampreys [407]. The APP family was probably involved in the evolution of a more capable brain, because of an anticipated function in neuronal migration, backed by severe cortical dysplasias in triple knockout mice as described above. The relationship among the APP family members is still controversial, although more studies result in a closer relationship of APLP2 and APP than of either to APLP1. For example, current results from Langeland “strongly support that APP and APLP2 are most closely related and APLP1 is an outgroup to them.” Further they “strongly indicate that all these duplications are vertebrate-specific--i.e. not shared with amphioxus (=lancelet). In other words, the amphioxus APLP is an outgroup to all vertebrate APPs and APLPs.” [personal communication in 2009 by Jim Langeland from Kalamazoo College, USA; unpublished data] Based on the amino acid sequences of available APPs and APLPs the chronology of gene duplications has been calculated. Coulson, in line with the sequence identities and the shared Kunitz Protease Inhibitor (KPI) domain, computed the first duplication event with an ancestral APLP leading to APLP1 and APLP2 and the second one with APP diverging from APLP2 [408]. The latter event was proposed to have occurred 550 mya and the first earlier but subsequent to the speciation of insects. The statement on the first event is corroborated by Drosophila melanogaster appl and Caenorhabditis elegans apl-1 being more similar to APLP1 than to APP [408]. Collin in contrast calculated that APP diverged from a common ancestor of both APLPs [409]. Also, Collin calculated a much higher evolutionary distance for APLP1 than for APLP2 to appl and apl-1, in comparison with a nearly equal distance as calculated by Coulson. Knowledge of the fourth member would maybe add more evidence to one or the other theory which was the reason for searching it in this study.

36 Introduction

2.2 Characteristics of processed pseudogenes During the search for the fourth APP family member, a genomic DNA region was detected on chromosome 3 in open reading frame 52 (C3orf52), which, in its translation, contained fragments with similarity to APP family members. Its length was similar to the coding region of APP family member mRNAs and therefore considered as potential APLP-like processed rather than as the fourth member. In order to verify or falsify the potential APLP-like processed pseudogene, knowledge on the characteristics of verified processed pseudogenes was necessary. Processed pseudogenes are a subtype of pseudogenes, the general term for non- functional genes, which became part of the genome by a collection of events called retrotransposition. In analogy to retroviral RNA, mRNA is here reverse transcribed and the transcript itself used as template to finally synthesize a so-called retrogene on the complementary strand [407, 411], which upon integration in the genome turns usually immediately into a processed pseudogene (Fig. 5) (=retropseudogene) because a nearby promoter with the correct orientation is missing [412]. Therefore processed pseudogenes are in general intronless and sometimes polyadenylated in addition to features which render the sequences non-functional like reading-frame- shifts and truncations [413].

Transcription of genomic DNA 5‟

Splicing of RNA transcript 5‟

5‟ Reverse-transcription of mRNA 5‟ 3‟ Integration into the genome 5‟ 3‟ Synthesis of complementary strand 5‟ 5‟ 3‟ Fig. 5: Principal steps in the creation of a processed pseudogene (red).

37 Introduction

In a whole genome survey, approximately 8000 processed pseudogenes were identified [413]. In average, they possess 75% and 86% sequence similarity for amino acids and for nucleotides, respectively, 94% completeness in coding regions and 740 bp [413]. Pseudogenic fragments have an average length of 370 bp [413]. So far, no APLP-like pseudogene has yet been reported, which is however not surprising, because negative results were obtained for 23000 other genes, corresponding to 90% of the proteome, after screening the whole genome [413]. Of genes coding for proteins most processed pseudogenes have been found from ribosomal protein genes [413]. Similarity to parent genes is correlated to the age of pseudogenes, since they tend to accumulate nucleotide substitutions [413], silent and non-silent equally, over time. This process is called compositional assimilation, begins usually with integration in the genome and proceeds up to the point when pseudogenes eventually “blend” with their region [414]. The rates of their nucleotide substitutions are anticipated to be close to rates of silent mutations in genes, which are calibrated by dividing the sum of different silent mutations in orthologous genes of species by the ages of their ancestor‟s fossils or by ages of biogeographical events as close as possible to the probable species divergence [415]. Fossil-based calibrations tend to overestimate the rate because poorly preserved fossil records result in the ages of calibration points being underestimated whereas biogeographical calibrations tend to overestimate the calibration point age and underestimate the substitution rate because colonization may occur anytime after the formation of islands, mountain ranges or land bridges [415]. Substitution rates for human sequences under no selection have been estimated to around 1.5∙10-9/site(=nucleotide)/year [416]. However, this value might only be applicable on relatively young processed pseudogenes, which were produced after the primate-rodent divergence 85-75 mya and comprise by far most known processed pseudogenes [413]. Different substitution rates due to different taxa is one of several reasons to be careful with calculated ages (using only the above primate substitution rate) and lead rather to over- than underestimation for time points of retrotransposition [412]. Rodents have in general higher substitution rates, for example 4.6∙10-9/site/year (based on mouse-rabbit globin pseudogene differences) [417]. For chicken substitution rates of 3.6∙10-9/site/year were assumed [418]. Also the type of DNA makes a difference, since vertebrate mtDNA in contrast changes

38 Introduction with around 2%/1 million years which translates to 2∙10-8/site/year [415]. Further, substitution rates might be different due to genomic locations of pseudogenes depending on the gc-content of the isochore (=homogenous 100-300 kbp region) because it is anticipated that chromosomes with lower gc-content have a slower DNA-turnover rate which reduces the substitution rate [413]. Technically, in addition to the 8000 processed pseudogenes mentioned above, more than one quarter of the consists of processed pseudogenes of the types Alu and LINE1. Alu copies are derivatives of the 7SL RNA gene with an integrated Pol III promoter (and are cleavable by a restriction enzyme of Arthrobacter luteus). LINE1 is a transposon and stands for Long Interspersed Nuclear Element 1. The processed pseudogenes share some features with Alu and/or LINE1 copies. For example, the average gc-contents of isochores from processed pseudogenes and Alu copies are 46% and 48% [413], while LINE1 copies have their highest density in isochores with less than 37% gc-content [413]. A shared property of LINE1 copies and processed pseudogenes are flanking short direct repeats of 5-15 bp, in case they have not decayed [413]. Further, 5‟-truncations are found in most LINE1 copies due to the LINE1 machinery [411] and also in 8% of Alu copies which led to suggest that retrotransposition in general is largely dependent on the LINE1-machinery [411] and could be confirmed in in vitro experiments by retrotransposition of a reporter gene [419]. It is anticipated that pre-retrotranspositional 5‟-truncation also occurred in a fraction of processed pseudogenes of which around 50% are 5‟-or 3‟-truncated [420]. Dependence of retrotransposition on LINE1 activity is backed by largely similar and/or complementary age profiles of the three groups as can be deduced from analysis of their sequence divergences to the respective parent sequences. The 0.5 million LINE1 copies comprise 17% of mammalian genomes and in human 30-60 of them are presently active [411]. They were presumably most frequently inserted from 150 to 50 mya [421] which is quite well paralleled by the sequence divergence of the copies to the active form from the beginning rise at 34-33% to the peak at 21% and further to the local minimum at 13% (Fig. 6) [413]. The in total around 1.2 million Alu copies, which contribute 10% of the human genome [422], have an average sequence divergence of 8% (Fig. 6) [413] and 85% of them were retroposed between 60 and 40 mya [423], which fits to substitution rates of 4∙10-9 and 1∙10-9 before and after 40 mya with an average age of 50 my

39 Introduction assumed. From 50 mya on, far less LINE1 copies were inserted which could suggest that the LINE1 machinery was by then strongly occupied with retrotransposition of Alu copies. The divergence of processed pseudogenes from parent genes rises strongly from 25- 24%, reaches its peak at 9%, drops sharply from 6-5% and has its average around 13% (Fig. 6)[413], which correspond to the time points 92.5, 52.5, 45 and 62.5 mya when the above Alu substitution rates are used. The Alu copies and most processed pseudogenes are therefore supposed to have been created after the rodent-primate divergence [413].

Fig. 6: Sequence divergence distribution of LINE1 copies, Alu copies and processed pseudogenes. from [413] For processed pseudogenes to become fixed in the genome, the LINE1-machinery has to be active in the germ-line, which is in fact supposed to provide a beneficial environment for retrotranspositional activity. More specifically, it was hypothesized that LINE1-mediated retrotransposition is most frequent during the prophase of meiosis I when many strand breaks present free 3‟-ends which are used by the LINE1 as primers to turn any randomly annealed RNA into a template and do not have to be created by the LINE1 endonuclease first [411]. The

40 Introduction germ-line mRNA transcription level was therefore expected to be the most dominant factor [413]. For some parent genes with identified processed pseudogenes, the expression during spermatogenesis was confirmed, however not particular at which stage [413]. Further, the duration of gametogenesis is supposed to be of high importance, based on the knowledge of far more processed pseudogenes in mammals than in birds and amphibians in combination to large differences in duration of oogenesis between mammals with up to several years and only several weeks in the others besides spermatogenesis being equally short [424]. Indeed, the high frequency of LINE1 retrotransposition beginning 150 mya correlates quite well to the age of 125 my for the oldest placental mammalian fossil Eomaia [425]. Processed pseudogenes are nothing but molecular fossils and in addition to present genes they can provide hints on ancestral genes. For this reason, the potential APLP-like processed pseudogenic sequence and especially its validity was intensively examined.

During this process, gene expression data indicating an involvement of C3orf52 in myeloid leukemia were noticed and because of possible medical importance compiled and analyzed.

41 Materials and Methods

3 Screen preparation and execution

The cells, APP-constructs, inhibitors, siRNAs and other reagents used for the screen as well as details on experimental execution and data-generation are summarized in this materials and methods section.

3.1 Cells

The cell types used were the cell lines HEK293 and SHSY5Y and the primary murine neurons of the CGC type.

HEK293 HEK293 (Human Embryonic Kidney 293) [71], not the SV40T substrain, were obtained from PD Elisa May and cultured with Dulbecco‟s Modified Eagle‟s Medium (DMEM, Gibco 31966) supplemented with 5% heat inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 0.1 mg/ml streptomycin (1% P/S, Gibco 15140) in T75 (75 cm2 surface) cell culture flasks (Nunc 156499) in a humidified atmosphere of 5%

CO2 at 37°C. After washing with PBS (g/l: 8 NaCl, 1.8 Na2HPO4·2H2O, 0.4 KH2PO4; pH 7.4), they were passaged every fourth day with 0.25% trypsin solution (Gibco 25200-056). At first, the medium was removed from the outgrown cells followed by a washing-step with 10 ml PBS-buffer. After the removal of the PBS, 1 ml of Trypsin- EDTA was added and the flask was incubated for 1 min at 37°C. 2 ml of DMEM (10% FBS) were added to stop trypsination process. The cell suspension was transferred into a 15 ml tube and centrifuged (500 x g / 1800 rpm for 5 min). Subsequently, the supernatant was discarded and the pelleted cells were resuspended in 2 ml DMEM (10% FBS). 400 µl of the suspension were transferred into a T75 flask containing 10 ml of DMEM (5% FBS). Remaining cells were either used for experiments or disposed. Cells were either transfected as singled cells after trypsinization or as adherent monolayer. HEK293 stably transfected with APPswe or SEAP-APPswe were obtained from Lundbeck A/S (Valby, Denmark) and kept in DMEM supplemented with 10% FBS, 1% P/S and 0.5 g/l G418.

42 Materials and Methods

SHSY5Y

SHSY5Y cells were obtained from Lundbeck A/S (Valby, Denmark) and cultured with DMEM supplemented with 10% FBS and 1% P/S in a humidified atmosphere of 5%

CO2 at 37°C. After washing with PBS, they were passaged three times per week with trypsin solution. SHSY5Y stably transfected with APPswe, SEAP-APPswe or SEAP-APPswe-EpoR were obtained from Lundbeck A/S (Valby, Denmark) and kept in DMEM supplemented with 10% FBS, 1% P/S and 0.25 g/l G418.

Cerebellar granule cells (CGCs)

CGCs were isolated from BALB/c mice according to a protocol based on the work of Messer [426]. BALB/c mice were obtained from the animal house (TFA=Tierforschungsanlage) of the University of Konstanz where they were kept and bred at a constant temperature of 22.0°C, 55% relative humidity, a twelve hour day/night rhythm with a light intensity of 100 Lux and free access to water and food (Altromin 1314) in Macrolon-standard cages. All experiments were performed according to the guidelines of the National Institute of Health, the European Commission (directive 86/609/EEC), the national German administration and the ethics commission of the University of Konstanz. Handling of the mice was conducted by professional staff, according to national and European ethics and legal guidelines. Five seven day old mice were decapitated and the heads sterilized with 70% ethanol. After several seconds, the skin was pushed over the skull from the neck to the eyes with one coarse forceps while another forceps, which was held against the snout, was then used to fix the skin at the position of the eyes. With a small curved pair of scissors a horizontal circular cut was then made only on one side of the skull, starting from the foramen magnum, where the spine enters the skull, and ending at bregma, close to the ear, and always as close as possible to skull to avoid cutting the brain stem because otherwise the brain sticks to the skull. With a forceps the skull was flipped over by grasping the skull along the cut and pulling it to the other, not cut, side. To minimize contamination the exposed brain was sprayed shortly with 70% ethanol and the cerebellum immediately removed with a very slender forceps by aligning the forceps parallel with the upper and lower boundary between cerebellum

43 Materials and Methods and other brain structures, pushing down the forceps gently into the boundaries some millimeters until it is not visible anymore and by squeezing and then lifting the forceps. The cerebelli were placed in a small vessel with pre-cooled HIB solution (g/l: 7 NaCl, 0.37 KCl, 5.96 HEPES, 1.6 Glucose; pH 7.4) until five have been removed. They were then chopped with a mechanical tissue chopper adjusted to 400 µm thick cuts three times, and the teflon disk turned 45° after each round. 4.5 ml of HIB solution were transferred to a sterile conical 50 ml-tube and appr. 1 ml of it aspirated into a pipette. The chopped cerebelli were transferred with the pipette and some of the HIB solution to the tube by adding the solution and immediately aspirating the clumps which are floating for a short while. When not using little volumes clumps were clinging to the pipette walls which could not be brought back to the solution. 0.5 ml of trypsin solution (0.25 g T0303, 32 ml PBS) was added, and left at 37 °C for 18 min while gently shaking. Immediately after the incubation 1 ml of trypsin stopper solution

(0.1875 g T9128, 0.185 g MgSO4·7H2O, 50 ml PBS) and DNAse 1 solution (0.1 g

DN25, 0.185 g MgSO4·7H2O, 50 ml PBS) was added, mixed, and the tube centrifuged for 5 min at 300xg. The supernatant was carefully removed and discarded. The pellet was resuspended in a small volume of Basal Medium with Earle‟s salts (Gibco 41010-026) supplemented with 10% FBS, 20 mM KCl, 2 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (1% P/S). The tissue pieces were triturated with a 10 ml syringe and a 2 inch 18 gauge needle by aspirating up and down the solution ten times and the cell suspension filtered through a 40 µm, 70 µm or 100 µm plastic mesh to remove remaining clumps. The cells were counted and usually 14 Mio cells per animal obtained. For transfection singled cells prior to plating were used. 0.2 Mio of the cells in 100 µl or 0.7 Mio in 500 µl were seeded per 96-well (Nunc 161093) or 24-well (Greiner 662160) which were pre-coated with Poly-L-Lysine (PLK, PM >300 kDa) by incubating with 50 μg/ml PLK

(200 mg P1524 autoclaved in 1 l H2O) for 2 h at 37°C, washing twice with Tyrodes buffer (g/l: 8 NaCl, 0.2 KCl, 0.2 CaCl2·2H2O, 0.1 MgCl2·6H2O, 0.05 NaH2PO4·H2O,

0.1 Na2HPO4·2H2O) and once with H2O. For culture on glass, also 0.7 Mio cells were seeded, however the glass was pre-coated with 200 µg/ml PLK. The medium was exchanged 45 min after seeding to remove astroglia and fibroblast contamination which are not adherent yet. After 30 h 10 μM (final) cytosine arabinoside (2.8 mg

C1768, 10 ml H2O) was added to the culture.

44 Materials and Methods

3.2 Molecular material

In the following the generation of the different APP-constructs by Lundbeck A/S, the used inhibitors, antibodies, primers and siRNAs are described.

3.2.1 APP-constructs

The APP-constructs used in this study were generated by Lundbeck A/S in a pcDNA3.1 vector backbone. The SEAP sequence substituted the N-terminal domain up to and including leucine 583 of APPswe or APPwt. In the APP-EpoR constructs, the C-terminal region downstream of glycine 605 of APPswe (K595M/N596L) or APPwt was replaced by the receptor (EpoR) transmembrane domain [427]. Cloning of SEAP-APPswe and SEAP-APP: SEAP-APPswe was cloned by fusing secreted alkaline phosphatase (SEAP 1-511, NM_001632, with linking region YSRVGAAGRFEQ) with an overlap PCR approach to human APPswe/wt, 13 amino acids N-terminal of its β-cleavage-site. Full-length SEAP and truncated human APPswe were amplified by PCR in two separate PCR reactions using either pSEAPbasic (Clontech, USA) and a Lundbeck in-house cloned human APPswe in the pcDNA3.1 vector. The SEAP fragment was amplified with the primers 5-GGTACCGAGCTCTTACGCGT-3‟ and 5‟-CTCCTCCGTCTTGATATTTGTCTGCTCGAAGCGGCC-3‟. The APPswe fragment was amplified with the primers 5‟-GGCCGCTTCGAGCAGACAAATATCAAGACGGAGGAG-3‟ and 5‟-TGAACTCCCACGTTCACATG-3‟. The purified PCR products were used as templates in a third PCR reaction with the primers 5‟-GGTACCGAGCTCTTACGCGT-3‟ and 5‟-TGAACTCCCACGTTCACATG- 3‟. Subsequently, the SEAP-APPswe PCR product was digested by NheI and ClaI and ligated into a NheI and ClaI digested pcDNA3.1 construct expressing human APPswe. For generating SEAP-APP the same cloning approach was used by fusing the human APP to SEAP. Cloning of SEAP-APPswe-AβK16V and SEAP-APPwt-AβK16V: In order to introduce the AβK16V (K612V in APP695 nomenclature) α-mutation into SEAP-APPswe, DpnI mediated site-directed mutagenesis was applied [428]. Human APPswe was used as

45 Materials and Methods template in a PCR reaction with sense and anti-sense primers harboring the mutations. Sense primer for AβK16V was 5‟-GAAGTTCATCATCAAGTATTGGTGTTCTTTGCA-3‟ and anti-sense primer was 5‟-TGCAAAGAACACCAATACTTGATGATGAACTTC-3‟. Subsequently, the methylated parental plasmid DNA was degraded by digestion with Dpn I. Finally, an aliquot of the digested PCR reaction was transformed into the highly competent E. coli DH5α strain for minipreparation of mutant plasmid DNA. For generating SEAP-APP-AβK16V the same cloning approach was used. Cloning of SEAP-APPswe-EpoR and SEAP-APP-EpoR: SEAP-APPswe-EpoR was cloned by an overlap PCR approach. Briefly, SEAP-APPswe, truncated 9 amino acids downstream of the β-cleavage site, was amplified from pSEAP-APPswe with the following primers: 5‟-GCGCCTGCTGAGCAGGAA-3‟ and 5‟- TCCAGAAGCCGCCTGAGTCATGTCGGAATTCT-3‟. Fifty-two amino acids covering parts of the extracellular, the transmembrane domain and the intracellular domain of human EpoR (amino acids 231-282; accession no.: NM_000121.2) were amplified by RT-PCR from human polyA+ RNA with 5‟-ACATGACTCAGGCGGCTTCTGGAGCGCCT-3‟ and 5‟-GGAAGCTCTAGATCTTCTGCTTCAGAGCCC-3‟. The purified PCR products were used as templates in a third PCR reaction with the primers 5‟-GCGCCTGCTGAGCAGGAA-3‟ and 5‟-GGAAGCTCTAGATCTTCTGCTTCAGAGCCC-3‟. Subsequently, the PCR product was digested with Sac2 and Not1 and the 834 bp fragment was ligated into the above mentioned pSEAP-APPswe. All constructs have been sequence-verified. For generating SEAP-APP-EpoR the same cloning approach was used. Plasmids were reproduced in E. coli DH-5α which were transiently transfected by electroporation at 1800 V with an Eppendorf Elektroporator 2510 from Prof. Hartig‟s group and purified with a Qiagen Maxi-Prep Kit. Sequences of the APP-constructs:

APPswe: MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTI QNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCP LAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTES VEEVVRVPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQ QLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSL SLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEP VDARPAADRGLTTRPGSGLTNIKTEEISEVNLDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHH GVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN

46 Materials and Methods

APP: MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTI QNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCP LAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTES VEEVVRVPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQ QLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSL SLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEP VDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHH GVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN

SEAP: MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYV ALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSD ADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHL MGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHS HVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQ EQTFIAHVMAFAACLEPYTACDLAPPAGTTDAAHPGYSRVGAAGRFEQT

SEAP-APPswe: MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYV ALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSD ADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHL MGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHS HVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQ EQTFIAHVMAFAACLEPYTACDLAPPAGTTDAAHPGYSRVGAAGRFEQ TNIKTEEISEVNLDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKM QQNGYENPTYKFFEQMQN

SEAP-APP: MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYV ALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSD ADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHL MGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHS HVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQ EQTFIAHVMAFAACLEPYTACDLAPPAGTTDAAHPGYSRVGAAGRFEQ TNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKM QQNGYENPTYKFFEQMQN

SEAP-APPswe-AβK16V: MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYV ALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSD ADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHL MGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHS HVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQ EQTFIAHVMAFAACLEPYTACDLAPPAGTTDAAHPGYSRVGAAGRFEQ TNIKTEEISEVNLDAEFRHDSGYEVHHQVLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKM QQNGYENPTYKFFEQMQN SEAP-APP-AβK16V: MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYV ALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSD ADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHL MGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHS HVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQ EQTFIAHVMAFAACLEPYTACDLAPPAGTTDAAHPGYSRVGAAGRFEQ TNIKTEEISEVKMDAEFRHDSGYEVHHQVLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKM QQNGYENPTYKFFEQMQN SEAP-APPswe-EpoR: MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYV ALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSD ADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHL MGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHS HVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQ EQTFIAHVMAFAACLEPYTACDLAPPAGTTDAAHPGYSRVGAAGRFEQ TNIKTEEISEVNLDAEFRHDSG GFWSAWSEPVSLLTPSDLDPLILTLSLILVVILVLLTVLALLSHRRALKQKI Sequence: SEAP, 955 to 2508 APPswe, 2509 to 2574 EpoR, 2575 to 2730 SEAP-APP-EpoR: MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYV ALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSD ADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHL MGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHS HVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQ EQTFIAHVMAFAACLEPYTACDLAPPAGTTDAAHPGYSRVGAAGRFEQ TNIKTEEISEVKMDAEFRHDSG GFWSAWSEPVSLLTPSDLDPLILTLSLILVVILVLLTVLALLSHRRALKQKI

47 Materials and Methods

APP-citrin was another used APP-construct. For the group of Prof. Claudia Stürmer (University of Konstanz), the effect of a GFP- fusion protein with a dominant-negative mutant of Reggie R1EA was examined.

3.2.2 Inhibitors and other compounds

The BACE1 inhibitor IPAD (Fig. 7A) [398] was purchased as β-secretase inhibitor 4 from Merck/Calbiochem (Darmstadt, Germany, cat. no. 565788). AQD (Fig. 7B) [399] was synthesized by Lundbeck A/S following the schemes provided by Johnson & Johnson Pharmaceutical R&D (Johnson & Johnson, Beerse, Belgium). The γ- secretase inhibitor LY450139 (Fig. 7C) [429] was synthesized by Lundbeck A/S following the schemes provided by Lilly Research Labs (Indianapolis, IN, USA). The α-secretase inhibitor GM6001 was purchased from Merck/Calbiochem (cat. no. 364205). The Casein kinase 1 δ/ε inhibitor PF-670462 [430] was purchased from Tocris (cat. no. 3316), the Phospho-Inositide 3-Kinase (PIK3) inhibitor LY294002 (440202-5MG), the c-Jun N-terminal Kinase JNK-inhibitor 2 (420119) and the p38-MAPK-inhibitor (506126) from Calbiochem. The mixed lineage kinase inhibitor and inhibitor of JNK activation Cep-1347 was kindly provided by Lundbeck A/S. Simvastatin is a statin leading to cholesterol depletion and was purchased from Calbiochem (567020). The prodrug Simvastatin was converted to the active compound Simvastatinic-hydroxy acid by opening the lactone-ring [431]. Briefly, 4 mg of Simvastatin was dissolved in 0.1 ml of ethanol and then 0.15 ml of 0.1 M NaOH. After being heated at 50°C for two hours, the resulting solution was neutralized with HCl to a pH of approximately 7.2 and brought up to a volume of 1 ml with distilled water. Lipofectamine2000 (Invitrogen, San Diego, CA, USA) was purchased from Fisher Scientific GmbH (Ulm, Germany). Botulinum Neurotoxin C (BoNT/C) was dissolved in 10 mM HEPES, 150 mM NaCl and 0.1% BSA, on ice and sterile, to 50 µg/ml, aliquoted and frozen in liquid nitrogen.

48 Materials and Methods

A B IPAD: AQD: Isophthalamide-Derivative Aminoquinazoline-Derivative

LY450139 = Semagacestat C

Fig. 7: Lewis-structures of the BACE1-inhibitors IPAD and AQD and the γ-secretase inhibitor LY450139.

3.2.3 Antibodies

Immunostaining was performed using the following antibodies: for huAPPswe: clone 6E10, dil. 1:500, secondary (sec.) anti-mouse antibody with Alexa 488 (Invitrogen, Carlsbad, CA, USA) or sec. goat-anti-mouse antibody with

A594; for SEAP: clone 8B6, mouse-anti-human IgG1 (GeneTex, San Antonio, TX, USA), dil. 1:100, sec. goat-anti-mouse antibody with A594; for BACE1: rabbit-anti- human anti-BACE1-N-terminus, dil. 1:100, sec. donkey-anti-rabbit with A488 or goat- anti-human anti-BACE1-Ectodomain, dil. 1:200, sec. chicken-anti-goat with A647; for Thy1: rat-anti-mouse, dil. 1:500, sec. anti-rat with A488. Western blotting was performed using the following antibodies: for human APP (cell lysates): clone 6E10, recognizes human Aβ5-9, mouse IgG1 (Calbiochem, San Diego, CA, USA), dil. 1:500; for Aβ: clone 6E10, recognizes Aβ5-9, mouse IgG1 (Calbiochem), dil. 1:500; for sAPPβswe and sAPPβwt (medium samples): Ab generated at H. Lundbeck A/S by immunization of rabbits with conjugates of KLH and 6 C-terminal amino acids of sAPPβswe and sAPPβwt, respectively, dil. 1:50; for sAPPα: (medium samples) 6E10 dil. 1:500; secondary Abs: anti-mouse or anti-rabbit HRP conjugate, dil. 1:5000.

49 Materials and Methods

3.2.4 Primers

Most primer sequences were obtained from the qPrimer-depot (NCBI) with intron- spanning amplicons of 100-150 bp and an annealing temperature of 60°C. Hs Actin B forward: 5‟-GCACAGAGCCTCGCCTT-3‟ Hs Actin B reverse: 5‟-GTTGTCGACGACGAGCG-3‟ Hs APP forward: 5‟-GGTTTGGCACTGCTCCTG-3‟ Hs APP reverse: 5‟-CAGTCTGCCACAGAACATGG-3‟ Hs BACE1 forward: 5‟-CCGGCGGGAGTGGTATTATGA-3‟ Hs BACE1 reverse: 5‟-CCACTGTCCACAATGCTCTTGTC-3‟ Hs CSNK1D forward: 5‟-CAGCTCCACATTGAGAGCAA-3‟ Hs CSNK1D reverse: 5‟-ACCATGACGTTGTAGTCCCC-3‟ Hs CSNK1E forward: 5‟-AGCTACGTGTGGGGA-3‟ Hs CSNK1E reverse: 5‟-AGCTTGATGGCGACTTCC-3‟ Hs GAPDH forward: 5‟-GAAGGTGAAGGTCGGAGTCA-3‟ Hs GAPDH reverse: 5‟-TTGAGGTCAATGAAGGGGTC-3‟ Hs MAP2K1IP1 forward: 5‟-CGGATGACCTAAAGCGATTC-3‟ Hs MAP2K1IP1 reverse: 5‟-TCTGGAGCATTGTCATTTGC-3‟ Hs MAP3K3 forward: 5‟-GAAGCAGGCACCTCTCTGTC-3‟ Hs MAP3K3 reverse: 5‟-CCTCACTGTTGATGCTGGTG-3‟ Hs SEAP forward: 5‟-ATCGCTACGCAGCTCATCTC-3‟ Hs SEAP reverse: 5‟-CCCACCTTGGCTGTAGTCAT-3‟ Hs TRAD forward: 5‟-TTGCTAAGTGCTGCTGTTGC-3‟ Hs TRAD reverse: 5‟-ACTGTGGATGGAGTTCAGGG-3‟

3.2.5 small interfering RNA (siRNA)

The following siRNAs were purchased from MWG (Ebersberg, Germany). They were extended by dTdT at the 3‟-end. 5'-AGGUAGUGUAAUCGCCUUG-3' as non-specific control, “Con”; 5'-GCAAGCUGACCCUGAAGUU-3' for jellyfish GFP, Clontech C1eGFP, modified from [432], “GFP”; 5'-GCCAAGAACCUCAUCAUCU-3' for human SEAP, NM_031313 [433, 434], “SEAP”; 5'-UGAAGUGAAUCUGGAUGCA-3' for human APP, NM_201414, with Swedish mutation [435], “APPswe”;

50 Materials and Methods

5'-GCUUUGUGGAGAUGGUGGA-3' for human BACE1, NM_012104 [436], “BACE1”; 5'-UGGACUGCAAGGAGUACAA-3' for human BACE1, NM_012104 [436], “BACE1Kao3”; 5'-GACUGUGGCUACAACAUUC-3' for human BACE1, NM_012104 [437], “BACE1Singer6”; 5'-GUCCACUUCGUAUGCUGGU-3' for human PSEN1, NM_000021, “PSEN1”; 5'-UGGCCAAGAAGUACCGGGA-3' for human CSNK1D/E, NM_001893 (CSNK1D) [438], “CSNK1”; The following kinase siRNAs with sequences from the Ambion Kinome siRNA library (below) were purchased from MWG (Ebersberg, Germany) or Microsynth (Switzerland) in order to avoid unequal use of the library. They were extended by dTdT at the 3‟-end unless otherwise indicated in the sequence. “BCR”: plate 14 & well C11, ”CSNK1D”: 5-C10, “IRAK3”: 12-B7, “MAP2K1IP1”: 22- F11, “MAP2K1IP1#2”: 24-B8, “MAP2K1IP1#3”: 25-F5, “MAP3K3”: 8-F11, “MAP3K3#2”: 8-C2, “PFKM”: 21-A1, “non-hit RIPK4”: 12-A6, “hit RIPK4”: 12-E4, “STK10”: 8-A3, “STK29”: 4-D8, “TRAD”: 5-C1, “PRKCA”: GGCAGAAGAACGUGCACGATT and UCGUGCACGUUCUUCUGCCTC. For the following kinase target sequences were siRNAs purchased on a 96-well-plate from Qiagen (Hilden, Germany) and “validated RIPK4”: A3 reordered from MWG. A3 RIPK4 NM_020639 AAGCCTGATGACGAAGTGAAA A4 PRPF4B NM_003913 AAGGGTTTGAGTAAATACAAA A5 CHEK2 NM_001005735 AGGACTGTCTTATAAAGATTA B3 RIPK3 NM_006871 ACCGCTCGTTAACATATACAA B4 FN3K NM_022158 CCAGCTGTTTAACTACCTGAA B5 MPP5 NM_022474 AAGCCAGTTCATCATAAGGAA C3 ZAK NM_016653 CTATGATTACATTAACAGTAA C4 ERN2 NM_033266 AAGGATGAAACTGGCTTCTAT C5 PIK3R2 NM_005027 CCGCGAGTATGACCAGCTTTA D3 TLK1 NM_001136554 CAGAGAGTATAGAATACACAA D4 CSNK1D NM_001893 CCGGTCTAGGATCGAAATGTT D5 GFP as above AACTTCAGGGTCAGCTTGC E2 TSSK1B NM_052841 ATGGCAAGTGCTCTCCAATAA E3 FN3KRP NM_024619 ACGAGTGTTCGTGAAAGTGAA E4 MOS NM_005372 CGCGAACATCTTGATCAGTGA F3 MPP7 NM_173496 CACCGTATCGGCGACAAACTA F4 PIK4CB NM_002651 TCGGCTGATAGTGGCATGATT G3 PKLR NM_000298 AAGGGCAACTTCCCTGTGGAA G4 CSF1R NM_005211 CTGGAAGATCATCGAGAGCTA H3 TEX14 NM_032028 CCAGCTTTCCTTGGCCATCAA H4 PIK3C2A NM_002645 CAAGATGGTCGAATCAAGGAA

51 Materials and Methods

The Ambion Kinome siRNA library, Version 3, was a generous gift from Prof. Pierluigi Nicotera (now head of DZNE, Heidelberg, Germany) and the 1357 siRNAs in Tab. 3 below were used in this screen. plate & gene symbol accession sense antisense 4position-A1 HUNK NM_014586number GGACACCUAUGUCACCAAATT UUUGGUGACAUAGGUGUCCTT 4-A2 KIAA0999 NM_025164 GGAAAUAAACAGGAAGGAATT UUCCUUCCUGUUUAUUUCCTT 4-A3 KIAA1811 NM_032430 GGUCGCCAUCAAGAUCGUGTT CACGAUCUUGAUGGCGACCTT 4-A4 C14orf20 NM_174944 GGUCACUUUCCCAGCUAACTT GUUAGCUGGGAAAGUGACCTC 4-A5 MAPKAPK2 NM_004759 GGAAGUGCCUGCUGAUUGUTT ACAAUCAGCAGGCACUUCCTC 4-A6 MAPKAPK3 NM_004635 GGUAGACCAUCACUGGCAGTT CUGCCAGUGAUGGUCUACCTC 4-A7 MAPKAPK5 NM_003668 GGUACGUCUGCACAUGAUGTT CAUCAUGUGCAGACGUACCTC 4-A8 MARK1 NM_018650 GGCUGAAAACCUUCUCCUUTT AAGGAGAAGGUUUUCAGCCTT 4-A9 MARK2 NM_004954 GGUAUUUGAUUACCUAGUGTT CACUAGGUAAUCAAAUACCTC 4-A10 MARK3 NM_002376 GGAGCUCGGUGUAGAAACUTT AGUUUCUACACCGAGCUCCTG 4-A11 MARK4 NM_031417 GGUUGCCAUCAAGAUUAUCTT GAUAAUCUUGAUGGCAACCTC 4-B1 MELK NM_014791 GGCCUUGAAGAACCUGAGATT UCUCAGGUUCUUCAAGGCCTC 4-B2 MGC42105 NM_153361 GAAACUGACACAGGACAUGTT CAUGUCCUGUGUCAGUUUCTC 4-B3 MGC45428 NM_152619 GGAAGUUUCAGAGGAUUAATT UUAAUCCUCUGAAACUUCCTG 4-B4 MGC8407 NM_024046 GGAGUUUUGUGAAAUCUUCTT GAAGAUUUCACAAAACUCCTC 4-B5 MKNK1 NM_003684 GCACGAAGAGAACGAACUATT UAGUUCGUUCUCUUCGUGCTG 4-B6 MKNK2 NM_017572 GGCCACAUUCGGAGCAGGGTT CCCUGCUCCGAAUGUGGCCTG 4-B7 MYLK NM_005965 GGAAAAGCCUCAUGUAAAATT UUUUACAUGAGGCUUUUCCTC 4-B8 MYLK2 NM_033118 GGCAGAAGAAGGAAAGAACTT GUUCUUUCCUUCUUCUGCCTT 4-B9 PASK NM_015148 GGUCUGAACCAGUGGAUGUTT ACAUCCACUGGUUCAGACCTG 4-B10 PHKG1 NM_006213 GGUCACCUUGAGUGAGAAGTT CUUCUCACUCAAGGUGACCTT 4-B11 PHKG2 NM_000294 GAUCCGACUUUCAGAUUUCTT GAAAUCUGAAAGUCGGAUCTG 4-C1 PIM1 NM_002648 GGUUUUCUUCAGGCAGAGGTT CCUCUGCCUGAAGAAAACCTG 4-C2 PIM2 NM_006875 GGGAUAGAUGGACAUUUGUTT ACAAAUGUCCAUCUAUCCCTG 4-C3 PRKAA1 NM_006251 GGGAUUGGAACAUGAUUUATT UAAAUCAUGUUCCAAUCCCTG 4-C4 PRKAA2 NM_006252 GGUGAUCAGCACUCCAACATT UGUUGGAGUGCUGAUCACCTG 4-C5 PRKCM NM_002742 GGGUGUGGUCUGAAUUACCTT GGUAAUUCAGACCACACCCTT 4-C6 PRKCN NM_005813 GGAUCUUGUGUGCUCCAUATT UAUGGAGCACACAAGAUCCTT 4-C7 PRKD2 NM_016457 CGACCAACAGAUACUAUAATT UUAUAGUAUCUGUUGGUCGTG 4-C8 PSKH1 NM_006742 GGACUUCAUUGACCGCCUGTT CAGGCGGUCAAUGAAGUCCTT 4-C9 PSKH2 NM_033126 GGGUUGUCAGGGUAGAGCATT UGCUCUACCCUGACAACCCTG 4-C10 SNARK NM_030952 GGACAAAAUCAAAGAUGAGTT CUCAUCUUUGAUUUUGUCCTT 4-C11 SNF1LK NM_173354 GGUUAUGGAAACAAAGGACTT GUCCUUUGUUUCCAUAACCTG 4-D1 SNRK NM_017719 GGGUAAAAUUCACACAUACTT GUAUGUGUGAAUUUUACCCTT 4-D2 SSTK NM_032037 GCUCAUGGCGAAGAGACUUTT AAGUCUCUUCGCCAUGAGCTC 4-D3 STK17A NM_004760 GGAUGGAAAAGGCACUAGATT UCUAGUGCCUUUUCCAUCCTG 4-D4 STK17B NM_004226 CCAACAGCAGAGAUAUGCCTT GGCAUAUCUCUGCUGUUGGTC 4-D5 STK22B NM_053006 GAAAACACCUACUGACUUUTT AAAGUCAGUAGGUGUUUUCTT 4-D6 STK22C NM_052841 GGUGUAUGAGAUGCUGGAGTT CUCCAGCAUCUCAUACACCTG 4-D7 STK22D = TSSK1B NM_032028 GGCACAGCCUGAGACAAAATT UUUUGUCUCAGGCUGUGCCTG 4-D8 STK29 NM_003957 GGAGGCUCGGAAGUUCUUCTT GAAGAACUUCCGAGCCUCCTT 4-D9 STK33 NM_030906 GGAUUGAGAAUGGAGCUGCTT GCAGCUCCAUUCUCAAUCCTT 4-D10 TRAD NM_007064 GGAUGAAAUGUCCUCCUCUTT AGAGGAGGACAUUUCAUCCTG 4-D11 TRIO NM_007118 GGAUCUCAGGAGACUCAUUTT AAUGAGUCUCCUGAGAUCCTC 4-E1 TTN NM_003319 GGUGAGCUGGUUUAGGGAUTT AUCCCUAAACCAGCUCACCTC 4-E2 CHEK1 NM_001274 GGAGAGAAGGCAAUAUCCATT UGGAUAUUGCCUUCUCUCCTG 4-E3 STK11 NM_000455 GGAGGGCCGUCAAGAUCCUTT AGGAUCUUGACGGCCCUCCTG 4-E4 MGC4796 NM_032017 GGCAUAGAGAGCCAGGAAGTT CUUCCUGGCUCUCUAUGCCTT 4-E5 LOC91807 NM_182493 GGAGAAUAAAGAGCGAGUGTT CACUCGCUCUUUAUUCUCCTC 4-E6 APEG1 NM_005876 GAAAUUUGGAAACACCUAUTT AUAGGUGUUUCCAAAUUUCTC 4-E7 ARK5 NM_014840 GGAUAAGUUCUUACAAACGTT CGUUUGUAAGAACUUAUCCTT 4-E8 TRIB3 NM_021158 GGCAGAAGCCUGUGUGGAGTT CUCCACACAGGCUUCUGCCTT 4-E9 TRIB1 NM_025195 GGAGGACAGUGACAUUAGUTT ACUAAUGUCACUGUCCUCCTG 4-E10 CAMK1 NM_003656 GGACCCAGAGAAAAGAUUCTT GAAUCUUUUCUCUGGGUCCTT 4-E11 CAMK1G NM_020439 GGACUUUAUUUGCCACUUGTT CAAGUGGCAAAUAAAGUCCTT 4-F1 CAMK2A NM_015981 GGAUCUGAUCAAUAAGAUGTT CAUCUUAUUGAUCAGAUCCTT 4-F2 CAMK2B NM_001220 GGAGAUCAUUAAGACCACGTT CGUGGUCUUAAUGAUCUCCTG 4-F3 CAMK2D NM_001221 GGAGUCAACUGAGAGUUCATT UGAACUCUCAGUUGACUCCTT 4-F4 CAMK2G NM_001222 GGAGAUCAUUAAGAUUACATT UGUAAUCUUAAUGAUCUCCTG 4-F5 CAMK4 NM_001744 GGGAUGCGCUGAGCGAUUUTT AAAUCGCUCAGCGCAUCCCTG 4-F6 CASK NM_003688 GGUAUUGGAAGAAAUUUCATT UGAAAUUUCUUCCAAUACCTC 4-F7 CHEK2 NM_007194 GCUAAAUCAUCCUUGCAUCTT GAUGCAAGGAUGAUUUAGCTT 4-F8 CAMK1D NM_020397 GGCAACUGGCAAGCUCUUUTT AAAGAGCUUGCCAGUUGCCTT 4-F9 DAPK1 NM_004938 GGAAGCAAGUUUUCUUCCATT UGGAAGAAAACUUGCUUCCTT 4-F10 DAPK2 NM_014326 GGCACUUAAAUUAAAAUGATT UCAUUUUAAUUUAAGUGCCTT 4-F11 DAPK3 NM_001348 GCGCAUCGCACACUUUGACTT GUCAAAGUGUGCGAUGCGCTT 4-G1 DCAMKL1 NM_004734 GGGAUUGUGUAUGCCAUCUTT AGAUGGCAUACACAAUCCCTT 4-G2 TRIB2 NM_021643 GGUGUUUGAUAUCAGCUGCTT GCAGCUGAUAUCAAACACCTT 4-G3 HUNK NM_014586 GGCUAUUGGGUUUUACUAGTT CUAGUAAAACCCAAUAGCCTC 4-G4 KIAA0999 NM_025164 GGAUGGCUUCCAAGACAGUTT ACUGUCUUGGAAGCCAUCCTT 4-G5 KIAA1811 NM_032430 GGAGAACCAAGAAAAGAUGTT CAUCUUUUCUUGGUUCUCCTC 4-G6 C14orf20 NM_174944 GGUCUUGGGCUAAAAAUCUTT AGAUUUUUAGCCCAAGACCTC 4-G7 MAPKAPK2 NM_004759 GGCCAUCCAGUAUCUGCAUTT AUGCAGAUACUGGAUGGCCTC 4-G8 MAPKAPK3 NM_004635 GGGUCAUUCUUUUAACAAATT UUUGUUAAAAGAAUGACCCTG 4-G9 MAPKAPK5 NM_003668 GGCAUCAGAAGGAGAAAUCTT GAUUUCUCCUUCUGAUGCCTT 4-G10 MARK1 NM_018650 GGAAAAAAUCUUCAACUAUTT AUAGUUGAAGAUUUUUUCCTC 4-G11 MARK2 NM_004954 GGAUGAAAGAAAAAGAGGCTT GCCUCUUUUUCUUUCAUCCTG 52 Materials and Methods

4-H1 MARK3 NM_002376 GGUGAAGUAUUUGACUAUUTT AAUAGUCAAAUACUUCACCTC 4-H2 MARK4 NM_031417 GGGAAACUGAGGAAAUCUUTT AAGAUUUCCUCAGUUUCCCTC 4-H3 MELK NM_014791 GGGUAACAAGGAUUACCAUTT AUGGUAAUCCUUGUUACCCTT 4-H4 MGC42105 NM_153361 GUAAGCAAAAAAGGUGAAATT UUUCACCUUUUUUGCUUACTG 4-H5 MGC45428 NM_152619 GGCCUUUUCCUUAUUUAAGTT CUUAAAUAAGGAAAAGGCCTT 4-H6 MGC8407 NM_024046 GGUGGAAGAAGAUGAUUAUTT AUAAUCAUCUUCUUCCACCTC 4-H7 MKNK1 NM_003684 CGAAGAGAACGAACUAGCATT UGCUAGUUCGUUCUCUUCGTG 4-H8 MKNK2 NM_017572 GGACUGGUCAGAACCGUUATT UAACGGUUCUGACCAGUCCTC 4-H9 MYLK NM_005965 GGACGGGAACUGCUCUUUATT UAAAGAGCAGUUCCCGUCCTC 4-H10 MYLK2 NM_033118 GGUGGUGAAUUAUGACCAATT UUGGUCAUAAUUCACCACCTC 4-H11 PASK NM_015148 GGACCAGCAAAUCACUGCCTT GGCAGUGAUUUGCUGGUCCTT 5-A1 PHKG1 NM_006213 GGAAACCAGAAAGAUCAUGTT CAUGAUCUUUCUGGUUUCCTT 5-A2 PHKG2 NM_000294 GGGAGACUCUGCUGCUAUATT UAUAGCAGCAGAGUCUCCCTC 5-A3 PIM1 NM_002648 GGAACAACAUUUACAACUCTT GAGUUGUAAAUGUUGUUCCTG 5-A4 PIM2 NM_006875 GGGACCAGGAGAUUCUGGATT UCCAGAAUCUCCUGGUCCCTC 5-A5 PRKAA1 NM_006251 GGCAUCCUCAUAUAAUUAATT UUAAUUAUAUGAGGAUGCCTG 5-A6 PRKAA2 NM_006252 GGCAGAUUGUAUGCAGGUCTT GACCUGCAUACAAUCUGCCTG 5-A7 PRKCM NM_002742 GGUCAAAUUCUCAAUCAUATT UAUGAUUGAGAAUUUGACCTC 5-A8 PRKCN NM_005813 GGAGACCUAGUGGAAGUGGTT CCACUUCCACUAGGUCUCCTT 5-A9 PRKD2 NM_016457 GGAAAUUCCGCUGUCAGAATT UUCUGACAGCGGAAUUUCCTT 5-A10 PSKH1 NM_006742 GGUGUGACAGAGUAGAGGUTT ACCUCUACUCUGUCACACCTG 5-A11 PSKH2 NM_033126 GGAAGAUUCUGAAAGGCAATT UUGCCUUUCAGAAUCUUCCTG 5-B1 SNARK NM_030952 GGUGUGCAGCUUCUUCAAGTT CUUGAAGAAGCUGCACACCTT 5-B2 SNF1LK NM_173354 GGAAAACUGUGAACUUUCUTT AGAAAGUUCACAGUUUUCCTT 5-B3 SNRK NM_017719 GGUGGCAGUAAAAGUUAUUTT AAUAACUUUUACUGCCACCTT 5-B4 SSTK NM_032037 GACAAUUGAAAAGUCAAGUTT ACUUGACUUUUCAAUUGUCTC 5-B5 STK17A NM_004760 GGUCAUUCUGUGCCUGAAATT UUUCAGGCACAGAAUGACCTT 5-B6 STK17B NM_004226 GGAUUGUCGGGCAGAAAUUTT AAUUUCUGCCCGACAAUCCTG 5-B7 STK22B NM_053006 GAUUCCUUCCUCGGGAGAUTT AUCUCCCGAGGAAGGAAUCTC 5-B8 STK22C NM_052841 GGGCUUCAACCUGAAGCUGTT CAGCUUCAGGUUGAAGCCCTG 5-B9 STK22D = TSSK1B NM_032028 GCCGUCGACUAUGGAGACATT UGUCUCCAUAGUCGACGGCTT 5-B10 STK29 NM_003957 GGAGAAGAUGAUUUACUUCTT GAAGUAAAUCAUCUUCUCCTG 5-B11 STK33 NM_030906 GGAAAUCUAUACCUUUGGATT UCCAAAGGUAUAGAUUUCCTC 5-C1 TRAD NM_007064 GGUUCAUAUAAAAGGGCAATT UUGCCCUUUUAUAUGAACCTC 5-C2 TRIO NM_007118 GGACUAAUUUUGGCAGUUCTT GAACUGCCAAAAUUAGUCCTC 5-C3 TTN NM_003319 GGAACAAUUUACUCUCUGATT UCAGAGAGUAAAUUGUUCCTT 5-C4 CHEK1 NM_001274 GGAGAGCUUUUUGACAGAATT UUCUGUCAAAAAGCUCUCCTC 5-C5 STK11 NM_000455 GGAAAUUCAACUACUGAGGTT CCUCAGUAGUUGAAUUUCCTT 5-C6 MGC4796 NM_032017 GGGAGACUGUGGUAAUCUUTT AAGAUUACCACAGUCUCCCTC 5-C7 LOC91807 NM_182493 GGACAUCAAAUUAAGAUCATT UGAUCUUAAUUUGAUGUCCTG 5-C8 ARK5 NM_014840 GGCACCUACGGCAAAGUCATT UGACUUUGCCGUAGGUGCCTT 5-C9 CSNK1A1 NM_001892 GGAAAAAGACUACAAUGUATT UACAUUGUAGUCUUUUUCCTG 5-C10 CSNK1D NM_001893 GGAGAAGAGGUUGCCAUCATT UGAUGGCAACCUCUUCUCCTG 5-C11 CSNK1E NM_001894 GGACCUGUUCAACUUCUGUTT ACAGAAGUUGAACAGGUCCTC 5-D1 CSNK1G1 NM_022048 GGGUUGGCAAGAAGAUAGGTT CCUAUCUUCUUGCCAACCCTG 5-D2 CSNK1G2 NM_001319 GGUGGAGGUGGCCGAUGAATT UUCAUCGGCCACCUCCACCTC 5-D3 CSNK1G3 NM_004384 GGAACAGCUAGAUAUAUGATT UCAUAUAUCUAGCUGUUCCTG 5-D4 VRK3 NM_016440 GGAGCAUGUAGGGUCCCAGTT CUGGGACCCUACAUGCUCCTC 5-D5 CSNK1A1L NM_145203 GGAAGUAGCAGUGAAGCUGTT CAGCUUCACUGCUACUUCCTC 5-D6 TTBK2 NM_173500 GGGAAAAUGUUGCACUGAATT UUCAGUGCAACAUUUUCCCTG 5-D7 VRK1 NM_003384 GGAAUGGAAAGUAGGAUUATT UAAUCCUACUUUCCAUUCCTT 5-D8 VRK2 NM_006296 GGAAAAUCCUAGAAAAGGCTT GCCUUUUCUAGGAUUUUCCTG 5-D9 CSNK1A1 NM_001892 GGAAGUGGCAGUGAAGCUATT UAGCUUCACUGCCACUUCCTC 5-D10 CSNK1D NM_001893 GGUUGCCAUCAAGCUUGAATT UUCAAGCUUGAUGGCAACCTC 5-D11 CSNK1E NM_001894 GGCUAUCCCUCCGAAUUCUTT AGAAUUCGGAGGGAUAGCCTT 5-E1 CSNK1G1 NM_022048 GGUGAAGGUCUCCCACAGGTT CCUGUGGGAGACCUUCACCTG 5-E2 CSNK1G2 NM_001319 GGUGGCCGAUGAAACCAAATT UUUGGUUUCAUCGGCCACCTC 5-E3 CSNK1G3 NM_004384 GGCUUAAAGGCUGACACAUTT AUGUGUCAGCCUUUAAGCCTT 5-E4 VRK3 NM_016440 GGCUCAAAGAGAGGGCUGATT UCAGCCCUCUCUUUGAGCCTT 5-E5 CSNK1A1L NM_145203 GGAAAAAGACAACAAUGUGTT CACAUUGUUGUCUUUUUCCTG 5-E6 TTBK2 NM_173500 GGUGGAAUCAGCUCAACAATT UUGUUGAGCUGAUUCCACCTT 5-E7 VRK1 NM_003384 GGAGGCUUUGGCUGUAUAUTT AUAUACAGCCAAAGCCUCCTT 5-E8 VRK2 NM_006296 GGCAAUCAGUGGGUACUGGTT CCAGUACCCACUGAUUGCCTT 5-E9 CSNK1A1 NM_001892 GGUUUCUAAGCAUGAAUUGTT CAAUUCAUGCUUAGAAACCTT 5-E10 CSNK1D NM_001893 GGAUUAGCGAGAAGAAAAUTT AUUUUCUUCUCGCUAAUCCTT 5-E11 CSNK1E NM_001894 GGAAGGUGAGUAUGAGGCUTT AGCCUCAUACUCACCUUCCTC 5-F1 CSNK1G1 NM_022048 GGCACCUAAACAGAUUCUUTT AAGAAUCUGUUUAGGUGCCTC 5-F2 CSNK1G2 NM_001319 GCUCCGCCUAGGAAAGAAUTT AUUCUUUCCUAGGCGGAGCTC 5-F3 CSNK1G3 NM_004384 GGAGAGGUAUCAGAAAAUUTT AAUUUUCUGAUACCUCUCCTT 5-F4 VRK3 NM_016440 GGACAUCAUGAAGCAAAAATT UUUUUGCUUCAUGAUGUCCTC 5-F5 CSNK1A1L NM_145203 GGACUAAAGGCUAUGACAATT UUGUCAUAGCCUUUAGUCCTT 5-F6 TTBK2 NM_173500 GGAAAGACCAUGUUUGUAGTT CUACAAACAUGGUCUUUCCTT 5-F7 VRK1 NM_003384 GGUGAAAUUGCCAAAUACATT UGUAUUUGGCAAUUUCACCTG 5-F8 VRK2 NM_006296 GGGAAGAAGUUACAGAUUUTT AAAUCUGUAACUUCUUCCCTT 5-F9 ALS2CR7 NM_139158 GGCCACGGAGUAACAGUGATT UCACUGUUACUCCGUGGCCTT 5-F10 CCRK NM_012119 GGCUGGUUUACCAACAUCUTT AGAUGUUGGUAAACCAGCCTC 5-F11 CDC2 NM_001786 GGUUAUAUCUCAUCUUUGATT UCAAAGAUGAGAUAUAACCTG 5-G1 CDC2L1 NM_001787 GAAAAAGAAAGAGAGCACGTT CGUGCUCUCUUUCUUUUUCTT 5-G2 CDC2L5 NM_003718 GGCACAGAUUGUCUAGAUCTT GAUCUAGACAAUCUGUGCCTG 5-G3 CDK2 NM_001798 GGUGGUGUGGCCAGGAGUUTT AACUCCUGGCCACACCACCTC 5-G4 CDK3 NM_001258 GGGAGAUCUCGCUGCUCAATT UUGAGCAGCGAGAUCUCCCTG

5-G5 SCGB2A1 NM_002407 GGUAACCCUGGUGUUUGAGTT CUCAAACACCAGGGUUACCTT

53 Materials and Methods

5-G6 CDK5 NM_004935 GGAGCUGAAGCACAAGAACTT GUUCUUGUGCUUCAGCUCCTT 5-G7 CDK6 NM_001259 GGAUAUGAUGUUUCAGCUUTT AAGCUGAAACAUCAUAUCCTT 5-G8 CDK7 NM_001799 GGCACUGAAAAUGAAGUAUTT AUACUUCAUUUUCAGUGCCTG 5-G9 CDK8 NM_001260 GGCACUUAUGGUCACGUCUTT AGACGUGACCAUAAGUGCCTC 5-G10 CDK9 NM_001261 GGAGAAUUUUACUGUGUUUTT AAACACAGUAAAAUUCUCCTG 5-G11 CDKL1 NM_004196 GGCUUCACCUGGUGUUUGATT UCAAACACCAGGUGAAGCCTC 5-H1 CDKL2 NM_003948 GGUUUAUACUGAUUAUGUGTT CACAUAAUCAGUAUAAACCTC 5-H2 CDKL3 NM_016508 GGAAUUACUAAGCUCUGUGTT CACAGAGCUUAGUAAUUCCTG 5-H3 CDKL5 NM_003159 GGAGCCUAUGGAGUUGUACTT GUACAACUCCAUAGGCUCCTT 5-H4 CLK1 NM_004071 GGAAAAGAACCAGGAGUGUTT ACACUCCUGGUUCUUUUCCTT 5-H5 CLK2 NM_001291 GCAUAAGCGACGAAGAAGUTT ACUUCUUCGUCGCUUAUGCTT 5-H6 CLK3 NM_001292 GGAGGUCCUACAGUCGGGATT UCCCGACUGUAGGACCUCCTC 5-H7 CLK4 NM_020666 GGAAAAGAUCCAGGAGUAUTT AUACUCCUGGAUCUUUUCCTT 5-H8 CRK7 NM_016507 GGACCGGAUAUCGGGAAGUTT ACUUCCCGAUAUCCGGUCCTT 5-H9 DYRK1A NM_001396 GGUUUCUGCCUUAUCAUAUTT AUAUGAUAAGGCAGAAACCTG 5-H10 DYRK1B NM_004714 GGUAUUGCCUGAUGUGCGGTT CCGCACAUCAGGCAAUACCTG 5-H11 DYRK2 NM_003583 GGGAUCAUAUGUGCAGGUGTT CACCUGCACAUAUGAUCCCTG 6-A1 DYRK3 NM_001004 GGAGAUCAUACUCAGCACUTT AGUGCUGAGUAUGAUCUCCTG 6-A2 DYRK4 NM_003845 GGCAGAGGAGAAGUCACCATT UGGUGACUUCUCCUCUGCCTT 6-A3 ERK8 NM_139021 GGUCGUGGCCAUCAAGAAATT UUUCUUGAUGGCCACGACCTC 6-A4 HIPK4 NM_144685 GGAGCUGGCUAUCAUCCACTT GUGGAUGAUAGCCAGCUCCTT 6-A5 GSK3A NM_019884 GGGAACUAGUCGCCAUCAATT UUGAUGGCGACUAGUUCCCTG 6-A6 GSK3B NM_002093 GGACGGCAGCAAGGUGACATT UGUCACCUUGCUGCCGUCCTT 6-A7 HIPK2 NM_022740 GGGAGCGACAUGUUGGUAGTT CUACCAACAUGUCGCUCCCTT 6-A8 HIPK3 NM_005734 GGAAAGAAACUAUCCACGGTT CCGUGGAUAGUUUCUUUCCTG 6-A9 ICK NM_014920 GGUUAAGUCUUUAAAGAAGTT CUUCUUUAAAGACUUAACCTC 6-A10 NLK NM_016231 GGGUCUUCCGGGAAUUGAATT UUCAAUUCCCGGAAGACCCTT 6-A11 MAK NM_005906 GGAUGAAGAGAAAGUUCUATT UAGAACUUUCUCUUCAUCCTT 6-B1 MAPK1 NM_002745 GGGUUCCUGACAGAAUAUGTT CAUAUUCUGUCAGGAACCCTG 6-B2 MAPK10 NM_002753 GGGAUUUAAAACCAAGUAATT UUACUUGGUUUUAAAUCCCTG 6-B3 MAPK11 NM_002751 GGACCUGAGCAGCAUCUUCTT GAAGAUGCUGCUCAGGUCCTT 6-B4 MAPK12 NM_002969 GGCCAAGAACAACAUGAAGTT CUUCAUGUUGUUCUUGGCCTC 6-B5 MAPK13 NM_002754 GGAGAAGAUCCAGUACCUGTT CAGGUACUGGAUCUUCUCCTC 6-B6 MAPK14 NM_001315 GGUCUCUGGAGGAAUUCAATT UUGAAUUCCUCCAGAGACCTT 6-B7 MAPK4 NM_002747 GGGUGAGCUGUUCAAGUUCTT GAACUUGAACAGCUCACCCTG 6-B8 MAPK6 NM_002748 GGCUUUUCAUGUAUCAGCUTT AGCUGAUACAUGAAAAGCCTG 6-B9 MAPK7 NM_002749 GGCUCGGCUUGGAUUAUUCTT GAAUAAUCCAAGCCGAGCCTC 6-B10 MAPK8 NM_002750 GGAGCUCAAGGAAUAGUAUTT AUACUAUUCCUUGAGCUCCTG 6-B11 MAPK9 NM_002752 GGGAUUGUUUGUGCUGCAUTT AUGCAGCACAAACAAUCCCTT 6-C1 HIPK1 NM_152696 GGCAGACCGAAGAGAAUACTT GUAUUCUCUUCGGUCUGCCTT 6-C2 PCTK1 NM_006201 GGAGAUCAGACUGGAACAUTT AUGUUCCAGUCUGAUCUCCTT 6-C3 PCTK2 NM_002595 GGUGUUUGUCUCAGAAAUCTT GAUUUCUGAGACAAACACCTG 6-C4 PFTK1 NM_012395 GCAGCACAUUCUCAAGAGCTT GCUCUUGAGAAUGUGCUGCTT 6-C5 PRPF4B NM_003913 GGUUAUUGAUGCUUCUGAUTT AUCAGAAGCAUCAAUAACCTC 6-C6 RAGE NM_014226 GGGAUCAGGAAUACCUCUATT UAGAGGUAUUCCUGAUCCCTT 6-C7 SRPK1 NM_003137 GGAGGUUAUCAUCUUGUGATT UCACAAGAUGAUAACCUCCTT 6-C8 STK23 NM_014370 GGAUCUCAGGAGUCAAUGGTT CCAUUGACUCCUGAGAUCCTG 6-C9 SRPK2 NM_003138 GGAAUAUAAUCUUGAUGAGTT CUCAUCAAGAUUAUAUUCCTC 6-C10 CDK11 NM_015076 GGCGGAAAGAUGGAAAAGATT UCUUUUCCAUCUUUCCGCCTC 6-C11 ALS2CR7 NM_139158 GGAUCUGAGGCAGGGUUUUTT AAAACCCUGCCUCAGAUCCTC 6-D1 CCRK NM_012119 GGAGAUGGAGGACAAUCAGTT CUGAUUGUCCUCCAUCUCCTG 6-D2 CDC2 NM_001786 GGAACUUCGUCAUCCAAAUTT AUUUGGAUGACGAAGUUCCTT 6-D3 CDC2L5 NM_003718 GGUAACGAAGGUGGAAAAUTT AUUUUCCACCUUCGUUACCTG 6-D4 CDK2 NM_001798 GGAAGUUUCAGUAUUAGAUTT AUCUAAUACUGAAACUUCCTT 6-D5 CDK3 NM_001258 GGAAGCUCUAUCUGGUGUUTT AACACCAGAUAGAGCUUCCTC 6-D6 CDK4 NM_000075 GUUCGUGAGGUGGCUUUACTT GUAAAGCCACCUCACGAACTG 6-D7 CDK5 NM_004935 GGACCUGAAGAAGUAUUUUTT AAAAUACUUCUUCAGGUCCTG 6-D8 CDK6 NM_001259 GGUCUGGACUUUCUUCAUUTT AAUGAAGAAAGUCCAGACCTC 6-D9 CDK7 NM_001799 GGAGCUAAGUCAUCCAAAUTT AUUUGGAUGACUUAGCUCCTG 6-D10 CDK8 NM_001260 GGAACUGGGAUCUCUAUGUTT ACAUAGAGAUCCCAGUUCCTT 6-D11 CDK9 NM_001261 GGUGCUGAUGGAAAACGAGTT CUCGUUUUCCAUCAGCACCTT 6-E1 CDKL1 NM_004196 GGAAAAUCGGAUGUGGAUCTT GAUCCACAUCCGAUUUUCCTG 6-E2 CDKL2 NM_003948 GGCUGUUGAUGUGUGGGCCTT GGCCCACACAUCAACAGCCTT 6-E3 CDKL3 NM_016508 GGAUAUCAUCUAGUGAUCUTT AGAUCACUAGAUGAUAUCCTG 6-E4 CDKL5 NM_003159 GGAAACACAUGAAAUUGUGTT CACAAUUUCAUGUGUUUCCTT 6-E5 CLK1 NM_004071 GGCGUAUAAUCCCAAAAUATT UAUUUUGGGAUUAUACGCCTC 6-E6 CLK2 NM_001291 GAAGUCGCUCCUGGUCAAGTT CUUGACCAGGAGCGACUUCTT 6-E7 CLK3 NM_001292 GGUCCUACAGUCGGGAACATT UGUUCCCGACUGUAGGACCTC 6-E8 CLK4 NM_020666 GGCAUUGUAAACCACAUCATT UGAUGUGGUUUACAAUGCCTG 6-E9 CRK7 NM_016507 GGUAGCCAAAAGCAGCAGCTT GCUGCUGCUUUUGGCUACCTG 6-E10 DYRK1A NM_001396 GGUUUACUAUGCAAAAAAGTT CUUUUUUGCAUAGUAAACCTC 6-E11 DYRK1B NM_004714 GGUAUACUAUGCGAAGAAGTT CUUCUUCGCAUAGUAUACCTC 6-F1 DYRK2 NM_003583 GGUAUGAGGUCCUCAAGGUTT ACCUUGAGGACCUCAUACCTG 6-F2 DYRK3 NM_001004 GGGUAAAAGUUCAGAUUGCTT GCAAUCUGAACUUUUACCCTG 6-F3 DYRK4 NM_003845 GAGGAGAAGUCACCAAAGATT UCUUUGGUGACUUCUCCUCTG 6-F4 ERK8 NM_139021 GGGACAUUUACCUGGUGUUTT AACACCAGGUAAAUGUCCCTG 6-F5 HIPK4 NM_144685 GGGUCAAGGUGAUUGACUUTT AAGUCAAUCACCUUGACCCTG 6-F6 GSK3A NM_019884 GGUUCUCCAGGACAAGAGGTT CCUCUUGUCCUGGAGAACCTT 6-F7 GSK3B NM_002093 GGUGACAACAGUGGUGGCATT UGCCACCACUGUUGUCACCTT 6-F8 HIPK2 NM_022740 CCAGUACCCUUACAUAUAATT UUAUAUGUAAGGGUACUGGTT 6-F9 HIPK3 NM_005734 GGUCAAAUAGAAGUGAGCATT UGCUCACUUCUAUUUGACCTT

6-F10 ICK NM_014920 GGAAUGCAUGAACCUUCGGTT CCGAAGGUUCAUGCAUUCCTC

54 Materials and Methods

6-F11 NLK NM_016231 GGUUUGAAAUAUCUCCAUUTT AAUGGAGAUAUUUCAAACCTC 6-G1 MAK NM_005906 GGGCUGGCUUUUAUCCAUATT UAUGGAUAAAAGCCAGCCCTT 6-G2 MAPK1 NM_002745 GGAAAAGCUCAAAGAACUATT UAGUUCUUUGAGCUUUUCCTT 6-G3 MAPK10 NM_002753 GGCACAAGCUUCAUGAUGATT UCAUCAUGAAGCUUGUGCCTG 6-G4 MAPK11 NM_002751 GCACCUGAAGCACGAGAACTT GUUCUCGUGCUUCAGGUGCTT 6-G5 MAPK12 NM_002969 GCUCAUGAAACAUGAGAAGTT CUUCUCAUGUUUCAUGAGCTT 6-G6 MAPK13 NM_002754 GGCCUUAAGUACAUCCACUTT AGUGGAUGUACUUAAGGCCTT 6-G7 MAPK14 NM_001315 GGAAUUCAAUGAUGUGUAUTT AUACACAUCAUUGAAUUCCTC 6-G8 MAPK4 NM_002747 GGUCGCUGUGAAGAAGAUUTT AAUCUUCUUCACAGCGACCTT 6-G9 MAPK6 NM_002748 GGCAAUGGCUUGGUUUUUUTT AAAAAACCAAGCCAUUGCCTC 6-G10 MAPK7 NM_002749 GGCAAAAACUAUGUACACCTT GGUGUACAUAGUUUUUGCCTG 6-G11 MAPK8 NM_002750 GGAAUAGUAUGCGCAGCUUTT AAGCUGCGCAUACUAUUCCTT 6-H1 MAPK9 NM_002752 GGUUAUUCACAUGGAGCUGTT CAGCUCCAUGUGAAUAACCTG 6-H2 HIPK1 NM_152696 GGAUGGAUAAUGCUGUACCTT GGUACAGCAUUAUCCAUCCTC 6-H3 PCTK1 NM_006201 GGCAAAAGCAAGCUCACAGTT CUGUGAGCUUGCUUUUGCCTT 6-H4 PCTK2 NM_002595 GGAUAAUGAGCCUAUUGUGTT CACAAUAGGCUCAUUAUCCTT 6-H5 PFTK1 NM_012395 CCCAUUUGAGAAACCAGCUTT AGCUGGUUUCUCAAAUGGGTC 6-H6 PRPF4B NM_003913 GGGUAUGUCUCCAGCAAAATT UUUUGCUGGAGACAUACCCTC 6-H7 RAGE NM_014226 GGAAUACCUCUACUAACAATT UUGUUAGUAGAGGUAUUCCTG 6-H8 SRPK1 NM_003137 GGAAUUUUUCACCAAAAAATT UUUUUUGGUGAAAAAUUCCTT 6-H9 STK23 NM_014370 GGAGUCAAUGGAGUCCAUGTT CAUGGACUCCAUUGACUCCTG 6-H10 SRPK2 NM_003138 GGUCCUUCAAGGGUUAGAUTT AUCUAACCCUUGAAGGACCTG 6-H11 CDK11 NM_015076 GGAAUAUGCAUUGAAGCAATT UUGCUUCAAUGCAUAUUCCTT 8-A1 PAK6 NM_020168 GGCUAUUCCGAAGCAUGUUTT AACAUGCUUCGGAAUAGCCTG 8-A2 SLK NM_014720 GGCCCAGAAUAAAGAGACCTT GGUCUCUUUAUUCUGGGCCTT 8-A3 STK10 NM_005990 GGCCAAGAAUAAGGAGACGTT CGUCUCCUUAUUCUUGGCCTT 8-A4 STK24 NM_003576 GGCAGACCCAGAAGAGCUUTT AAGCUCUUCUGGGUCUGCCTT 8-A5 STK25 NM_006374 GGUCGUUUGUUUUUGUUCUTT AGAACAAAAACAAACGACCTT 8-A6 STK3 NM_006281 GGAUAGUUUUUCAAAUAGGTT CCUAUUUGAAAAACUAUCCTG 8-A7 STK39 NM_013233 GGAGGUUAUCGGCAGUGGATT UCCACUGCCGAUAACCUCCTG 8-A8 STK4 NM_006282 GGAACUAUGAAAAGAAGGGTT CCCUUCUUUUCAUAGUUCCTT 8-A9 TAO1 NM_004783 CGGAAACCACCGCUCUUUATT UAAAGAGCGGUGGUUUCCGTT 8-A10 PAK7 NM_020341 GAGCAGAAGUUUACCGGCCTT GGCCGGUAAACUUCUGCUCTT 8-A11 MAP2K7 NM_005043 GGAGAUCAUGAAGCAGACGTT CGUCUGCUUCAUGAUCUCCTG 8-B1 ALS2CR2 NM_018571 GGACCUAUUUUCCUGAAGGTT CCUUCAGGAAAAUAGGUCCTC 8-B2 FLJ23074 NM_025052 GGAAAUAAUGUUAUGCUCATT UGAGCAUAACAUUAUUUCCTT 8-B3 LYK5 NM_001003 GGACCUGAUGACUGUGAAUTT AUUCACAGUCAUCAGGUCCTC 8-B4 JIK NM_016281 GGUGGUGGCAAUUAAGAAGTT CUUCUUAAUUGCCACCACCTC 8-B5 MAP2K1 NM_002755 GGCCUUUCUUACCCAGAAGTT CUUCUGGGUAAGAAAGGCCTC 8-B6 MAP2K2 NM_030662 GGAAGCUGAUCCACCUUGATT UCAAGGUGGAUCAGCUUCCTG 8-B7 MAP2K3 NM_002756 GGAUCUACGGAUAUCCUGCTT GCAGGAUAUCCGUAGAUCCTT 8-B8 MAP2K4 NM_003010 GGAGCUUAUGGUUCUGUCATT UGACAGAACCAUAAGCUCCTC 8-B9 MAP2K5 NM_002757 GCCCUCCAAUAUGCUAGUATT UACUAGCAUAUUGGAGGGCTT 8-B10 MAP2K6 NM_002758 GGCUUGCAUUUCUAUUGGATT UCCAAUAGAAAUGCAAGCCTT 8-B11 MAP3K14 NM_003954 GCUCCGUCUACAAGCUUGATT UCAAGCUUGUAGACGGAGCTC 8-C1 MAP3K2 NM_006609 GGAAACCAGAAAAGCAAAATT UUUUGCUUUUCUGGUUUCCTG 8-C2 MAP3K3 NM_002401 GGACAGAAACCAUAACAGUTT ACUGUUAUGGUUUCUGUCCTG 8-C3 MAP3K4 NM_005922 GGAGAAAAAGAUCCGAGCATT UGCUCGGAUCUUUUUCUCCTG 8-C4 MAP3K5 NM_005923 GGAAAGCUCGUAAUUUAUATT UAUAAAUUACGAGCUUUCCTG 8-C5 MAP3K6 NM_004672 GGCUUUUGACGUAGAGCCCTT GGGCUCUACGUCAAAAGCCTT 8-C6 MAP3K8 NM_005204 CCCAGUCUAAUGACCAUGUTT ACAUGGUCAUUAGACUGGGTT 8-C7 MAP4K1 NM_007181 GGACAUCUACCAAGUGACATT UGUCACUUGGUAGAUGUCCTG 8-C8 MAP4K2 NM_004579 GGAGAUUUACCAUGCCACUTT AGUGGCAUGGUAAAUCUCCTG 8-C9 MAP4K3 NM_003618 GGCACGGAAUGUUAACACUTT AGUGUUAACAUUCCGUGCCTT 8-C10 MAP4K4 NM_004834 GGAUAAGUUACGUGUCUACTT GUAGACACGUAACUUAUCCTT 8-C11 MAP4K5 NM_006575 GGGCUCUCUUCUUAAUGUCTT GACAUUAAGAAGAGAGCCCTC 8-D1 MINK NM_015716 GGAGAUCAACAUGCUGAAATT UUUCAGCAUGUUGAUCUCCTG 8-D2 MST4 NM_016542 GGAAGCCGAAGAUGAAAUATT UAUUUCAUCUUCGGCUUCCTC 8-D3 MYO3A NM_017433 GGCCAGACGUGAACGUAUUTT AAUACGUUCACGUCUGGCCTT 8-D4 MYO3B NM_138995 GGUAACUAACAAGAGAGAUTT AUCUCUCUUGUUAGUUACCTT 8-D5 OSR1 NM_005109 GGUUCUGUUCUGGAUAUUATT UAAUAUCCAGAACAGAACCTC 8-D6 PAK1 NM_002576 GGACCGAUUUUACCGAUCCTT GGAUCGGUAAAAUCGGUCCTT 8-D7 PAK2 NM_002577 GGUAGUAGCUAAAAUUAGATT UCUAAUUUUAGCUACUACCTG 8-D8 PAK3 NM_002578 GGACAAGAGGUGGCCAUAATT UUAUGGCCACCUCUUGUCCTG 8-D9 PAK4 NM_005884 GGGACUACCAGCACGAGAATT UUCUCGUGCUGGUAGUCCCTC 8-D10 PAK6 NM_020168 GGACACAGGUGUUGUGACATT UGUCACAACACCUGUGUCCTC 8-D11 SLK NM_014720 GGUGGAGCAGUAGAUGCUGTT CAGCAUCUACUGCUCCACCTG 8-E1 STK10 NM_005990 GGUGUAAUUAUUUGUCACCTT GGUGACAAAUAAUUACACCTC 8-E2 STK24 NM_003576 GGGCUCCUUUGGAGAGGUGTT CACCUCUCCAAAGGAGCCCTT 8-E3 STK25 NM_006374 GGGCAUCGAUAACCACACATT UGUGUGGUUAUCGAUGCCCTT 8-E4 STK3 NM_006281 GGACAUGCAAAAUUGGCAGTT CUGCCAAUUUUGCAUGUCCTT 8-E5 STK39 NM_013233 GGAACUUAAUGACAUACGATT UCGUAUGUCAUUAAGUUCCTT 8-E6 STK4 NM_006282 GGGACUUGAAUACCUUCAUTT AUGAAGGUAUUCAAGUCCCTT 8-E7 TAO1 NM_004783 CCGCUCUUUAACAUGAAUGTT CAUUCAUGUUAAAGAGCGGTG 8-E8 PAK7 NM_020341 GCAGAAGUUUACCGGCCUUTT AAGGCCGGUAAACUUCUGCTC 8-E9 MAP2K7 NM_005043 GGCAGAAAUCAACGACCUGTT CAGGUCGUUGAUUUCUGCCTG 8-E10 ALS2CR2 NM_018571 GGAUUUACAUGGGUAUAAUTT AUUAUACCCAUGUAAAUCCTG 8-E11 FLJ23074 NM_025052 GGUACUCUGCUCAUAGAAATT UUUCUAUGAGCAGAGUACCTG 8-F1 LYK5 NM_001003 GCCUCAUUCUAUUUAACUUTT AAGUUAAAUAGAAUGAGGCTG 8-F2 JIK NM_016281 GGAAUUUAAGGAGAGGUAUTT AUACCUCUCCUUAAAUUCCTC 8-F3 MAP2K1 NM_002755 GGCUGAAUUACAGUGAAAUTT AUUUCACUGUAAUUCAGCCTG

8-F4 MAP2K2 NM_030662 GGUGGAAGAAGUGGAUUUUTT AAAAUCCACUUCUUCCACCTC

55 Materials and Methods

8-F5 MAP2K3 NM_002756 GGGCUACAAUGUCAAGUCCTT GGACUUGACAUUGUAGCCCTT 8-F6 MAP2K4 NM_003010 GGGUGACUGUUGGAUCUGUTT ACAGAUCCAACAGUCACCCTC 8-F7 MAP2K5 NM_002757 GGUUAAGCUGUGUGAUUUUTT AAAAUCACACAGCUUAACCTG 8-F8 MAP2K6 NM_002758 GGAACAGAAACGGCUACUGTT CAGUAGCCGUUUCUGUUCCTG 8-F9 MAP3K14 NM_003954 GCUAUUUCAAUGGUGUGAATT UUCACACCAUUGAAAUAGCTT 8-F10 MAP3K2 NM_006609 GGCUACUAAUUUAGAACCATT UGGUUCUAAAUUAGUAGCCTG 8-F11 MAP3K3 NM_002401 GGAAUACUCAGAUCGGGAATT UUCCCGAUCUGAGUAUUCCTG 8-G1 MAP3K4 NM_005922 GGACUGUUCAAAAGAUUCATT UGAAUCUUUUGAACAGUCCTT 8-G2 MAP3K5 NM_005923 GGUAUACAUGAGUGGAAUUTT AAUUCCACUCAUGUAUACCTT 8-G3 MAP3K6 NM_004672 GGUGGGUAUGUACAAGGUCTT GACCUUGUACAUACCCACCTG 8-G4 MAP3K8 NM_005204 GCCUUUAUUGGUAAAUUCUTT AGAAUUUACCAAUAAAGGCTC 8-G5 MAP4K1 NM_007181 GGAAAAGAUGAAGAGAAAGTT CUUUCUCUUCAUCUUUUCCTT 8-G6 MAP4K2 NM_004579 GGGAGAUGUCAAACUGGCUTT AGCCAGUUUGACAUCUCCCTG 8-G7 MAP4K3 NM_003618 GGCGAGAUAAGCUUUGGAUTT AUCCAAAGCUUAUCUCGCCTG 8-G8 MAP4K4 NM_004834 GGGUCGACAUGUUAAAACGTT CGUUUUAACAUGUCGACCCTT 8-G9 MAP4K5 NM_006575 GGACAAAACAAAAUGGUCATT UGACCAUUUUGUUUUGUCCTT 8-G10 MINK NM_015716 GGAGGAAGAGAUCAAACAGTT CUGUUUGAUCUCUUCCUCCTC 8-G11 MST4 NM_016542 GGUCUUUCAAAGUCAUUCUTT AGAAUGACUUUGAAAGACCTG 8-H1 MYO3A NM_017433 GGAUAACAAAGACUCGAAATT UUUCGAGUCUUUGUUAUCCTT 8-H2 MYO3B NM_138995 GGAAUGUUACCUUCAAUUGTT CAAUUGAAGGUAACAUUCCTG 8-H3 OSR1 NM_005109 GGAGAAAGUGGCAAUCAAATT UUUGAUUGCCACUUUCUCCTT 8-H4 PAK1 NM_002576 GGGUGGUUUAUGAUUAAGGTT CCUUAAUCAUAAACCACCCTC 8-H5 PAK2 NM_002577 GGAUCUGUUAAGCUCACUGTT CAGUGAGCUUAACAGAUCCTT 8-H6 PAK3 NM_002578 GGGAAAAUAAGAACCCUAATT UUAGGGUUCUUAUUUUCCCTC 8-H7 PAK4 NM_005884 GGUGAACAUGUAUGAGUGUTT ACACUCAUACAUGUUCACCTC 8-H8 PAK6 NM_020168 GGACUUUUUUUUAAGGGUCTT GACCCUUAAAAAAAAGUCCTT 8-H9 SLK NM_014720 GGCUACUUAUGUCUUGGUUTT AACCAAGACAUAAGUAGCCTC 8-H10 STK10 NM_005990 GGUUUACAAGGCCAAGAAUTT AUUCUUGGCCUUGUAAACCTT 8-H11 STK24 NM_003576 GGGUUUGUCAUUAAUAAUUTT AAUUAUUAAUGACAAACCCTG 9-A1 STK25 NM_006374 GGUCUACAAGGGCAUCGAUTT AUCGAUGCCCUUGUAGACCTC 9-A2 STK3 NM_006281 GGAAUCCGGUCAAGUUGUCTT GACAACUUGACCGGAUUCCTT 9-A3 STK39 NM_013233 GGUUAUCGGCAGUGGAGCUTT AGCUCCACUGCCGAUAACCTC 9-A4 STK4 NM_006282 GGAGAUAAUCAAAGAAAUCTT GAUUUCUUUGAUUAUCUCCTG 9-A5 TAO1 NM_004783 GCGGACCUACAAACUUCGCTT GCGAAGUUUGUAGGUCCGCTT 9-A6 PAK7 NM_020341 GCCUUUGAAAUCCGAUUUUTT AAAAUCGGAUUUCAAAGGCTT 9-A7 MAP2K7 NM_005043 GGACAGUUUCCCUACAAGATT UCUUGUAGGGAAACUGUCCTG 9-A8 ABL1 NM_005157 GGUCCAUCUCGCUGAGAUATT UAUCUCAGCGAGAUGGACCTC 9-A9 ABL2 NM_005158 GGCACUAAAUGAGGCUAUCTT GAUAGCCUCAUUUAGUGCCTG 9-A10 ACK1 NM_005781 GGAUUGACGAACUGUAUCUTT AGAUACAGUUCGUCAAUCCTG 9-A11 ALK NM_004304 GGAAGAGUCUGGCAGUUGATT UCAACUGCCAGACUCUUCCTC 9-B1 AXL NM_001699 GGUACAUUGGCUUCGGGAUTT AUCCCGAAGCCAAUGUACCTC 9-B2 BLK NM_001715 GGUCACUCGUCACAGGAAGTT CUUCCUGUGACGAGUGACCTG 9-B3 BMX NM_001721 GGAUGUACCCUCUGGGAAGTT CUUCCCAGAGGGUACAUCCTG 9-B4 BTK NM_000061 GGCAGUAAGAAGGGUUCAATT UUGAACCCUUCUUACUGCCTC 9-B5 CSF1R NM_005211 GGGCCAAGUUCAUUCAGAGTT CUCUGAAUGAACUUGGCCCTG 9-B6 CSK NM_004383 GGCGGGUACCAAACUCAGCTT GCUGAGUUUGGUACCCGCCTT 9-B7 DDR1 NM_001954 GGGACAUUUUGAUCCUGCCTT GGCAGGAUCAAAAUGUCCCTT 9-B8 DDR2 NM_006182 CCCAAACAUCAUCCAUCUATT UAGAUGGAUGAUGUUUGGGTC 9-B9 STYK1 NM_018423 GGACAUGGAGGAAAUGUGGTT CCACAUUUCCUCCAUGUCCTG 9-B10 EGFR NM_005228 GGGCAAAUACAGCUUUGGUTT ACCAAAGCUGUAUUUGCCCTC 9-B11 EPHA1 NM_005232 GGAAGUUACUCUGAUGGACTT GUCCAUCAGAGUAACUUCCTT 9-C1 EPHA2 NM_004431 GGGUGGGACCUGAUGCAGATT UCUGCAUCAGGUCCCACCCTT 9-C2 EPHA3 NM_005233 GGACUUACCAGGUGUGCAATT UUGCACACCUGGUAAGUCCTG 9-C3 EPHA4 NM_004438 GGGAGAACUUGGGUGGAUATT UAUCCACCCAAGUUCUCCCTG 9-C4 EPHA5 NM_004439 GGUGCUUCCAGAAUCUUCATT UGAAGAUUCUGGAAGCACCTT 9-C5 EPHA7 NM_004440 GGAAUAUAAGAGAAAACCUTT AGGUUUUCUCUUAUAUUCCTG 9-C6 EPHA8 NM_020526 GCGUGUGCUUAUCCGUUAATT UUAACGGAUAAGCACACGCTC 9-C7 EPHB1 NM_004441 GGUGUGCAAUGUCUUCGAGTT CUCGAAGACAUUGCACACCTG 9-C8 EPHB2 NM_004442 GGUGUGCAACGUGUUUGAGTT CUCAAACACGUUGCACACCTG 9-C9 EPHB3 NM_004443 GGCCAUGAAUCCCAUCCGCTT GCGGAUGGGAUUCAUGGCCTC 9-C10 EPHB4 NM_004444 GGACGUGAUCAAUGCCAUUTT AAUGGCAUUGAUCACGUCCTG 9-C11 EPHB6 NM_004445 GGCAGAAGACGAAUCCCACTT GUGGGAUUCGUCUUCUGCCTG 9-D1 ERBB2 NM_004448 GGACAUCUUCCACAAGAACTT GUUCUUGUGGAAGAUGUCCTT 9-D2 ERBB3 NM_001982 GGCAGUGUGUCCUGGGACUTT AGUCCCAGGACACACUGCCTG 9-D3 ERBB4 NM_005235 GGAUCGAUAUGCCUUGGCATT UGCCAAGGCAUAUCGAUCCTC 9-D4 FER NM_005246 GGAUAUUUCCAAAAUCAUGTT CAUGAUUUUGGAAAUAUCCTC 9-D5 FGFR1 NM_000604 GGAGAAAGAAACAGAUAACTT GUUAUCUGUUUCUUUCUCCTC 9-D6 FGFR2 NM_000141 GGAUACCACAUUAGAGCCATT UGGCUCUAAUGUGGUAUCCTC 9-D7 FGFR3 NM_000142 GGUUUAUCCCGCCGAUAGATT UCUAUCGGCGGGAUAAACCTT 9-D8 FGFR4 NM_002011 GGAUGGACAGGCCUUUCAUTT AUGAAAGGCCUGUCCAUCCTT 9-D9 FGR NM_005248 GGGCGAGAAGUUCCACAUCTT GAUGUGGAACUUCUCGCCCTT 9-D10 FLT1 NM_002019 GGAUCUAGUUCAGGUUCAATT UUGAACCUGAACUAGAUCCTG 9-D11 FLT3 NM_004119 GGGAACAUUUCCUGUCUCUTT AGAGACAGGAAAUGUUCCCTG 9-E1 FLT4 NM_002020 GGUGUUGCUGCUGCACGAGTT CUCGUGCAGCAGCAACACCTT 9-E2 FRK NM_002031 GGCAGACAAGUCAACCGUGTT CACGGUUGACUUGUCUGCCTC 9-E3 FYN NM_002037 GGACUCACCGUCUUUGGAGTT CUCCAAAGACGGUGAGUCCTT 9-E4 HCK NM_002110 GGCUCUGAGGACAUCAUCGTT CGAUGAUGUCCUCAGAGCCTG 9-E5 IGF1R NM_000875 GGAAUACAGGAAGUAUGGATT UCCAUACUUCCUGUAUUCCTG 9-E6 INSR NM_000208 GGAGUGUGGAGACAUCUGUTT ACAGAUGUCUCCACACUCCTC 9-E7 ITK NM_005546 GGUGGUGCAUGACAACUACTT GUAGUUGUCAUGCACCACCTG 9-E8 JAK1 NM_002227 GGUUCUAUUUCACCAAUUGTT CAAUUGGUGAAAUAGAACCTC

9-E9 JAK2 NM_004972 GGAGUUUGUAAAAUUUGGATT UCCAAAUUUUACAAACUCCTG

56 Materials and Methods

9-E10 JAK3 NM_000215 GGAUUUGGCCAGUGCUAUCTT GAUAGCACUGGCCAAAUCCTT 9-E11 KDR NM_002253 GGCUCAGCAUACAAAAAGATT UCUUUUUGUAUGCUGAGCCTG 9-F1 KIT NM_000222 GGCUCUUCUCAACCAUCUGTT CAGAUGGUUGAGAAGAGCCTG 9-F2 LMTK2 NM_014916 GGACCCAGAAAUAGACUUUTT AAAGUCUAUUUCUGGGUCCTT 9-F3 LTK NM_002344 GGUAGAGAUCCGAAGGCACTT GUGCCUUCGGAUCUCUACCTC 9-F4 LYN NM_002350 GGAACAAGGAGACAUUGUGTT CACAAUGUCUCCUUGUUCCTC 9-F5 MATK NM_002378 GGUGGCCGUGAAGAAUAUCTT GAUAUUCUUCACGGCCACCTT 9-F6 MERTK NM_006343 GGCAAGGGAAGAAGCCAAGTT CUUGGCUUCUUCCCUUGCCTC 9-F7 MET NM_000245 GGCACUAGCAAAGUCCGAGTT CUCGGACUUUGCUAGUGCCTC 9-F8 MST1R NM_002447 GGGCGACAGAAAUGAGAGUTT ACUCUCAUUUCUGUCGCCCTC 9-F9 MUSK NM_005592 GGGAUUAAAAGCAGUCCUATT UAGGACUGCUUUUAAUCCCTC 9-F10 NTRK1 NM_002529 GAGAACCUGACUGAGCUCUTT AGAGCUCAGUCAGGUUCUCTG 9-F11 NTRK2 NM_006180 GGUUAGAAAUCAUCAACGATT UCGUUGAUGAUUUCUAACCTT 9-G1 NTRK3 NM_002530 GGGCAGGAUUCAGGGAACATT UGUUCCCUGAAUCCUGCCCTT 9-G2 PDGFRA NM_006206 GGUUGUGCAGCUGAAUUCATT UGAAUUCAGCUGCACAACCTT 9-G3 PDGFRB NM_002609 GGAACUAUUCAUCUUUCUCTT GAGAAAGAUGAAUAGUUCCTC 9-G4 PTK2 NM_005607 GGAGUGGAAAUAUGAAUUGTT CAAUUCAUAUUUCCACUCCTC 9-G5 PTK2B NM_004103 GGUCUGCUUCUAUAGCAACTT GUUGCUAUAGAAGCAGACCTT 9-G6 PTK6 NM_005975 GGUGGCCAUUAAGGUGAUUTT AAUCACCUUAAUGGCCACCTG 9-G7 PTK7 NM_002821 GGUCACACUUCGUUGCCACTT GUGGCAACGAAGUGUGACCTG 9-G8 RET NM_000323 GGGAUGCUUACUGGGAGAATT UUCUCCCAGUAAGCAUCCCTC 9-G9 ROR1 NM_005012 GGCUCUCCUUUCGGUCCACTT GUGGACCGAAAGGAGAGCCTC 9-G10 ROR2 NM_004560 GGUGAAGUGGAGGUUCUGGTT CCAGAACCUCCACUUCACCTG 9-G11 ROS1 NM_002944 GGAUGUCACUUUUGGAACUTT AGUUCCAAAAGUGACAUCCTT 9-H1 RYK NM_002958 GGAUUGAACUGGAUGACAGTT CUGUCAUCCAGUUCAAUCCTT 9-H2 SRC NM_005417 GGCUGAGGAGUGGUAUUUUTT AAAAUACCACUCCUCAGCCTG 9-H3 SRMS NM_080823 GGCCAACUGGAAGCUGAUCTT GAUCAGCUUCCAGUUGGCCTT 9-H4 SYK NM_003177 GGAGGCAGAAGAUUACCUGTT CAGGUAAUCUUCUGCCUCCTC 9-H5 TEC NM_003215 GGGUCGAGCAGAGAAGAAATT UUUCUUCUCUGCUCGACCCTC 9-H6 TEK NM_000459 GGUGCCAUGGACUUGAUCUTT AGAUCAAGUCCAUGGCACCTT 9-H7 TIE NM_005424 GGUCACACACACUGUGAACTT GUUCACAGUGUGUGUGACCTT 9-H8 TNK1 NM_003985 GGGCACUUCGACUUUGUAATT UUACAAAGUCGAAGUGCCCTG 9-H9 TXK NM_003328 GGCCGUGGCUCAGCCAAUUTT AAUUGGCUGAGCCACGGCCTG 9-H10 TYK2 NM_003331 GGAAGUCUGCAUCCACAUUTT AAUGUGGAUGCAGACUUCCTC 9-H11 TYRO3 NM_006293 GGUGUGCCAUUUUUCACAGTT CUGUGAAAAAUGGCACACCTT 12-A1 ACVR2 NM_001616 GGACUGAUUGUGUAGAAAATT UUUUCUACACAAUCAGUCCTG 12-A2 ACVR2B NM_001106 GGCCCAGCUCAUGAAUGACTT GUCAUUCAUGAGCUGGGCCTT 12-A3 ACVRL1 NM_000020 GGACUAUAGACCACCCUUCTT GAAGGGUGGUCUAUAGUCCTC 12-A4 ACVR1C NM_145259 GGACUGAAGUGUGUAUGUCTT GACAUACACACUUCAGUCCTG 12-A5 AMHR2 NM_020547 GGCGAACCUGUGUGUUCUUTT AAGAACACACAGGUUCGCCTG 12-A6 RIPK4 NM_020639 GGGAGCGCAUGGAGCUUUUTT AAAAGCUCCAUGCGCUCCCTG 12-A7 ARAF1 NM_001654 GGACUCCUCUCUUUCUUCATT UGAAGAAAGAGAGGAGUCCTT 12-A8 BMPR1A NM_004329 GGACAGAAUCUGGAUAGUATT UACUAUCCAGAUUCUGUCCTT 12-A9 BMPR1B NM_001203 GGUCUUGCGUUGUAAAUGCTT GCAUUUACAACGCAAGACCTT 12-A10 BMPR2 NM_001204 GGUCUUCACAGUAUGAACATT UGUUCAUACUGUGAAGACCTT 12-A11 BRAF NM_004333 GGAGGUGUGGAAUAUCAAATT UUUGAUAUUCCACACCUCCTC 12-B1 LRRK1 NM_024652 GGAAUCACUCACUGACUACTT GUAGUCAGUGAGUGAUUCCTG 12-B2 KSR2 NM_173598 GGUUUUCCACCAAGUACUGTT CAGUACUUGGUGGAAAACCTG 12-B3 FLJ34389 NM_152649 GGCAGUAUUUACCACCAAATT UUUGGUGGUAAAUACUGCCTC 12-B4 ILK NM_004517 GGGCAAUGACAUUGUCGUGTT CACGACAAUGUCAUUGCCCTG 12-B5 IRAK1 NM_001569 GGUUGUCCUUGAGUAAUAATT UUAUUACUCAAGGACAACCTG 12-B6 IRAK2 NM_001570 GGACUUCAGCACCUCCAUUTT AAUGGAGGUGCUGAAGUCCTT 12-B7 IRAK3 NM_007199 GGUAAAAGUGGAACAAGAGTT CUCUUGUUCCACUUUUACCTT 12-B8 IRAK4 NM_016123 GGAUGGAAGAAGUUAGCUGTT CAGCUAACUUCUUCCAUCCTT 12-B9 KIAA1804 NM_032435 GGACCACCAAAAUGAGCACTT GUGCUCAUUUUGGUGGUCCTG 12-B10 LIMK1 NM_002314 GGACAAGAGGCUCAACUUCTT GAAGUUGAGCCUCUUGUCCTT 12-B11 LIMK2 NM_005569 GGAUGGGAAGCUCUACUGCTT GCAGUAGAGCUUCCCAUCCTT 12-C1 TNNI3K NM_015978 GGAAAAAGAACUGACAGAATT UUCUGUCAGUUCUUUUUCCTT 12-C2 MAP3K10 NM_002446 GGAAGCAAACAGUGGUCAUTT AUGACCACUGUUUGCUUCCTT 12-C3 MAP3K11 NM_002419 GGUGGGCAUCUUCCCGUCCTT GGACGGGAAGAUGCCCACCTG 12-C4 MAP3K12 NM_006301 GGAGCUGAGUGACAAGAGCTT GCUCUUGUCACUCAGCUCCTT 12-C5 MAP3K13 NM_004721 GGGAACACGAUGAAUCAGATT UCUGAUUCAUCGUGUUCCCTT 12-C6 MAP3K7 NM_003188 GGUCCUCAACUUUGAAGAGTT CUCUUCAAAGUUGAGGACCTG 12-C7 MFHAS1 NM_004225 GGAACUGGCUCAUUCCCAGTT CUGGGAAUGAGCCAGUUCCTT 12-C8 RAF1 NM_002880 GGCUUAACAGUGAAAAUUGTT CAAUUUUCACUGUUAAGCCTT 12-C9 RIPK1 NM_003804 GGCUUUGGGAAGGUGUCUCTT GAGACACCUUCCCAAAGCCTC 12-C10 RIPK2 NM_003821 GGAGAAGAAUUUGCCAAAGTT CUUUGGCAAAUUCUUCUCCTT 12-C11 RIPK3 NM_006871 GGCCACAGGGUUGGUAUAATT UUAUACCAACCCUGUGGCCTG 12-D1 TESK1 NM_006285 GGUUCAUGGGAGUCUGUGUTT ACACAGACUCCCAUGAACCTT 12-D2 TESK2 NM_007170 GGUACGACACCGAGCUUCUTT AGAAGCUCGGUGUCGUACCTT 12-D3 TGFBR1 NM_004612 GGCAUCAAAAUGUAAUUCUTT AGAAUUACAUUUUGAUGCCTT 12-D4 TGFBR2 NM_003242 GGACAUCUUCUCAGACAUCTT GAUGUCUGAGAAGAUGUCCTT 12-D5 ZAK NM_016653 GGUGCGAAAUUGAGGCAACTT GUUGCCUCAAUUUCGCACCTC 12-D6 Ksr NM_013571 GGCAAAGCUGGUGAAAUACTT GUAUUUCACCAGCUUUGCCTC 12-D7 ANKK1 NM_178510 GGUUCCGCAUCAUCCAUGATT UCAUGGAUGAUGCGGAACCTG 12-D8 ACVR1 NM_001105 GGUGGUGAAUUUUUAAUCATT UGAUUAAAAAUUCACCACCTC 12-D9 ACVR1B NM_004302 GGAUCUUGUCUACGAUCUCTT GAGAUCGUAGACAAGAUCCTG 12-D10 ACVR2 NM_001616 GGAGUGUCUUUUCUUUAAUTT AUUAAAGAAAAGACACUCCTG 12-D11 ACVR2B NM_001106 GGUGUACUUCUGCUGCUGUTT ACAGCAGCAGAAGUACACCTG 12-E1 ACVRL1 NM_000020 GGAGCACCUGAUUCCUUUCTT GAAAGGAAUCAGGUGCUCCTG 12-E2 ACVR1C NM_145259 GGAGCAUGUUGGGCAUCAGTT CUGAUGCCCAACAUGCUCCTT

12-E3 AMHR2 NM_020547 GGUGGAAAUGCAAGGAUGCTT GCAUCCUUGCAUUUCCACCTG

57 Materials and Methods

12-E4 RIPK4 NM_020639 GGACCUGUGUGAAAAGCCUTT AGGCUUUUCACACAGGUCCTC 12-E5 ARAF1 NM_001654 GGAGCUCAUUGUCGAGGUCTT GACCUCGACAAUGAGCUCCTC 12-E6 BMPR1A NM_004329 GGAUACCUUGCCUUUUUUATT UAAAAAAGGCAAGGUAUCCTC 12-E7 BMPR1B NM_001203 GGCUCAGAUUUUCAGUGUCTT GACACUGAAAAUCUGAGCCTT 12-E8 BMPR2 NM_001204 GGAUGUUGGUCUCACAUUGTT CAAUGUGAGACCAACAUCCTT 12-E9 BRAF NM_004333 GGCCCUAUUGGACAAAUUUTT AAAUUUGUCCAAUAGGGCCTC 12-E10 LRRK1 NM_024652 GGACUCAGACAUGCUACAUTT AUGUAGCAUGUCUGAGUCCTC 12-E11 KSR2 NM_173598 GGGAUGCUUUUUGGCCUCATT UGAGGCCAAAAAGCAUCCCTT 12-F1 FLJ34389 NM_152649 GUAUUUACCACCAAAAUGCTT GCAUUUUGGUGGUAAAUACTG 12-F2 ILK NM_004517 GGACAUGACUGCCCGAAUUTT AAUUCGGGCAGUCAUGUCCTC 12-F3 IRAK1 NM_001569 GGAGUACAUCAAGACGGGATT UCCCGUCUUGAUGUACUCCTC 12-F4 IRAK2 NM_001570 GGAAAAACUUUUGAGCUUGTT CAAGCUCAAAAGUUUUUCCTG 12-F5 IRAK3 NM_007199 GGAGAUGGGACAUCGUCGATT UCGACGAUGUCCCAUCUCCTG 12-F6 IRAK4 NM_016123 GGAGAUUUGAAGCAUUACUTT AGUAAUGCUUCAAAUCUCCTT 12-F7 KIAA1804 NM_032435 GGUAAAGAAGAGGAAGGGCTT GCCCUUCCUCUUCUUUACCTT 12-F8 LIMK1 NM_002314 GGAUCUAUGAUGGCCAGUATT UACUGGCCAUCAUAGAUCCTC 12-F9 LIMK2 NM_005569 GGUGAUCAUUGAGGAUGGGTT CCCAUCCUCAAUGAUCACCTT 12-F10 TNNI3K NM_015978 GGAAUAUAUUUGGCUCUGATT UCAGAGCCAAAUAUAUUCCTT 12-F11 MAP3K10 NM_002446 GGUGCUUUCCCAAGACUGUTT ACAGUCUUGGGAAAGCACCTG 12-G1 MAP3K11 NM_002419 GGAGGUGAUCGGCAUUGGATT UCCAAUGCCGAUCACCUCCTC 12-G2 MAP3K12 NM_006301 GGUCGACAUCUGGUCCUUUTT AAAGGACCAGAUGUCGACCTT 12-G3 MAP3K13 NM_004721 GGCCUGUAUGGAAUAUCAUTT AUGAUAUUCCAUACAGGCCTT 12-G4 MAP3K7 NM_003188 GGAGAUCGAGGUGGAAGAGTT CUCUUCCACCUCGAUCUCCTT 12-G5 MFHAS1 NM_004225 GGAGAAAUGUCUGGACAUUTT AAUGUCCAGACAUUUCUCCTC 12-G6 RAF1 NM_002880 GGUAAAAAAGCACGCUUAGTT CUAAGCGUGCUUUUUUACCTT 12-G7 RIPK1 NM_003804 GGCGAAGAUGAUGAACAGATT UCUGUUCAUCAUCUUCGCCTC 12-G8 RIPK2 NM_003821 GGAGGGACAAUUAUCUAUATT UAUAGAUAAUUGUCCCUCCTT 12-G9 RIPK3 NM_006871 GGCCAUGGCAAGUCUGGAUTT AUCCAGACUUGCCAUGGCCTT 12-G10 TESK1 NM_006285 GGCUGAUGUCUUUGCCUUCTT GAAGGCAAAGACAUCAGCCTT 12-G11 TESK2 NM_007170 GGUGGUGGAGGAGAAGGAATT UUCCUUCUCCUCCACCACCTC 12-H1 TGFBR1 NM_004612 GGUUCUGGCUCAGGUUUACTT GUAAACCUGAGCCAGAACCTG 12-H2 TGFBR2 NM_003242 GGUCGCUUUGCUGAGGUCUTT AGACCUCAGCAAAGCGACCTT 12-H3 ZAK NM_016653 GGACAAGGAGGUGGCUGUATT UACAGCCACCUCCUUGUCCTG 12-H4 Ksr NM_013571 GGUUGGAGUUCAACAGAUGTT CAUCUGUUGAACUCCAACCTG 12-H5 ANKK1 NM_178510 GGACCUAAAUAUGAUGUGUTT ACACAUCAUAUUUAGGUCCTG 12-H6 ACVR1 NM_001105 GGCUGCUUCCAGGUUUAUGTT CAUAAACCUGGAAGCAGCCTT 12-H7 ACVR1B NM_004302 GGGAAGCAGAGAUAUACCATT UGGUAUAUCUCUGCUUCCCTG 12-H8 ACVR2 NM_001616 GGGCAAUAUGUGUAAUGAATT UUCAUUACACAUAUUGCCCTC 12-H9 ACVR2B NM_001106 GGGCUGCUGGCUAGAUGACTT GUCAUCUAGCCAGCAGCCCTT 12-H10 ACVRL1 NM_000020 GGUAGUGUGAGUGUGGUGUTT ACACCACACUCACACUACCTC 12-H11 ACVR1C NM_145259 GGAAGGAAGGAAAUAUUUUTT AAAAUAUUUCCUUCCUUCCTC 14-A1 RIOK2 NM_018343 GGAGAGAAGACACUCUUGATT UCAAGAGUGUCUUCUCUCCTG 14-A2 ADCK4 NM_024876 GGGAAGCGGUUAACUUAUCTT GAUAAGUUAACCGCUUCCCTG 14-A3 FRAP1 NM_004958 GGAAAUGGGUUGAUGAACUTT AGUUCAUCAACCCAUUUCCTC 14-A4 HSPB8 NM_014365 GGUGUCUGGCAAACAUGAATT UUCAUGUUUGCCAGACACCTC 14-A5 HAK NM_052947 GGAAAAUGCAUCAUUAGCUTT AGCUAAUGAUGCAUUUUCCTT 14-A6 LAK NM_025144 GGUCGCCAAAGGUCUCCACTT GUGGAGACCUUUGGCGACCTG 14-A7 ADCK5 NM_174922 GGCUACUUGGAGGUGAUGUTT ACAUCACCUCCAAGUAGCCTG 14-A8 ADCK1 NM_020421 GGUCCACAAGGCAGUGCUGTT CAGCACUGCCUUGUGGACCTG 14-A9 ADCK2 NM_052853 GGCCCAACCUACAUCAAACTT GUUUGAUGUAGGUUGGGCCTG 14-A10 MIDORI NM_020778 GGAGCACCUUCUGCUCCAUTT AUGGAGCAGAAGGUGCUCCTT 14-A11 PDK1 NM_002610 GGACAAAAGUGCUGAGGAUTT AUCCUCAGCACUUUUGUCCTT 14-B1 PDK2 NM_002611 GGUGGUCAAAGAUGCCUACTT GUAGGCAUCUUUGACCACCTC 14-B2 PDK3 NM_005391 GGUCUUGGAUAACUUUCUATT UAGAAAGUUAUCCAAGACCTG 14-B3 PDK4 NM_002612 GGAAUCAUAGAGUAUAAAGTT CUUUAUACUCUAUGAUUCCTT 14-B4 PRKDC NM_006904 GGUUUCUAAUAUGGUGGCGTT CGCCACCAUAUUAGAAACCTG 14-B5 PTK9 NM_002822 GGACAAACAACCAUGCUAUTT AUAGCAUGGUUGUUUGUCCTC 14-B6 PTK9L NM_007284 CCUCUCUUUUGCUGGGUACTT GUACCCAGCAAAAGAGAGGTC 14-B7 SMG1 NM_014006 GAAUCUGAUGAUUGUGCACTT GUGCACAAUCAUCAGAUUCTT 14-B8 STK19 NM_004197 GGUUCUCUUCCCUCCAUUCTT GAAUGGAGGGAAGAGAACCTG 14-B9 RIOK3 NM_003831 GGUUGACUGGCAGGAUACUTT AGUAUCCUGCCAGUCAACCTC 14-B10 TAF1 NM_004606 GGUGUUACGUUUUCUACGUTT ACGUAGAAAACGUAACACCTT 14-B11 TAF1L NM_153809 GGUGGCCCAUUUACUUUAGTT CUAAAGUAAAUGGGCCACCTC 14-C1 TIF1 NM_003852 GGACAACGCAGAAGCCAAUTT AUUGGCUUCUGCGUUGUCCTC 14-C2 TRIM28 NM_005762 GGAGAUGAUCCCUACUCAATT UUGAGUAGGGAUCAUCUCCTG 14-C3 TRIM33 NM_015906 GGUGCAGAAUAGGAUAAAATT UUUUAUCCUAUUCUGCACCTG 14-C4 TRPM6 NM_017662 GGGUAAAGAAAGUGAACAATT UUGUUCACUUUCUUUACCCTT 14-C5 TRPM7 NM_017672 GGUGUUUUUGUGGUCGCUUTT AAGCGACCACAAAAACACCTG 14-C6 TRRAP NM_003496 GGAGAAACCAGCACAGCAATT UUGCUGUGCUGGUUUCUCCTG 14-C7 RIOK1 NM_031480 GGGAUAUGGACAUAAUUAUTT AUAAUUAUGUCCAUAUCCCTC 14-C8 ATM NM_000051 GCCAGCAAAUUCUAGUGCCTT GGCACUAGAAUUUGCUGGCTC 14-C9 ATR NM_001184 GGCAGUUGUAUUGAAUUCATT UGAAUUCAAUACAACUGCCTT 14-C10 BCKDK NM_005881 GGAUGAAAAGCUCGUCCGCTT GCGGACGAGCUUUUCAUCCTC 14-C11 BCR NM_004327 GGAUCCAACGACCAAGAACTT GUUCUUGGUCGUUGGAUCCTT 14-D1 BRD2 NM_005104 CCAACUGUUACAUUUACAATT UUGUAAAUGUAACAGUUGGTG 14-D2 BRD3 NM_007371 CCUGCCGGAUUAUCAUAAATT UUUAUGAUAAUCCGGCAGGTT 14-D3 BRD4 NM_014299 GGAAGUGGAAGAGAAUAAATT UUUAUUCUCUUCCACUUCCTC 14-D4 BRDT NM_001726 GGAAACAACAGUUCAUUGGTT CCAAUGAACUGUUGUUUCCTT 14-D5 CABC1 NM_020247 GGGCAACUUUGUUUUCUUCTT GAAGAAAACAAAGUUGCCCTT 14-D6 EEF2K NM_013302 GGAAGCCUGGAAGCACGCATT UGCGUGCUUCCAGGCUUCCTT 14-D7 FASTK NM_006712 GCCCUGCACUUUGUUUUUUTT AAAAAACAAAGUGCAGGGCTC

14-D8 RIOK2 NM_018343 GGAAAUGCAGGCAGAUGAUTT AUCAUCUGCCUGCAUUUCCTT

58 Materials and Methods

14-D9 ADCK4 NM_024876 GGUUGGCCAGAUGCUCAGCTT GCUGAGCAUCUGGCCAACCTT 14-D10 FRAP1 NM_004958 GGAGCUCCAGCACUAUGUCTT GACAUAGUGCUGGAGCUCCTT 14-D11 HSPB8 NM_014365 GGAGUUGAUGGUGAAGACCTT GGUCUUCACCAUCAACUCCTC 14-E1 HAK NM_052947 GGGAGAAGGCAUGCAAGUUTT AACUUGCAUGCCUUCUCCCTT 14-E2 LAK NM_025144 GGAGGCAAAGGAAAUGAAGTT CUUCAUUUCCUUUGCCUCCTG 14-E3 ADCK5 NM_174922 GGAGGUGGAUGAGUUGUUCTT GAACAACUCAUCCACCUCCTG 14-E4 ADCK1 NM_020421 GGGAGGAAUGCUGAGAAGGTT CCUUCUCAGCAUUCCUCCCTT 14-E5 ADCK2 NM_052853 GGCAUCAAGUGGCUUAGCUTT AGCUAAGCCACUUGAUGCCTG 14-E6 MIDORI NM_020778 GGGUCUUGUUUCCCAAAAATT UUUUUGGGAAACAAGACCCTT 14-E7 PDK1 NM_002610 GGGUGUGAUUGAAUACAAGTT CUUGUAUUCAAUCACACCCTG 14-E8 PDK2 NM_002611 GGAAGAUCUGUCCAUCAAGTT CUUGAUGGACAGAUCUUCCTC 14-E9 PDK3 NM_005391 GGAUCCACAGGUCUUGGAUTT AUCCAAGACCUGUGGAUCCTC 14-E10 PDK4 NM_002612 GGAAAUUGAUAUCCUCCCGTT CGGGAGGAUAUCAAUUUCCTT 14-E11 PRKDC NM_006904 GGAGUUAUCUAUUGCUAUCTT GAUAGCAAUAGAUAACUCCTT 14-F1 PTK9 NM_002822 GGAGACUAUUUAGAGUCCATT UGGACUCUAAAUAGUCUCCTT 14-F2 PTK9L NM_007284 GGAAAGUGUUUCUGGGAGGTT CCUCCCAGAAACACUUUCCTG 14-F3 SMG1 NM_014006 GCUGAUCCAGAAAAAUCUUTT AAGAUUUUUCUGGAUCAGCTT 14-F4 STK19 NM_004197 GGGUUCUUUUCUCUACGUGTT CACGUAGAGAAAAGAACCCTG 14-F5 RIOK3 NM_003831 GGAAAGCUGCUUCAUUUUUTT AAAAAUGAAGCAGCUUUCCTT 14-F6 TAF1 NM_004606 GGAAGAAGAAGCACCGUGATT UCACGGUGCUUCUUCUUCCTC 14-F7 TAF1L NM_153809 GGAUGAUAAAGAGCCACAGTT CUGUGGCUCUUUAUCAUCCTT 14-F8 TIF1 NM_003852 GGCCUUGUUAAGUUAACACTT GUGUUAACUUAACAAGGCCTT 14-F9 TRIM28 NM_005762 GGAGCACAUUCUGCGCUUUTT AAAGCGCAGAAUGUGCUCCTG 14-F10 TRIM33 NM_015906 GGUCUUUCAUCUAACUUGUTT ACAAGUUAGAUGAAAGACCTT 14-F11 TRPM6 NM_017662 GGCAAGAAACACAAAGUAGTT CUACUUUGUGUUUCUUGCCTT 14-G1 TRPM7 NM_017672 GGCAAUAGAAGAAUGGUCUTT AGACCAUUCUUCUAUUGCCTG 14-G2 TRRAP NM_003496 GGACAGUGAGACUCGAACATT UGUUCGAGUCUCACUGUCCTC 14-G3 AAK1 NM_014911 GGACAAGCAAUGGGAUGAATT UUCAUCCCAUUGCUUGUCCTC 14-G4 ASB10 NM_080871 GGGAUUUCCGCUUCAACAUTT AUGUUGAAGCGGAAAUCCCTC 14-G5 BMP2K NM_017593 GGUGGAUUCUCCACAGUUUTT AAACUGUGGAGAAUCCACCTT 14-G6 BUB1 NM_004336 GGAAUUCAAAACCAGGCUGTT CAGCCUGGUUUUGAAUUCCTC 14-G7 BUB1B NM_001211 GGCUUCAGAAAUGUAACAATT UUGUUACAUUUCUGAAGCCTG 14-G8 TP53RK NM_033550 GGCUCAGUGACUGUUCGAGTT CUCGAACAGUCACUGAGCCTT 14-G9 CAMKK1 NM_032294 GGACCUGAUCCUGAAGAUGTT CAUCUUCAGGAUCAGGUCCTT 14-G10 CAMKK2 NM_006549 GGACUGUGUGCAGCUGAAUTT AUUCAGCUGCACACAGUCCTG 14-G11 CDC7 NM_003503 GGCUGAAGGCUCUUUAAAATT UUUUAAAGAGCCUUCAGCCTG 14-H1 CHUK NM_001278 GGCCUGUGAUGUUCCUGAATT UUCAGGAACAUCACAGGCCTT 14-H2 PLK3 NM_004073 GGAAGAAGACCAUCUGUGGTT CCACAGAUGGUCUUCUUCCTC 14-H3 CSNK2A1 NM_001895 GGUGGAAUAUUUCAUGGACTT GUCCAUGAAAUAUUCCACCTG 14-H4 CSNK2A2 NM_001896 GGCCAUUAAUAUCACCAACTT GUUGGUGAUAUUAAUGGCCTC 14-H5 EIF2AK3 NM_004836 GGUAUUUGGGAAUAAGAUGTT CAUCUUAUUCCCAAAUACCTC 14-H6 ERN1 NM_001433 GGACAGGCUCAAUCAAAUGTT CAUUUGAUUGAGCCUGUCCTC 14-H7 ERN2 NM_033266 GGUUCACACUCUCAGGCCATT UGGCCUGAGAGUGUGAACCTG 14-H8 FLJ10074 NM_017988 GGAUUCAACAUGCCCGUUATT UAACGGGCAUGUUGAAUCCTG 14-H9 PXK NM_017771 GGCCUAAGUCUACCUCUUCTT GAAGAGGUAGACUUAGGCCTG 14-H10 FLJ20574 NM_017886 GGCCUGAAAUAACCAACUGTT CAGUUGGUUAUUUCAGGCCTT 14-H11 FLJ23356 NM_032237 GGAUGACAACACUAUGCUUTT AAGCAUAGUGUUGUCAUCCTC 15-A1 FLJ32685 NM_152534 GGCGGUAUAUGAACCAGUCTT GACUGGUUCAUAUACCGCCTC 15-A2 GAK NM_005255 GGAAUACAACACCAAUGUATT UACAUUGGUGUUGUAUUCCTC 15-A3 GSG2 NM_031965 GGCAUCUGAUGCUGAAAAGTT CUUUUCAGCAUCAGAUGCCTT 15-A4 GUCY2C NM_004963 GGUGAGUCAGAACUGCCACTT GUGGCAGUUCUGACUCACCTG 15-A5 GUCY2D NM_000180 GGGUCACAGCAGUGAUCAUTT AUGAUCACUGCUGUGACCCTG 15-A6 GUCY2F NM_001522 GGUUGCUGCGCGAUUAGCCTT GGCUAAUCGCGCAGCAACCTC 15-A7 HRI NM_014413 GGAUAUUUCUCGUAUCCAGTT CUGGAUACGAGAAAUAUCCTC 15-A8 IKBKE NM_014002 GGUCUUCAACACUACCAGCTT GCUGGUAGUGUUGAAGACCTT 15-A9 KIS NM_144624 GGCAAUCAGGAUGUAAAGUTT ACUUUACAUCCUGAUUGCCTT 15-A10 LOC149420 NM_152835 GGAAUGCAUCCUACAAAAGTT CUUUUGUAGGAUGCAUUCCTC 15-A11 MGC16169 NM_033115 GGUAAUUGCACAGGGAAUUTT AAUUCCCUGUGCAAUUACCTC 15-B1 MOS NM_005372 GGGUCCAAUAGCCUAGGGATT UCCCUAGGCUAUUGGACCCTG 15-B2 NEK11 NM_024800 GGAGAGGAAUUAAAGGUACTT GUACCUUUAAUUCCUCUCCTC 15-B3 NEK2 NM_002497 GGAAGAGUGAUGGCAAGAUTT AUCUUGCCAUCACUCUUCCTC 15-B4 NEK3 NM_002498 GGCUUCCCAAGUCUUUCUCTT GAGAAAGACUUGGGAAGCCTT 15-B5 NEK4 NM_003157 GGAGUCAUGGGAAGGAGGATT UCCUCCUUCCCAUGACUCCTT 15-B6 NEK6 NM_014397 GGCAUCCCAACACGCUGUCTT GACAGCGUGUUGGGAUGCCTC 15-B7 NEK7 NM_133494 GUUUAUAGAGCAGCCUGUCTT GACAGGCUGCUCUAUAAACTT 15-B8 NEK9 NM_033116 GGAGUAAAGUACAAUUUCATT UGAAAUUGUACUUUACUCCTT 15-B9 NPR1 NM_000906 GGAUACCUGAAAAUUGAUATT UAUCAAUUUUCAGGUAUCCTG 15-B10 NPR2 NM_000907 GGUUCUGUUUGAACUCAAATT UUUGAGUUCAAACAGAACCTG 15-B11 NRBP NM_013392 GGCGAGAAGAGGUGAAUCATT UGAUUCACCUCUUCUCGCCTC 15-C1 PACE-1 NM_020423 GGACACCUAACACACAAUATT UAUUGUGUGUUAGGUGUCCTC 15-C2 PIK3R4 NM_014602 GGAACUGAAAAUCAGGCUUTT AAGCCUGAUUUUCAGUUCCTC 15-C3 PINK1 NM_032409 GGAGAUCCAGGCAAUUUUUTT AAAAAUUGCCUGGAUCUCCTG 15-C4 PKMYT1 NM_004203 GAACCUGGAUUCUCCCUCATT UGAGGGAGAAUCCAGGUUCTG 15-C5 PLK1 NM_005030 GGUUUUCGAUUGCUCCCAGTT CUGGGAGCAAUCGAAAACCTT 15-C6 PRKR NM_002759 GGGAGUAGUACUUAAAUAUTT AUAUUUAAGUACUACUCCCTG 15-C7 PRKWNK1 NM_018979 GGAUGAUAUCGAAGAGCUGTT CAGCUCUUCGAUAUCAUCCTG 15-C8 PRKWNK4 NM_032387 GGAAGAAAAGGAGGACAUGTT CAUGUCCUCCUUUUCUUCCTG 15-C9 RNASEL NM_021133 GGUCAAAGCCCUAAAAUUCTT GAAUUUUAGGGCUUUGACCTT 15-C10 SCYL1 NM_020680 GGAGAUUCAGAUCAAAGAGTT CUCUUUGAUCUGAAUCUCCTC 15-C11 PRKWNK2 NM_006648 GGAUGUACCUGCUUUUGUGTT CACAAAAGCAGGUACAUCCTG 15-D1 PLK2 NM_006622 GGUAUACAAUGCCGUCCUCTT GAGGACGGCAUUGUAUACCTT

15-D2 AURKB NM_004217 GGAGGAUCUACUUGAUUCUTT AGAAUCAAGUAGAUCCUCCTC

59 Materials and Methods

15-D3 AURKC NM_003160 GGAAAGCCAUUUCAUUGUGTT CACAAUGAAAUGGCUUUCCTT 15-D4 STK16 NM_003691 GGUACGCUGUGGAAUGAGATT UCUCAUUCCACAGCGUACCTC 15-D5 PLK4 NM_014264 GGUAUCUAAAGAAUAGAGUTT ACUCUAUUCUUUAGAUACCTG 15-D6 STK31 NM_031414 GGUACAUUGACUAUGGAAATT UUUCCAUAGUCAAUGUACCTC 15-D7 STK35 NM_080836 GGGCAAUCAAGACAACAAATT UUUGUUGUCUUGAUUGCCCTC 15-D8 STK36 NM_015690 GGGAGAGCUCUUUCAGAUCTT GAUCUGAAAGAGCUCUCCCTC 15-D9 STK6 NM_003600 GGCAACCAGUGUACCUCAUTT AUGAGGUACACUGGUUGCCTG 15-D10 TBK1 NM_013254 GGAGACAACAACAAGACAUTT AUGUCUUGUUGUUGUCUCCTC 15-D11 TEX14 NM_031272 GGAAAUGGGCAGUCAACCUTT AGGUUGACUGCCCAUUUCCTT 15-E1 TLK1 NM_012290 GGGUGGAAAUGGCUCAAGUTT ACUUGAGCCAUUUCCACCCTG 15-E2 TLK2 NM_006852 GGAGGGAAGAAUAGAUGAUTT AUCAUCUAUUCUUCCCUCCTT 15-E3 TOPK NM_018492 GGACCAUAGUUUCUUGUUATT UAACAAGAAACUAUGGUCCTC 15-E4 TTK NM_003318 GGAGGAAAAGAAGAAUUUATT UAAAUUCUUCUUUUCCUCCTC 15-E5 ULK1 NM_003565 GCUUUGUCAAUCACCCAAGTT CUUGGGUGAUUGACAAAGCTG 15-E6 ULK2 NM_014683 GGAAUUACCCAACUCUGUCTT GACAGAGUUGGGUAAUUCCTG 15-E7 NM_003390 GGUAUAUUCAUUCAAUGUCTT GACAUUGAAUGAAUAUACCTC 15-E8 NEK8 NM_178170 GGCAUCAUCAUGACAUUCGTT CGAAUGUCAUGAUGAUGCCTC 15-E9 PRKWNK3 NM_001002 GGAGAAAAACAGUACCUUCTT GAAGGUACUGUUUUUCUCCTT 15-E10 AAK1 NM_014911 GGUGUGCAAGAGAGAAAUCTT GAUUUCUCUCUUGCACACCTG 15-E11 ASB10 NM_080871 GACUCUGGUCUCUGACAUATT UAUGUCAGAGACCAGAGUCTC 15-F1 BMP2K NM_017593 GGUAGUGAAUCAAAUGAAUTT AUUCAUUUGAUUCACUACCTG 15-F2 BUB1 NM_004336 GGUUAUUUCAGACACGCCUTT AGGCGUGUCUGAAAUAACCTG 15-F3 BUB1B NM_001211 GGUGGGAAGGAGAGUAAUATT UAUUACUCUCCUUCCCACCTT 15-F4 TP53RK NM_033550 GGGUCUCUCCAACUUAGCCTT GGCUAAGUUGGAGAGACCCTG 15-F5 CAMKK1 NM_032294 GGAGGAGGUUAAGAACUCATT UGAGUUCUUAACCUCCUCCTC 15-F6 CAMKK2 NM_006549 GGAUGAAAUUGGAAAGGGCTT GCCCUUUCCAAUUUCAUCCTT 15-F7 CDC7 NM_003503 GGCUCUUUAAAAAAAAACGTT CGUUUUUUUUUAAAGAGCCTT 15-F8 CHUK NM_001278 GGAGAUCUCCGAAAGCUGCTT GCAGCUUUCGGAGAUCUCCTC 15-F9 PLK3 NM_004073 GCGCGAGAAGAUCCUAAAUTT AUUUAGGAUCUUCUCGCGCTG 15-F10 CSNK2A1 NM_001895 GGAAGGGAGGACCCAAUCUTT AGAUUGGGUCCUCCCUUCCTT 15-F11 CSNK2A2 NM_001896 GGUUAAGAUUCUGGAGAACTT GUUCUCCAGAAUCUUAACCTC 15-G1 EIF2AK3 NM_004836 GGUCAUUAGUAAUUAUCAGTT CUGAUAAUUACUAAUGACCTG 15-G2 ERN1 NM_001433 GGCUCAAUCAAAUGGACUUTT AAGUCCAUUUGAUUGAGCCTG 15-G3 ERN2 NM_033266 GGACCAAUGUACGUCACAGTT CUGUGACGUACAUUGGUCCTT 15-G4 FLJ10074 NM_017988 GGACGGGUUACAGAAUAAATT UUUAUUCUGUAACCCGUCCTC 15-G5 PXK NM_017771 GGAGGAAAGAAAAAAAAGATT UCUUUUUUUUCUUUCCUCCTC 15-G6 FLJ20574 NM_017886 GGUGGUUCCUUAAAAACAGTT CUGUUUUUAAGGAACCACCTG 15-G7 FLJ23356 NM_032237 GGAUAGGACAGAUGAAAAATT UUUUUCAUCUGUCCUAUCCTG 15-G8 FLJ32685 NM_152534 GGUAUUUUAUGGAAGCCAATT UUGGCUUCCAUAAAAUACCTT 15-G9 GAK NM_005255 GGGUUUGCAUUUGUGUAUGTT CAUACACAAAUGCAAACCCTC 15-G10 GSG2 NM_031965 GGCUGGAGAGAACUAGAUCTT GAUCUAGUUCUCUCCAGCCTG 15-G11 GUCY2C NM_004963 GGCCUCGACCUACUCAGGATT UCCUGAGUAGGUCGAGGCCTT 15-H1 GUCY2D NM_000180 GGCACCGGCUACUUCACAUTT AUGUGAAGUAGCCGGUGCCTC 15-H2 GUCY2F NM_001522 GGGCUCUCUCCAGUUUCAUTT AUGAAACUGGAGAGAGCCCTC 15-H3 HRI NM_014413 GGUGUUUAAGCUACUUUGCTT GCAAAGUAGCUUAAACACCTG 15-H4 IKBKE NM_014002 GGUACUGGUGAUGGAGUACTT GUACUCCAUCACCAGUACCTT 15-H5 KIS NM_144624 GGGUCACAGAAACAUCGUGTT CACGAUGUUUCUGUGACCCTG 15-H6 LOC149420 NM_152835 GGAUGGGAUGGUGCAAAAGTT CUUUUGCACCAUCCCAUCCTT 15-H7 MGC16169 NM_033115 GGAUACAGAGUACCAACUATT UAGUUGGUACUCUGUAUCCTT 15-H8 MOS NM_005372 GGACAGUUAAGUUUGGGAATT UUCCCAAACUUAACUGUCCTC 15-H9 NEK11 NM_024800 GGUACUUAAGGAAAUAUCUTT AGAUAUUUCCUUAAGUACCTT 15-H10 NEK2 NM_002497 GGAUGUUAAACUUAAAGGATT UCCUUUAAGUUUAACAUCCTC 15-H11 NEK3 NM_002498 GGAGGCUGUUCUUUUAGCCTT GGCUAAAAGAACAGCCUCCTT 17-A1 RNASEL NM_021133 GGGAGGACAUUGUGGAACUTT AGUUCCACAAUGUCCUCCCTG 17-A2 SCYL1 NM_020680 GGAUUUCUGUCGGCACAAGTT CUUGUGCCGACAGAAAUCCTC 17-A3 PRKWNK2 NM_006648 GGAUGCUGACAUAAAGAAGTT CUUCUUUAUGUCAGCAUCCTC 17-A4 PLK2 NM_006622 GGGAAAAGAUUGACAAAGATT UCUUUGUCAAUCUUUUCCCTT 17-A5 AURKB NM_004217 GGUCCUCUUCAAGUCCCAGTT CUGGGACUUGAAGAGGACCTT 17-A6 AURKC NM_003160 GGUAGAUGUGAGGUUUCCATT UGGAAACCUCACAUCUACCTT 17-A7 STK16 NM_003691 GGCUGAAGGACAAAGGCAATT UUGCCUUUGUCCUUCAGCCTT 17-A8 PLK4 NM_014264 GGACACAUCAAAAAAUGCCTT GGCAUUUUUUGAUGUGUCCTT 17-A9 STK31 NM_031414 GGUGCUUAAGAAAGGAUUUTT AAAUCCUUUCUUAAGCACCTC 17-A10 STK35 NM_080836 GGCAAAGAGGGCAAUCAAGTT CUUGAUUGCCCUCUUUGCCTC 17-A11 STK36 NM_015690 GGUAAAAGUAGUAGAUUGGTT CCAAUCUACUACUUUUACCTT 17-B1 STK6 NM_003600 GGAACUGGCAUCAAAACAGTT CUGUUUUGAUGCCAGUUCCTC 17-B2 TBK1 NM_013254 GGCAUUUUUGAAUAAGUCATT UGACUUAUUCAAAAAUGCCTC 17-B3 TEX14 NM_031272 GGUACACUUAAAAUGGAAATT UUUCCAUUUUAAGUGUACCTT 17-B4 TLK1 NM_012290 GGACCUGGAAAAGAAGGAGTT CUCCUUCUUUUCCAGGUCCTG 17-B5 TLK2 NM_006852 GGAAAGGAUAAAUUCACAGTT CUGUGAAUUUAUCCUUUCCTG 17-B6 TOPK NM_018492 GGUGAAAAGUCUCUAAAUGTT CAUUUAGAGACUUUUCACCTC 17-B7 TTK NM_003318 GGAAAAGAAGAAUUUAUCATT UGAUAAAUUCUUCUUUUCCTC 17-B8 ULK1 NM_003565 GCGAAUUUUGUGUGAUUUCTT GAAAUCACACAAAAUUCGCTT 17-B9 ULK2 NM_014683 GGUUUAUUGAUGAAAUCAATT UUGAUUUCAUCAAUAAACCTC 17-B10 WEE1 NM_003390 GGCUGGAUGGAUGCAUUUATT UAAAUGCAUCCAUCCAGCCTC 17-B11 NEK8 NM_178170 GGAGCCUCUGCUGAGUAUATT UAUACUCAGCAGAGGCUCCTG 17-C1 PRKWNK3 NM_001002 GGAAACCUGUAAAGAAAAUTT AUUUUCUUUACAGGUUUCCTG 17-C2 ADK NM_001123 GGAACUGUUUGAUGAACUUTT AAGUUCAUCAAACAGUUCCTT 17-C3 AK1 NM_000476 GGGCACCCAGUGUGAGAAGTT CUUCUCACACUGGGUGCCCTT 17-C4 AK3 NM_013410 GGAAGAUGUGGUCAUUCAUTT AUGAAUGACCACAUCUUCCTG 17-C5 AK3L1 NM_016282 GGUUAAGAAAUGUGUUUUATT UAAAACACAUUUCUUAACCTG 17-C6 AK5 NM_012093 GGCACACAGUGUGAAAAGCTT GCUUUUCACACUGUGUGCCTT

17-C7 C9orf12 NM_022755 GGUCGUUCAGCUACCUUUATT UAAAGGUAGCUGAACGACCTC

60 Materials and Methods

17-C8 CALM3 NM_005184 CCAAUUGAUUGACUGAGAATT UUCUCAGUCAAUCAAUUGGTG 17-C9 CARKL NM_013276 GGAUGUGAGUAGAAUCCUCTT GAGGAUUCUACUCACAUCCTG 17-C10 CDK5R1 NM_003885 GGAGCAUUUUGUGUCUUAATT UUAAGACACAAAAUGCUCCTG 17-C11 CDKN1A NM_000389 GGCCCGCUCUACAUCUUCUTT AGAAGAUGUAGAGCGGGCCTT 17-D1 CDKN3 NM_005192 GGACUUGGGAGAUCUUGUCTT GACAAGAUCUCCCAAGUCCTC 17-D2 CERK NM_022766 GGUAGCAGUGGGUCACUUUTT AAAGUGACCCACUGCUACCTC 17-D3 CHKA NM_001277 GGUACUUUGGUUAAUGUUGTT CAACAUUAACCAAAGUACCTG 17-D4 CKB NM_001823 CCCAGAUUGAAACUCUCUUTT AAGAGAGUUUCAAUCUGGGTG 17-D5 CKM NM_001824 GGUACUGACCCUUGAACUCTT GAGUUCAAGGGUCAGUACCTT 17-D6 CKMT1 NM_020990 GGAGACCUAUGAGGUAUUUTT AAAUACCUCAUAGGUCUCCTC 17-D7 CKMT2 NM_001825 GGAGUCCUAUGAGGUGUUUTT AAACACCUCAUAGGACUCCTC 17-D8 CSF1R NM_005211 GGGCCAAGUUCAUUCAGAGTT CUCUGAAUGAACUUGGCCCTG 17-D9 DCK NM_000788 GGAACUUACAAUGUCUCAGTT CUGAGACAUUGUAAGUUCCTC 17-D10 DGKA NM_001345 GGAUUUAGAGAUGAGUAAATT UUUACUCAUCUCUAAAUCCTT 17-D11 DGKB NM_004080 GGAUGUUCUUGAAGAAUUCTT GAAUUCUUCAAGAACAUCCTT 17-E1 DGKD NM_003648 GGAAGCUCAUCUUGUGUGCTT GCACACAAGAUGAGCUUCCTG 17-E2 DGKE NM_003647 GGAGAUUAUGCUCAAGAAUTT AUUCUUGAGCAUAAUCUCCTT 17-E3 DGKG NM_001346 GGGUGGGAGCCUCAAACAATT UUGUUUGAGGCUCCCACCCTC 17-E4 DGKI NM_004717 GGAGAAGCUGAGGAACUUATT UAAGUUCCUCAGCUUCUCCTC 17-E5 DGKQ NM_001347 GGCUGCACAACAAGGGUGUTT ACACCCUUGUUGUGCAGCCTG 17-E6 DGKZ NM_003646 GGAACGACUUCUGUAAGCUTT AGCUUACAGAAGUCGUUCCTC 17-E7 DGUOK NM_001929 GGCUCUCCAUCGAAGGCAATT UUGCCUUCGAUGGAGAGCCTT 17-E8 DKFZP586B1621 NM_015533 GGACUAUGCUGGAUUCUCUTT AGAGAAUCCAGCAUAGUCCTG 17-E9 DTYMK NM_012145 GGAAAAGUUGAGCCAGGGCTT GCCCUGGCUCAACUUUUCCTT 17-E10 ETNK1 NM_018638 GGAAGUAAAGAGUUUUCGATT UCGAAAACUCUUUACUUCCTC 17-E11 ETNK2 NM_018208 GGUUAAUCGCCUUAGAAAUTT AUUUCUAAGGCGAUUAACCTG 17-F1 FLJ10842 NM_018238 GGUGUUUGGCAAUCAACUCTT GAGUUGAUUGCCAAACACCTG 17-F2 RFK NM_018339 GGUGAUAUUGAAGAAGCUATT UAGCUUCUUCAAUAUCACCTT 17-F3 FN3KRP NM_024619 GGAGUCUUCAUUGCUUCUGTT CAGAAGCAAUGAAGACUCCTG 17-F4 FLJ12476 NM_022784 GGCUGUGUGACAUCUUAGATT UCUAAGAUGUCACACAGCCTC 17-F5 FLJ13052 NM_023018 GGAGUUCAGGAGGACACGCTT GCGUGUCCUCCUGAACUCCTT 17-F6 PIP5K2C NM_024779 GGCCAGCUCCAAGAUCAAGTT CUUGAUCUUGGAGCUGGCCTT 17-F7 C9orf98 NM_152572 GGAUUGUAAUAUUAGGUCCTT GGACCUAAUAUUACAAUCCTG 17-F8 FN3K NM_022158 GGCCCAGUGUUCGUCAAAGTT CUUUGACGAACACUGGGCCTG 17-F9 FUK NM_145059 GGCCUUAACUAGCAAAACUTT AGUUUUGCUAGUUAAGGCCTG 17-F10 GALK1 NM_000154 GGGAGUGAUUCAGUACUACTT GUAGUACUGAAUCACUCCCTT 17-F11 GALK2 NM_001001 GGAACUGCCAAGUUGAUAGTT CUAUCAACUUGGCAGUUCCTT 17-G1 GCK NM_000162 GGACCUGAAGAAGGUGAUGTT CAUCACCUUCUUCAGGUCCTC 17-G2 GK NM_000167 GGAUGUUUCUUACUAUGUATT UACAUAGUAAGAAACAUCCTG 17-G3 GK2 NM_033214 GGAAAUAGUAACUUCGUCATT UGACGAAGUUACUAUUUCCTG 17-G4 GNE NM_005476 GGACCAUGAUCGCAUCCUUTT AAGGAUGCGAUCAUGGUCCTC 17-G5 GUK1 NM_000858 GGAAAUCAAGAAAGCUCAATT UUGAGCUUUCUUGAUUUCCTC 17-G6 HK1 NM_000188 GGUAUGAGAAGAUGAUCAGTT CUGAUCAUCUUCUCAUACCTT 17-G7 HK2 NM_000189 GGUUGACCAGUAUCUCUACTT GUAGAGAUACUGGUCAACCTT 17-G8 HK3 NM_002115 GGUGACAAGGGCACAGCUATT UAGCUGUGCCCUUGUCACCTT 17-G9 IHPK1 NM_153273 GGCGUGGUAUCUGUCUGUUTT AACAGACAGAUACCACGCCTT 17-G10 IHPK3 NM_054111 GGUUGAGAGGAAGAGCUUCTT GAAGCUCUUCCUCUCAACCTG 17-G11 ITPK1 NM_014216 GGUUCCAGGAGUACAUCGATT UCGAUGUACUCCUGGAACCTG 17-H1 ITPKA NM_002220 GGACGUGGGUCAGAAAAACTT GUUUUUCUGACCCACGUCCTC 17-H2 ITPKB NM_002221 GGAAGAUAAAAAACAUGGUTT ACCAUGUUUUUUAUCUUCCTC 17-H3 ITPKC NM_025194 GGCAGAAUUCUGGACAGACTT GUCUGUCCAGAAUUCUGCCTG 17-H4 KHK NM_000221 GGACUCGGAGAUAAGGUGUTT ACACCUUAUCUCCGAGUCCTC 17-H5 KIAA0626 NM_021647 GGCAUUUGAGAUCGCCAAGTT CUUGGCGAUCUCAAAUGCCTT 17-H6 LCK NM_005356 GGGAGAGGUGGUGAAACAUTT AUGUUUCACCACCUCUCCCTG 17-H7 MGC26597 NM_152700 GGUGUUCACCUUGGUCGUUTT AACGACCAAGGUGAACACCTG 17-H8 C6orf199 NM_145025 GGAAUUUUUUGUUGUCCAATT UUGGACAACAAAAAAUUCCTT 17-H9 MRC2 NM_006039 GGUCUACACCAUCCAGGGATT UCCCUGGAUGGUGUAGACCTC 17-H10 MVK NM_000431 GGAGGAUUUGGAGCUAAUUTT AAUUAGCUCCAAAUCCUCCTT 17-H11 NAGK NM_017567 GGAUGGGAAGAUCCUGGCATT UGCCAGGAUCUUCCCAUCCTC 18-A1 COASY NM_025233 GGUUCUUGAUUUCAUCACGTT CGUGAUGAAAUCAAGAACCTC 18-A2 NME6 NM_005793 GGCUGUUCAUCAGCAGAUUTT AAUCUGCUGAUGAACAGCCTC 18-A3 NME1 NM_000269 GGAUUCCGCCUUGUUGGUCTT GACCAACAAGGCGGAAUCCTT 18-A4 NME2 NM_002512 CCAAUCCAGCAGAUUCAAATT UUUGAAUCUGCUGGAUUGGTC 18-A5 NME3 NM_002513 GAGGAAGGGCUUCAAGUUGTT CAACUUGAAGCCCUUCCUCTC 18-A6 NME4 NM_005009 GGAGUUGAACAGUAAAGAGTT CUCUUUACUGUUCAACUCCTT 18-A7 NME7 NM_013330 GGAAAGAAGCAUUGGAUUUTT AAAUCCAAUGCUUCUUUCCTT 18-A8 CDADC1 NM_030911 GGCUUUCUAAAGUCAACCUTT AGGUUGACUUUAGAAAGCCTT 18-A9 NYD-SP25 NM_033516 GGAUGACCAUCUCCACUUUTT AAAGUGGAGAUGGUCAUCCTC 18-A10 P15RS NM_018170 GGAAGCUUACUUUUCUCUATT UAGAGAAAAGUAAGCUUCCTG 18-A11 PACSIN1 NM_020804 GGAGGUGAAGAACAAUCUGTT CAGAUUGUUCUUCACCUCCTG 18-B1 PANK1 NM_138316 GGUGUCAGAUAGUAAUAUCTT GAUAUUACUAUCUGACACCTC 18-B2 PAPSS1 NM_005443 GGAGUACCUGGUUUGUCAUTT AUGACAAACCAGGUACUCCTC 18-B3 PAPSS2 NM_004670 GGUAUUGAUUCUGAUUAUGTT CAUAAUCAGAAUCAAUACCTG 18-B4 PDXK NM_003681 GGCUGAACAACAUGAAUAATT UUAUUCAUGUUGUUCAGCCTC 18-B5 PFKFB1 NM_002625 GGCAAGACCUAUAUCUCCATT UGGAGAUAUAGGUCUUGCCTC 18-B6 PFKFB2 NM_006212 GGAGGGACAUGAUUUUGAATT UUCAAAAUCAUGUCCCUCCTC 18-B7 PFKFB3 NM_004566 GGAGACACAUGAUCCUUCATT UGAAGGAUCAUGUGUCUCCTC 18-B8 PFKFB4 NM_004567 GGGCCUGAAAAUCAGGAAGTT CUUCCUGAUUUUCAGGCCCTC 18-B9 PFKL NM_001002 GGAAAGAGGAAGUGACCUCTT GAGGUCACUUCCUCUUUCCTG 18-B10 PFKM NM_000289 GGAAUGAAAAGUGCAAUGATT UCAUUGCACUUUUCAUUCCTT 18-B11 PFKP NM_002627 GGCUGGGAGGAGCAGAUGUTT ACAUCUGCUCCUCCCAGCCTT

18-C1 PGK1 NM_000291 GGAACCCUUAAACAGUUGCTT GCAACUGUUUAAGGGUUCCTG

61 Materials and Methods

18-C2 PGK2 NM_138733 GGAUCAAGGCUUCCAUCCCTT GGGAUGGAAGCCUUGAUCCTC 18-C3 PI4K2B NM_018323 GGUUCAAGUGGAAGUUACUTT AGUAACUUCCACUUGAACCTT 18-C4 PI4KII NM_018425 GGAUCAUUGCUGUCUUCAATT UUGAAGACAGCAAUGAUCCTC 18-C5 PIK4CA NM_002650 GGCUGGAUCAACACAUACCTT GGUAUGUGUUGAUCCAGCCTT 18-C6 PIK4CB NM_002651 GGAGGUGUUGGAGAAAGUCTT GACUUUCUCCAACACCUCCTG 18-C7 PIP5K1A NM_003557 GCUGCUCCUCCAUCUUCUUTT AAGAAGAUGGAGGAGCAGCTC 18-C8 PIP5K2A NM_005028 GGUGGACAAUCACCUUUUUTT AAAAAGGUGAUUGUCCACCTT 18-C9 PIP5K2B NM_003559 GGUGGACAAUCAUCUCUUCTT GAAGAGAUGAUUGUCCACCTT 18-C10 CDC42BPA NM_003607 GGAUGACAAUAACUUAUACTT GUAUAAGUUAUUGUCAUCCTG 18-C11 PRKD2 NM_016457 GGAAAUUCCGCUGUCAGAATT UUCUGACAGCGGAAUUUCCTT 18-D1 PKLR NM_000298 GGAGAUGAUCAAGGCCGGGTT CCCGGCCUUGAUCAUCUCCTT 18-D2 PKM2 NM_002654 GGGAAAGAACAUCAAGAUUTT AAUCUUGAUGUUCUUUCCCTT 18-D3 PMVK NM_006556 GGAGCAUGGCUUGAACUUCTT GAAGUUCAAGCCAUGCUCCTG 18-D4 PNKP NM_007254 GGAGUUCUUUCUCAAGUGGTT CCACUUGAGAAAGAACUCCTC 18-D5 PRKAR2A NM_004157 GGAGGACGAGGACUUGGAATT UUCCAAGUCCUCGUCCUCCTC 18-D6 PRKRA NM_003690 GGGAAAACACCGAUUCAGGTT CCUGAAUCGGUGUUUUCCCTG 18-D7 PRPF4B NM_003913 GGUUAUUGAUGCUUCUGAUTT AUCAGAAGCAUCAAUAACCTC 18-D8 PRPS1 NM_002764 CGCAUGCUUUGAGGCAGUATT UACUGCCUCAAAGCAUGCGTT 18-D9 PRPS2 NM_002765 GGCGACAACUUUCAAGUAUTT AUACUUGAAAGUUGUCGCCTC 18-D10 ALDH18A1 NM_002860 GGUCUUCACGUUAAACUUGTT CAAGUUUAACGUGAAGACCTT 18-D11 RBKS NM_022128 GGUGCCAACCAGUGUGUCCTT GGACACACUGGUUGGCACCTT 18-E1 SPHK1 NM_021972 GGCUGAAAUCUCCUUCACGTT CGUGAAGGAGAUUUCAGCCTC 18-E2 SPHK2 NM_020126 GGAUUGCGCUCGCUUUCAUTT AUGAAAGCGAGCGCAAUCCTG 18-E3 SEPHS1 NM_012247 GGGAAAGGGAUAAAGUGAUTT AUCACUUUAUCCCUUUCCCTG 18-E4 SEPHS2 NM_012248 GGUUGCUGUCAAUGCCCACTT GUGGGCAUUGACAGCAACCTG 18-E5 TK1 NM_003258 GGUGAUUCUCGGGCCGAUGTT CAUCGGCCCGAGAAUCACCTG 18-E6 TK2 NM_004614 GGCAUACUCGUCCUCAGGUTT ACCUGAGGACGAGUAUGCCTG 18-E7 TPK1 NM_022445 GGUGCCAACCGCUUAUAUGTT CAUAUAAGCGGUUGGCACCTC 18-E8 UCK1 NM_031432 GGCCAAGGCCUUGAAAGGATT UCCUUUCAAGGCCUUGGCCTT 18-E9 UGP2 NM_001001 GGCCCUAAAAGUCUGAUUGTT CAAUCAGACUUUUAGGGCCTT 18-E10 UMP-CMPK NM_016308 GGAAGAACCCAGAUUCACATT UGUGAAUCUGGGUUCUUCCTT 18-E11 UCK2 NM_012474 GGAGGUACGAGACCUGUUCTT GAACAGGUCUCGUACCUCCTG 18-F1 UCKL1 NM_017859 GGUGCUGACUGAGCAGCAGTT CUGCUGCUCAGUCAGCACCTT 18-F2 XYLB NM_005108 GGAAAGUGUGCAUUUUGACTT GUCAAAAUGCACACUUUCCTC 18-F3 ADK NM_001123 GGAACUUGUGCUGCAUGCATT UGCAUGCAGCACAAGUUCCTG 18-F4 AK1 NM_000476 GGCUUCCUGAUUGAUGGCUTT AGCCAUCAAUCAGGAAGCCTT 18-F5 AK3 NM_013410 GGGUAUAUAACCUGGACUUTT AAGUCCAGGUUAUAUACCCTT 18-F6 AK3L1 NM_016282 GGCUUUCAUUGACCAAGGGTT CCCUUGGUCAAUGAAAGCCTT 18-F7 AK5 NM_012093 GGAACUGGCAUCAGAAUCUTT AGAUUCUGAUGCCAGUUCCTC 18-F8 C9orf12 NM_022755 GGCUGAAUGAUAGAGAUAUTT AUAUCUCUAUCAUUCAGCCTT 18-F9 CALM3 NM_005184 GGUCAAUUAUGAAGAGUUUTT AAACUCUUCAUAAUUGACCTG 18-F10 CARKL NM_013276 GGCUGUGAAUGGACAGAGGTT CCUCUGUCCAUUCACAGCCTT 18-F11 CDK5R1 NM_003885 GGUCUUCUCCGACCUGAAGTT CUUCAGGUCGGAGAAGACCTG 18-G1 CDKN1A NM_000389 GGCGGUUAUGAAAUUCACCTT GGUGAAUUUCAUAACCGCCTG 18-G2 CDKN3 NM_005192 GAUGAACAGACUCCAAUUCTT GAAUUGGAGUCUGUUCAUCTT 18-G3 CERK NM_022766 GGCUAGUUUUAGCUUCCUUTT AAGGAAGCUAAAACUAGCCTT 18-G4 CHKA NM_001277 GGACGAGUUCCACAUCAGUTT ACUGAUGUGGAACUCGUCCTC 18-G5 CKB NM_001823 GGACUAUGAGUUCAUGUGGTT CCACAUGAACUCAUAGUCCTT 18-G6 CKM NM_001824 GGAGACUCCAUCUGGCUUCTT GAAGCCAGAUGGAGUCUCCTT 18-G7 CKMT1 NM_020990 GGUAUUUGCUGACCUGUUUTT AAACAGGUCAGCAAAUACCTC 18-G8 CKMT2 NM_001825 GGGUGAUGAAGCACACAACTT GUUGUGUGCUUCAUCACCCTG 18-G9 CSF1R NM_005211 GGACUAUCAAUGCAGUGCCTT GGCACUGCAUUGAUAGUCCTG 18-G10 DCK NM_000788 GGGAAGUCAACAUUUGUGATT UCACAAAUGUUGACUUCCCTG 18-G11 DGKA NM_001345 GGAUGGCGAGAUGGCUAAATT UUUAGCCAUCUCGCCAUCCTC 18-H1 DGKB NM_004080 GGGAAACAAGACAUUCUUATT UAAGAAUGUCUUGUUUCCCTT 18-H2 DGKD NM_003648 GGAAUGAACAUGAGUGUUUTT AAACACUCAUGUUCAUUCCTT 18-H3 DGKE NM_003647 GGUGCAUUUGGUGCCAGAATT UUCUGGCACCAAAUGCACCTG 18-H4 DGKG NM_001346 GGCCAACAGCGCAGAUACUTT AGUAUCUGCGCUGUUGGCCTC 18-H5 DGKI NM_004717 GGAACUUAACUUUCCGGAATT UUCCGGAAAGUUAAGUUCCTC 18-H6 DGKQ NM_001347 GGAGGUGGAGCUGCCCAGUTT ACUGGGCAGCUCCACCUCCTG 18-H7 DGKZ NM_003646 GGGAUUCCAGCAGAAGUUCTT GAACUUCUGCUGGAAUCCCTT 18-H8 DGUOK NM_001929 GGAAGCCAGUACAGAUCUUTT AAGAUCUGUACUGGCUUCCTG 18-H9 DKFZP586B1621 NM_015533 GGAGCUGAUCUGUUACAAGTT CUUGUAACAGAUCAGCUCCTG 18-H10 DTYMK NM_012145 GAUCAACUGAAAUCGGCAATT UUGCCGAUUUCAGUUGAUCTT 18-H11 ETNK1 NM_018638 GGAUGUAGUCCUGGUGAGATT UCUCACCAGGACUACAUCCTC 21-A1 PFKM NM_000289 GGACUUUCGGGAACGAGAATT UUCUCGUUCCCGAAAGUCCTT 21-A2 PFKP NM_002627 GGAAAAAAAUUUACCACCGTT CGGUGGUAAAUUUUUUUCCTC 21-A3 PGK1 NM_000291 GGAAGAAGGGAAGGGAAAATT UUUUCCCUUCCCUUCUUCCTC 21-A4 PGK2 NM_138733 GGAACUAGAUUACUUUGCUTT AGCAAAGUAAUCUAGUUCCTT 21-A5 PI4K2B NM_018323 GGAAAAUUAUUGGUGUGUUTT AACACACCAAUAAUUUUCCTC 21-A6 PI4KII NM_018425 GGACCUAUAUGAACUCUUCTT GAAGAGUUCAUAUAGGUCCTC 21-A7 PIK4CA NM_002650 GGUUUAAGAACACAGAAGCTT GCUUCUGUGUUCUUAAACCTG 21-A8 PIK4CB NM_002651 GGCUGUUUGAGUCAAAACUTT AGUUUUGACUCAAACAGCCTC 21-A9 PIP5K1A NM_003557 GGCACAAGUGACAACAAAGTT CUUUGUUGUCACUUGUGCCTT 21-A10 PIP5K2A NM_005028 GGUCUUCCUGGAAAAACUATT UAGUUUUUCCAGGAAGACCTT 21-A11 PIP5K2B NM_003559 GGAGAGUAAAAAGAACUUCTT GAAGUUCUUUUUACUCUCCTC 21-B1 CDC42BPA NM_003607 GGUUUCAGUUUCCAGCCCATT UGGGCUGGAAACUGAAACCTC 21-B2 PRKD2 NM_016457 CGACCAACAGAUACUAUAATT UUAUAGUAUCUGUUGGUCGTG 21-B3 PKLR NM_000298 GGCCUAUCUGAGACUAUAATT UUAUAGUCUCAGAUAGGCCTC 21-B4 PKM2 NM_002654 GGACCUGAGAUCCGAACUGTT CAGUUCGGAUCUCAGGUCCTT 21-B5 PMVK NM_006556 GGAACAGUAUGCUCAGGAGTT CUCCUGAGCAUACUGUUCCTT

21-B6 PNKP NM_007254 GGUGGCUGGCUUUGAUCUGTT CAGAUCAAAGCCAGCCACCTT

62 Materials and Methods

21-B7 PRKAR2A NM_004157 GGCUGAUAGCUUUUACAUCTT GAUGUAAAAGCUAUCAGCCTT 21-B8 PRKRA NM_003690 GGACCUGCUCAUAAGAGAGTT CUCUCUUAUGAGCAGGUCCTC 21-B9 PRPF4B NM_003913 GGCAGGCAAUUGUUCAGAATT UUCUGAACAAUUGCCUGCCTT 21-B10 PRPS1 NM_002764 CGGAUUGAGAUUAACUGCUTT AGCAGUUAAUCUCAAUCCGTC 21-B11 PRPS2 NM_002765 GGAGACCAGCGUGGAGAUUTT AAUCUCCACGCUGGUCUCCTG 21-C1 ALDH18A1 NM_002860 GGCUAUGUUUACCCAGUACTT GUACUGGGUAAACAUAGCCTC 21-C2 RBKS NM_022128 GGAACUGCUUCUAUAAUUGTT CAAUUAUAGAAGCAGUUCCTG 21-C3 SPHK1 NM_021972 GGGCAGGCAUAUGGAGUAUTT AUACUCCAUAUGCCUGCCCTT 21-C4 SPHK2 NM_020126 GUGGUGUAAGAACCACGUGTT CACGUGGUUCUUACACCACTG 21-C5 SEPHS1 NM_012247 GGAUAGCGUGUGCCAAUGUTT ACAUUGGCACACGCUAUCCTG 21-C6 SEPHS2 NM_012248 GGAAGCCAUGUUCAAUAUGTT CAUAUUGAACAUGGCUUCCTG 21-C7 TK1 NM_003258 GGAACAACAGCAUCUUUCATT UGAAAGAUGCUGUUGUUCCTG 21-C8 TK2 NM_004614 GGUGUUAACGGAGCCUGUGTT CACAGGCUCCGUUAACACCTC 21-C9 TPK1 NM_022445 GGUAACAUUAUAAAUGACATT UGUCAUUUAUAAUGUUACCTG 21-C10 UCK1 NM_031432 GGACAGUACAAUUUUGACCTT GGUCAAAAUUGUACUGUCCTT 21-C11 UGP2 NM_001001 GGAUUAAUAAAGAAUCUUUTT AAAGAUUCUUUAUUAAUCCTC 21-D1 UMP-CMPK NM_016308 GGAGAGCUGCUUCGUGAUGTT CAUCACGAAGCAGCUCUCCTG 21-D2 UCK2 NM_012474 GGGAUCUUGAGCAGAUUUUTT AAAAUCUGCUCAAGAUCCCTG 21-D3 UCKL1 NM_017859 GGACUGUUGAAUACAAAGATT UCUUUGUAUUCAACAGUCCTG 21-D4 XYLB NM_005108 GGUUCGAGCACUAAUUGAATT UUCAAUUAGUGCUCGAACCTC 21-D5 CBL NM_005188 GCUCGGCUCCAGAAAUUCATT UGAAUUUCUGGAGCCGAGCTT 21-D6 COL4A3BP NM_005713 CCACAUGACUUACUCAUUATT UAAUGAGUAAGUCAUGUGGTT 21-D7 DAB2 NM_001343 GGCCCUAAGGAUUCUAGAUTT AUCUAGAAUCCUUAGGGCCTC 21-D8 DKFZP434C131 NM_015518 GGUCAAGAUGAGGGAAUCUTT AGAUUCCCUCAUCUUGACCTG 21-D9 NRK NM_198465 CCUCUACAAAUGCAGAUUATT UAAUCUGCAUUUGUAGAGGTT 21-D10 FLJ22761 NM_025130 GGAAUCAUUGCAUGCUUAATT UUAAGCAUGCAAUGAUUCCTG 21-D11 GULP1 NM_016315 GCAGGCAGUAUGACACCUATT UAGGUGUCAUACUGCCUGCTG 21-E1 LOC340371 NM_178564 CCAGAAAGGUCAUCCAGAUTT AUCUGGAUGACCUUUCUGGTC 21-E2 STK32A NM_145001 GCGCAAUGAAGUGAGAAAUTT AUUUCUCACUUCAUUGCGCTC 21-E3 MMAA NM_172250 GGACCACAAAUGAAGCUAUTT AUAGCUUCAUUUGUGGUCCTT 21-E4 MPP1 NM_002436 GCACAGCUCGAUUUUUGAUTT AUCAAAAAUCGAGCUGUGCTT 21-E5 MPP2 NM_005374 GCGGAAAGCAUUUGUCAAGTT CUUGACAAAUGCUUUCCGCTT 21-E6 MPP5 NM_022474 GCACCGAGAGAUGGCUGUUTT AACAGCCAUCUCUCGGUGCTT 21-E7 MPP6 NM_016447 GCUGUCAGUGACAAUAACUTT AGUUAUUGUCACUGACAGCTT 21-E8 MPP7 NM_173496 CCACUGGGAGCUACCAUUATT UAAUGGUAGCUCCCAGUGGTT 21-E9 PCDH7 NM_002589 CGUGUUUGUCAAUGAAAGUTT ACUUUCAUUGACAAACACGTG 21-E10 SITPEC NM_016581 GAUGCGGGAGUAUGGUGUCTT GACACCAUACUCCCGCAUCTT 21-E11 TXNDC6 NM_178130 GCUGGGUUUGAAAUUCUAATT UUAGAAUUUCAAACCCAGCTT 21-F1 TXNDC3 NM_016616 CGUCAAUAUACAACCAUUUTT AAAUGGUUGUAUAUUGACGTG 21-F2 CBL NM_005188 CGGUGGACAAGAAGAUGGUTT ACCAUCUUCUUGUCCACCGTC 21-F3 COL4A3BP NM_005713 CCUCACGAUUUUGAUGAAUTT AUUCAUCAAAAUCGUGAGGTG 21-F4 DAB2 NM_001343 GCAAAGAUAUCCUGUUAGUTT ACUAACAGGAUAUCUUUGCTT 21-F5 DKFZP434C131 NM_015518 GGACACUCGUGAAGUGGUATT UACCACUUCACGAGUGUCCTT 21-F6 NRK NM_198465 GGUGAGCAGAACUAAUGGATT UCCAUUAGUUCUGCUCACCTG 21-F7 FLJ22761 NM_025130 GCAUUGACAAGGGAACACUTT AGUGUUCCCUUGUCAAUGCTC 21-F8 GULP1 NM_016315 GCCAUACCUUAAAGAUAACTT GUUAUCUUUAAGGUAUGGCTT 21-F9 LOC340371 NM_178564 GGUACAAAAUCGUGAAUCUTT AGAUUCACGAUUUUGUACCTT 21-F10 STK32A NM_145001 GUGCGUGGAGCGCAAUGAATT UUCAUUGCGCUCCACGCACTT 21-F11 MMAA NM_172250 GCCGUUUAAUUCUCUUGGATT UCCAAGAGAAUUAAACGGCTG 21-G1 MPP1 NM_002436 CCGAGGACAUGUACACCAATT UUGGUGUACAUGUCCUCGGTC 21-G2 MPP2 NM_005374 CCAGGAUGAUGCCAACUGGTT CCAGUUGGCAUCAUCCUGGTT 21-G3 MPP5 NM_022474 GGUGAUAUACUUCAUGUGATT UCACAUGAAGUAUAUCACCTT 21-G4 MPP6 NM_016447 GGCUAGCCAUGUAAAAGAGTT CUCUUUUACAUGGCUAGCCTG 21-G5 MPP7 NM_173496 GCAUUUCCAGGAAAGGAGATT UCUCCUUUCCUGGAAAUGCTT 21-G6 PCDH7 NM_002589 GCACUACGGUGAUUGUGCATT UGCACAAUCACCGUAGUGCTG 21-G7 SITPEC NM_016581 CCUCUACUACCCGAUGCAGTT CUGCAUCGGGUAGUAGAGGTT 21-G8 TXNDC6 NM_178130 GGUUGAAGAUCACUGACCUTT AGGUCAGUGAUCUUCAACCTT 21-G9 TXNDC3 NM_016616 CCGGAUGCUGUGAUUAGUATT UACUAAUCACAGCAUCCGGTT 21-G10 CBL NM_005188 CCUUAUAUCUUAGACCUGCTT GCAGGUCUAAGAUAUAAGGTG 21-G11 COL4A3BP NM_005713 GCAAUGGAUAGAUGCCAUUTT AAUGGCAUCUAUCCAUUGCTG 21-H1 DAB2 NM_001343 CCGGGCAUUUGGUUACGUGTT CACGUAACCAAAUGCCCGGTT 21-H2 DKFZP434C131 NM_015518 CGGAAUAUCUCUCACCUGGTT CCAGGUGAGAGAUAUUCCGTT 21-H3 NRK NM_198465 CGCACACCGAGUAAUUCACTT GUGAAUUACUCGGUGUGCGTG 21-H4 FLJ22761 NM_025130 GGGCAAGAUCGAAACACGGTT CCGUGUUUCGAUCUUGCCCTT 21-H5 GULP1 NM_016315 GGCACAGUAUUUUGUCUCGTT CGAGACAAAAUACUGUGCCTT 21-H6 LOC340371 NM_178564 GCCACCUCUUUGACCCUUUTT AAAGGGUCAAAGAGGUGGCTC 21-H7 STK32A NM_145001 CGAACAUGAUACCUGGCUCTT GAGCCAGGUAUCAUGUUCGTC 21-H8 MMAA NM_172250 GGAAAAAGGAGUUAGCCCATT UGGGCUAACUCCUUUUUCCTG 21-H9 MPP1 NM_002436 GCCGUCUUCCUGCACUACATT UGUAGUGCAGGAAGACGGCTT 21-H10 MPP2 NM_005374 GCGAAUGAUGUAUUUGACCTT GGUCAAAUACAUCAUUCGCTT 21-H11 MPP5 NM_022474 GGAUGAUGCCAAUACGUCGTT CGACGUAUUGGCAUCAUCCTG 22-A1 MPP6 NM_016447 GGUUCAUAGUAUUGAAUCCTT GGAUUCAAUACUAUGAACCTG 22-A2 MPP7 NM_173496 CCCAAUGUGAAGGCUUUGCTT GCAAAGCCUUCACAUUGGGTT 22-A3 PCDH7 NM_002589 GCAGACUGACAGGUAUGAGTT CUCAUACCUGUCAGUCUGCTC 22-A4 SITPEC NM_016581 CGGAGUUCCUGCUGAUUCATT UGAAUCAGCAGGAACUCCGTC 22-A5 TXNDC6 NM_178130 GCUGGUACAUCACAUGUGCTT GCACAUGUGAUGUACCAGCTT 22-A6 TXNDC3 NM_016616 GCAUAAGACAGUGCUCACUTT AGUGAGCACUGUCUUAUGCTC 22-A7 AK2 NM_001625 GAUUGGCUGAAAACUUCUGTT CAGAAGUUUUCAGCCAAUCTG 22-A8 AK7 NM_152327 GCGGAAACAUCGGGAAGUUTT AACUUCCCGAUGUUUCCGCTG 22-A9 AKAP1 NM_003488 CCAAAGACCUACGUGAGCUTT AGCUCACGUAGGUCUUUGGTG 22-A10 AKAP10 NM_007202 GCUUCAAUAUCCGUACAUUTT AAUGUACGGAUAUUGAAGCTT

22-A11 AKAP11 NM_016248 CGGUUACUGGUCAUCAUUUTT AAAUGAUGACCAGUAACCGTC

63 Materials and Methods

22-B1 AKAP12 NM_005100 GCGGUUGUUCACGACAUCATT UGAUGUCGUGAACAACCGCTG 22-B2 AKAP2 NM_001004 GCUUUCUGAGGAUGAUAUCTT GAUAUCAUCCUCAGAAAGCTG 22-B3 AKAP28 NM_178813 GGAAAGACUUAAUUCACAGTT CUGUGAAUUAAGUCUUUCCTC 22-B4 AKAP3 NM_006422 CCGCCUCACGAAUCUAGUCTT GACUAGAUUCGUGAGGCGGTT 22-B5 AKAP4 NM_003886 GGGCGAAUAUCACAGAGCATT UGCUCUGUGAUAUUCGCCCTC 22-B6 AKAP5 NM_004857 GGAAAAGGCAUCCAUGCUUTT AAGCAUGGAUGCCUUUUCCTT 22-B7 AKAP6 NM_004274 CCCAUGGCCAUCAACAUGATT UCAUGUUGAUGGCCAUGGGTG 22-B8 AKAP7 NM_004842 GCGUGCUGUUUAAGUUAAGTT CUUAACUUAAACAGCACGCTG 22-B9 AKAP8 NM_005858 GCUGGCAAGGUUAUGAAAATT UUUUCAUAACCUUGCCAGCTG 22-B10 AKAP8L NM_014371 GGAUAACACCACCAACUAUTT AUAGUUGGUGGUGUUAUCCTG 22-B11 AKAP9 NM_005751 GCAGUAAACAUGAUGUGUCTT GACACAUCAUGUUUACUGCTT 22-C1 AKIP NM_017900 GGACUCAGCCUUUAAAAAATT UUUUUUAAAGGCUGAGUCCTC 22-C2 ASK NM_006716 GGUUAAACUAAGAAUCCAATT UUGGAUUCUUAGUUUAACCTG 22-C3 CALM1 NM_006888 GCAGAACUACGUCACGUCATT UGACGUGACGUAGUUCUGCTG 22-C4 CALM2 NM_001743 GCAUUCCGUGUGUUUGAUATT UAUCAAACACACGGAAUGCTT 22-C5 CDC42SE2 NM_020240 GGUUAUGGAGGUGGAAUGCTT GCAUUCCACCUCCAUAACCTC 22-C6 CDKN1B NM_004064 GCUUGCCCGAGUUCUACUATT UAGUAGAACUCGGGCAAGCTG 22-C7 CDKN1C NM_000076 GGUACACUGGUCCCAAAGUTT ACUUUGGGACCAGUGUACCTT 22-C8 CDKN2A NM_000077 CGUAGAUAUAUGCCUUCCCTT GGGAAGGCAUAUAUCUACGTT 22-C9 CDKN2B NM_004936 CCCACCUAAUUCGAUGAAGTT CUUCAUCGAAUUAGGUGGGTG 22-C10 CDKN2C NM_001262 GGAUAAUGAAGGGAACCUGTT CAGGUUCCCUUCAUUAUCCTC 22-C11 CDKN2D NM_001800 GCACAGUUUGUGGCUUAUATT UAUAAGCCACAAACUGUGCTC 22-D1 CHKB NM_005198 GCGUGAUGUUCGCCAUACUTT AGUAUGGCGAACAUCACGCTT 22-D2 CIB2 NM_006383 CCUCGUCCCAAUGGACUACTT GUAGUCCAUUGGGACGAGGTT 22-D3 CIB3 NM_054113 GGCUCUUCUAUCGCUACCATT UGGUAGCGAUAGAAGAGCCTC 22-D4 CNKSR1 NM_006314 GGCACUGGAAGGAAUGGUATT UACCAUUCCUUCCAGUGCCTC 22-D5 CRIM1 NM_016441 GCCGGGAAACCUGAACAUATT UAUGUUCAGGUUUCCCGGCTG 22-D6 CSNK2B NM_001320 GGCAGCCGAGAUGCUUUAUTT AUAAAGCAUCUCGGCUGCCTG 22-D7 CaMKIINalpha NM_018584 GCAAGCGGGUUGUUAUUGATT UCAAUAACAACCCGCUUGCTC 22-D8 DGKH NM_152910 GCACGAAAAAUGCUAACAATT UUGUUAGCAUUUUUCGUGCTT 22-D9 DLG1 NM_004087 GCCGUCUGAACCAAUUCAATT UUGAAUUGGUUCAGACGGCTT 22-D10 DLG3 NM_021120 CCUCAACGACAUGUACGCUTT AGCGUACAUGUCGUUGAGGTG 22-D11 DLG4 NM_001365 CGGUGUGGACCUCCGAAAUTT AUUUCGGAGGUCCACACCGTT 22-E1 DOK1 NM_001381 CCUCUCUAUUGGGACUUGUTT ACAAGUCCCAAUAGAGAGGTT 22-E2 ERF NM_006494 GGCUCAGUCAGUGACUGUATT UACAGUCACUGACUGAGCCTC 22-E3 FLT3LG NM_001459 GCUGUCUGACUACCUGCUUTT AAGCAGGUAGUCAGACAGCTC 22-E4 GCKR NM_001486 CCCUGUUAUUAGCAGCCCATT UGGGCUGCUAAUAACAGGGTT 22-E5 HGS NM_004712 CGACAAGAACCCACACGUCTT GACGUGUGGGUUCUUGUCGTT 22-E6 IHPK2 NM_016291 GGAACUUGUGUCUAAUAGCTT GCUAUUAGACACAAGUUCCTG 22-E7 IKBKAP NM_003640 CGGUGCUCAUUGGUUCAGATT UCUGAACCAAUGAGCACCGTC 22-E8 IKBKB NM_001556 GCUGGUUCAUAUCUUGAACTT GUUCAAGAUAUGAACCAGCTT 22-E9 IKBKG NM_003639 CCUUACGCUUCAGCUGUUGTT CAACAGCUGAAGCGUAAGGTG 22-E10 IPMK NM_152230 GGUCAGCAAGUACCCAUUATT UAAUGGGUACUUGCUGACCTG 22-E11 KIAA0551 XM_039796 CCGGAAUAUUGCUACAUACTT GUAUGUAGCAAUAUUCCGGTG 22-F1 KIAA1446 NM_020836 CGAGAUUGUUGCCCUCAACTT GUUGAGGGCAACAAUCUCGTG 22-F2 KIAA1765 XM_047355 GCUCGAGAAUCACAGAGCGTT CGCUCUGUGAUUCUCGAGCTT 22-F3 LIM NM_006457 GCAAAAUGGCCCACCAAGATT UCUUGGUGGGCCAUUUUGCTG 22-F4 LOC283846 NM_199284 GCAGGUAUCUGGUAAGUCUTT AGACUUACCAGAUACCUGCTG 22-F5 LOC375133 NM_199345 GCUCUGACCAAGUGGAGAUTT AUCUCCACUUGGUCAGAGCTG 22-F6 LOC375449 NM_198828 CGAGGAGCUUGACCACAUATT UAUGUGGUCAAGCUCCUCGTC 22-F7 LY6G5B NM_021221 GAAAAACGCAACACCUACUTT AGUAGGUGUUGCGUUUUUCTT 22-F8 MADD NM_003682 CCAUGUGGCGGAUCUUUACTT GUAAAGAUCCGCCACAUGGTG 22-F9 MAGI-3 NM_020965 CGAACGACAUCUGUCAGCATT UGCUGACAGAUGUCGUUCGTT 22-F10 MAGI1 NM_173515 CGAGAGAAGAUCAACGGCGTT CGCCGUUGAUCUUCUCUCGTT 22-F11 MAP2K1IP1 NM_021970 GGACUAAUUGUCAGCCUAGTT CUAGGCUGACAAUUAGUCCTG 22-G1 MAP3K1 XM_042066 GCACGAAUGGUUGGAAAGGTT CCUUUCCAACCAUUCGUGCTT 22-G2 MAP3K7IP1 NM_006116 GGAUGAGCUCUUCCGUCUUTT AAGACGGAAGAGCUCAUCCTC 22-G3 MAP3K7IP2 NM_015093 CGCACAGCAUUAGUGAUGGTT CCAUCACUAAUGCUGUGCGTT 22-G4 MAP3K9 NM_033141 GCCUUAUGGAGAUGGAGGATT UCCUCCAUCUCCAUAAGGCTG 22-G5 MAPK3 NM_002746 CCGGAUGUUAACCUUUAACTT GUUAAAGGUUAACAUCCGGTC 22-G6 MAPK8IP1 NM_005456 GGACACACUGAAUAAUAAUTT AUUAUUAUUCAGUGUGUCCTG 22-G7 MAPK8IP2 NM_012324 GGAUGACUUCCAGGAGUUUTT AAACUCCUGGAAGUCAUCCTG 22-G8 MAPK8IP3 NM_015133 GCACAUUGAGAGGUCCAAGTT CUUGGACCUCUCAAUGUGCTC 22-G9 MAPKAP1 NM_024117 CGGGAAUGUGUGAGAUGGUTT ACCAUCUCACACAUUCCCGTG 22-G10 MARCKS NM_002356 GGUACUGUUUUGGAGAACUTT AGUUCUCCAAAACAGUACCTG 22-G11 MAST3 XM_038150 GCCAUGCGAAAGCGACUUUTT AAAGUCGCUUUCGCAUGGCTT 22-H1 MLL2 NM_003482 GGCGUGUGGCUGACAGAAATT UUUCUGUCAGCCACACGCCTT 22-H2 NEK1 NM_012224 GCUGGCUCGAACUUGCAUATT UAUGCAAGUUCGAGCCAGCTC 22-H3 NIPA NM_016478 GCGGAACAACCUUCAUUGGTT CCAAUGAAGGUUGUUCCGCTT 22-H4 NJMU-R1 NM_022344 GCAGAUUGGCACAGCAACGTT CGUUGCUGUGCCAAUCUGCTT 22-H5 NME5 NM_003551 GGAGGAGAUACAAGAUAUUTT AAUAUCUUGUAUCUCCUCCTC 22-H6 NRGN NM_006176 GGGAAGAGGGUUGUUUUGGTT CCAAAACAACCCUCUUCCCTT 22-H7 OSRF NM_012382 GCACUACAGUUAACUCCAATT UUGGAGUUAACUGUAGUGCTT 22-H8 P101-PI3K NM_014308 GGCUUCCACGCUACGUGUUTT AACACGUAGCGUGGAAGCCTT 22-H9 PACSIN2 NM_007229 GCUGGCUAUCUCACGAGAATT UUCUCGUGAGAUAGCCAGCTT 22-H10 PACSIN3 NM_016223 GGCUUAUGCCCAGCAGUUGTT CAACUGCUGGGCAUAAGCCTT 22-H11 PANK2 NM_024960 GCAUUCGGAAGUACCUGACTT GUCAGGUACUUCCGAAUGCTT 23-A1 PANK3 NM_024594 CGUGGCAUAUGGAUCCACCTT GGUGGAUCCAUAUGCCACGTT 23-A2 PANK4 NM_018216 CCAAGCUGGCCUACUAUUCTT GAAUAGUAGGCCAGCUUGGTT 23-A3 PCM1 NM_006197 GGUAGAGCAACAGCUGCUATT UAGCAGCUGUUGCUCUACCTT 23-A4 PCTK3 NM_002596 GGUUGGAUACGGAUGGCAUTT AUGCCAUCCGUAUCCAACCTG

23-A5 PHKA1 NM_002637 GGAUAGCCUCCAUGCAAAGTT CUUUGCAUGGAGGCUAUCCTT

64 Materials and Methods

23-A6 PHKA2 NM_000292 GGCAAUUGAUGAACUGGACTT GUCCAGUUCAUCAAUUGCCTC 23-A7 PHKB NM_000293 CCUGAUAAUGAAACUCUCUTT AGAGAGUUUCAUUAUCAGGTC 23-A8 PIK3AP1 NM_152309 CCGUUAUCAGCUAUUAUACTT GUAUAAUAGCUGAUAACGGTT 23-A9 PIK3C2A NM_002645 GGAAGCUUACCUAUCUAUCTT GAUAGAUAGGUAAGCUUCCTT 23-A10 PIK3C2B NM_002646 GCUUCUAUCUGGGAUACCCTT GGGUAUCCCAGAUAGAAGCTC 23-A11 PIK3C2G NM_004570 GCAGCAAUACGAGUCUGGCTT GCCAGACUCGUAUUGCUGCTT 23-B1 PIK3C3 NM_002647 GGAACAACGGUUUCGCUCUTT AGAGCGAAACCGUUGUUCCTC 23-B2 PIK3CA NM_006218 CCAGUAGGCAACCGUGAAGTT CUUCACGGUUGCCUACUGGTT 23-B3 PIK3CB NM_006219 CCAUCGUAAGCUCAGAGGUTT ACCUCUGAGCUUACGAUGGTT 23-B4 PIK3CD NM_005026 CGACUUUCGCGCCAAGAUGTT CAUCUUGGCGCGAAAGUCGTT 23-B5 PIK3CG NM_002649 GGAAGCUUCAAUGCUGACATT UGUCAGCAUUGAAGCUUCCTT 23-B6 PIK3R1 NM_181504 CCUACUACUGUAGCCAACATT UGUUGGCUACAGUAGUAGGTT 23-B7 PIK3R2 NM_005027 GCUUAAGGUCUAUCACCAGTT CUGGUGAUAGACCUUAAGCTG 23-B8 PIK3R3 NM_003629 GCAGUGUCACACACAAGAATT UUCUUGUGUGUGACACUGCTC 23-B9 PIP5K1B NM_003558 CCUCUAAUAGAACUGUCUATT UAGACAGUUCUAUUAGAGGTT 23-B10 PIP5K1C NM_012398 GCACGUGUGUGCCGUUUUATT UAAAACGGCACACACGUGCTT 23-B11 PIP5KL1 NM_173492 GGACCUCAACUUUCAGGGCTT GCCCUGAAAGUUGAGGUCCTT 23-C1 PKIA NM_006823 GCACUGUUUUUAGCAUUACTT GUAAUGCUAAAAACAGUGCTT 23-C2 PKIB NM_032471 GGCUCAUAAUCUAUCAAGATT UCUUGAUAGAUUAUGAGCCTT 23-C3 PKIG NM_007066 GGGAUUUUGGCUCAAAACGTT CGUUUUGAGCCAAAAUCCCTT 23-C4 PNCK NM_198452 GCUGGAUGAUUCAUCUGUGTT CACAGAUGAAUCAUCCAGCTG 23-C5 PRKAB1 NM_006253 CCACCUAUACGCGCUGUCUTT AGACAGCGCGUAUAGGUGGTT 23-C6 PRKAB2 NM_005399 GCCAUAAUGACUUUGUUGCTT GCAACAAAGUCAUUAUGGCTC 23-C7 PRKAG1 NM_002733 GGUGAAGAAAGCUUUUUUUTT AAAAAAAGCUUUCUUCACCTG 23-C8 PRKAG2 NM_016203 GCCUGAACGGUUAGAGAAUTT AUUCUCUAACCGUUCAGGCTC 23-C9 PRKAG3 NM_017431 GGAAGAGGACACUAUGUCUTT AGACAUAGUGUCCUCUUCCTC 23-C10 PRKAR1A NM_002734 GGAGGAGGCAAAACAGAUUTT AAUCUGUUUUGCCUCCUCCTT 23-C11 PRKAR1B NM_002735 GGACUACAAAACCAUGACUTT AGUCAUGGUUUUGUAGUCCTT 23-D1 PRKAR2B NM_002736 GCUUUAUUGAGUCACUGCCTT GGCAGUGACUCAAUAAAGCTT 23-D2 PRKCABP NM_012407 GCUAUACAAAGGGAUGACGTT CGUCAUCCCUUUGUAUAGCTC 23-D3 PRKCBP1 NM_012408 CCUGGCUUACUGAACAGUATT UACUGUUCAGUAAGCCAGGTT 23-D4 PRKCDBP NM_145040 GAAGCUCUGCUCCAAAUGGTT CCAUUUGGAGCAGAGCUUCTT 23-D5 PRKCSH NM_001001 GGUCAACGAUGACUAUUGCTT GCAAUAGUCAUCGUUGACCTG 23-D6 PRKRIR NM_004705 CGUUGUUUUGUUCAAAAACTT GUUUUUGAACAAAACAACGTG 23-D7 SCAP1 NM_003726 GCCAGGUACUAUUGGGAUUTT AAUCCCAAUAGUACCUGGCTT 23-D8 SH3KBP1 NM_031892 GCUGAAAGUUGGCGACAUCTT GAUGUCGCCAACUUUCAGCTC 23-D9 SIK2 NM_015191 GCCAGGCGAAAAUUCUGGCTT GCCAGAAUUUUCGCCUGGCTT 23-D10 SKIV2L NM_006929 CGGAUCCCUGGUCUCUUUUTT AAAAGAGACCAGGGAUCCGTC 23-D11 SKP2 NM_005983 CCUUAGACCUCACAGGUAATT UUACCUGUGAGGUCUAAGGTC 23-E1 STK11IP NM_052902 CCACGUAUUUGAGCUGCACTT GUGCAGCUCAAAUACGUGGTT 23-E2 T3JAM NM_025228 GCAAACCAAUUACGGAGUUTT AACUCCGUAAUUGGUUUGCTG 23-E3 TSKS NM_021733 CCAACGUGUCACUGCUCAATT UUGAGCAGUGACACGUUGGTG 23-E4 AK2 NM_001625 GCUUGAUUCUGUGAUUGAATT UUCAAUCACAGAAUCAAGCTT 23-E5 AK7 NM_152327 GCAGAAUGCAGGUCAACUATT UAGUUGACCUGCAUUCUGCTC 23-E6 AKAP1 NM_003488 GGGCUUGAAGACUCUUGCATT UGCAAGAGUCUUCAAGCCCTG 23-E7 AKAP10 NM_007202 GCACUAAAAAUCAUGCCUUTT AAGGCAUGAUUUUUAGUGCTT 23-E8 AKAP11 NM_016248 GCUGCCUAAAAUUCCUGUGTT CACAGGAAUUUUAGGCAGCTC 23-E9 AKAP12 NM_005100 GCUAACACAACCCACUGAGTT CUCAGUGGGUUGUGUUAGCTC 23-E10 AKAP2 NM_001004 GCGAGGGAGACAACUAUAGTT CUAUAGUUGUCUCCCUCGCTT 23-E11 AKAP28 NM_178813 GUAGCUCUAGCUCUGGUUGTT CAACCAGAGCUAGAGCUACTT 23-F1 AKAP3 NM_006422 GCCUCAGUAAGAUAGCAUCTT GAUGCUAUCUUACUGAGGCTT 23-F2 AKAP4 NM_003886 GCAAGCGAACGGGCAAUUUTT AAAUUGCCCGUUCGCUUGCTT 23-F3 AKAP5 NM_004857 CGGGAACUCUAAUCCUUGATT UCAAGGAUUAGAGUUCCCGTT 23-F4 AKAP6 NM_004274 GGAGGUAUCUCAAGUAUCUTT AGAUACUUGAGAUACCUCCTC 23-F5 AKAP7 NM_004842 GCUAUCAAGUGCUAAGUUUTT AAACUUAGCACUUGAUAGCTG 23-F6 AKAP8 NM_005858 GGGAGGCUAGCAUGUGUUCTT GAACACAUGCUAGCCUCCCTT 23-F7 AKAP8L NM_014371 GGAACACUUUAAGUACGUATT UACGUACUUAAAGUGUUCCTT 23-F8 AKAP9 NM_005751 GCACACCAUGAUUUGAAUATT UAUUCAAAUCAUGGUGUGCTG 23-F9 AKIP NM_017900 GUGCAAAAACGUGCUGAAGTT CUUCAGCACGUUUUUGCACTG 23-F10 ASK NM_006716 GGAGCAGAAUUUCCUGUAUTT AUACAGGAAAUUCUGCUCCTT 23-F11 CALM1 NM_006888 GGAUAAACCGUUGAGACGUTT ACGUCUCAACGGUUUAUCCTT 23-G1 CALM2 NM_001743 GGUACUCGUACACUAUUUUTT AAAAUAGUGUACGAGUACCTG 23-G2 CDC42SE2 NM_020240 GUGGAAUGAAUUCAGUUAGTT CUAACUGAAUUCAUUCCACTG 23-G3 CDKN1B NM_004064 GCUUACUCUGUCCAUUUAUTT AUAAAUGGACAGAGUAAGCTT 23-G4 CDKN1C NM_000076 CCGCUGGGAUUACGACUUCTT GAAGUCGUAAUCCCAGCGGTT 23-G5 CDKN2A NM_000077 CCAGAGAGGCUCUGAGAAATT UUUCUCAGAGCCUCUCUGGTT 23-G6 CDKN2B NM_004936 GCCUGUCUGAGACUCACAGTT CUGUGAGUCUCAGACAGGCTT 23-G7 CDKN2C NM_001262 CGUUGCCUCUACUUUAUCATT UGAUAAAGUAGAGGCAACGTG 23-G8 CDKN2D NM_001800 GCCACCUAAACGGUUCAGUTT ACUGAACCGUUUAGGUGGCTG 23-G9 CHKB NM_005198 GUCGGCCAUUGAAAACUCATT UGAGUUUUCAAUGGCCGACTT 23-G10 CIB2 NM_006383 GGUUAGGACCCUUCCCACATT UGUGGGAAGGGUCCUAACCTT 23-G11 CIB3 NM_054113 CGACGACUACAUUUGUGCGTT CGCACAAAUGUAGUCGUCGTT 23-H1 CNKSR1 NM_006314 CCGAGACCCUCAGCUCAAUTT AUUGAGCUGAGGGUCUCGGTT 23-H2 CRIM1 NM_016441 GCCUGCGUAUUUAACAAUGTT CAUUGUUAAAUACGCAGGCTG 23-H3 CSNK2B NM_001320 GGAGACUUUGGUUACUGUCTT GACAGUAACCAAAGUCUCCTT 23-H4 CaMKIINalpha NM_018584 GAUGAUAGGAUUGAUGACGTT CGUCAUCAAUCCUAUCAUCTT 23-H5 DGKH NM_152910 GGUUCAUAUGACGAUGACATT UGUCAUCGUCAUAUGAACCTC 23-H6 DLG1 NM_004087 GCCUUAGCCCUAGUGUAGATT UCUACACUAGGGCUAAGGCTG 23-H7 DLG3 NM_021120 GGAAUGCAACUCAUGAGCATT UGCUCAUGAGUUGCAUUCCTC 23-H8 DLG4 NM_001365 GGUUAAAGGCCAAGGACUGTT CAGUCCUUGGCCUUUAACCTT 23-H9 DOK1 NM_001381 GGGCCUUUAUGAUCUGCCUTT AGGCAGAUCAUAAAGGCCCTG

23-H10 ERF NM_006494 GGACAUGAAACGGUACCUGTT CAGGUACCGUUUCAUGUCCTC

65 Materials and Methods

23-H11 FLT3LG NM_001459 GAUUACCCAGUCACCGUGGTT CCACGGUGACUGGGUAAUCTT 24-A1 GCKR NM_001486 GCGUGAGCUAAGCACCAAATT UUUGGUGCUUAGCUCACGCTG 24-A2 HGS NM_004712 CCCACACGUCGCCUUGUAUTT AUACAAGGCGACGUGUGGGTT 24-A3 IHPK2 NM_016291 GCUCCUAAGGUGGACAACATT UGUUGUCCACCUUAGGAGCTT 24-A4 IKBKAP NM_003640 GGUCAGAGUGUGGAACCGATT UCGGUUCCACACUCUGACCTT 24-A5 IKBKB NM_001556 GCAAACCGAGUUUGGGAUCTT GAUCCCAAACUCGGUUUGCTC 24-A6 IKBKG NM_003639 CCACUUGCCUCGGGCUAAUTT AUUAGCCCGAGGCAAGUGGTT 24-A7 IPMK NM_152230 GGGUUUAUCAUGUUCAUUCTT GAAUGAACAUGAUAAACCCTC 24-A8 KIAA0551 XM_039796 GGCAUGGAUGACCAACUUUTT AAAGUUGGUCAUCCAUGCCTG 24-A9 KIAA1446 NM_020836 CGAGCUCUAUAGGAAGGACTT GUCCUUCCUAUAGAGCUCGTT 24-A10 KIAA1765 XM_047355 GCAUUAUGAGACUGGCCGGTT CCGGCCAGUCUCAUAAUGCTT 24-A11 LIM NM_006457 GGACACUUUAGUGCAAAGATT UCUUUGCACUAAAGUGUCCTG 24-B1 LOC283846 NM_199284 GGAAAUUACUAGUAGGUGGTT CCACCUACUAGUAAUUUCCTC 24-B2 LOC375133 NM_199345 GUCAACCCAAACCCUGUCCTT GGACAGGGUUUGGGUUGACTT 24-B3 LOC375449 NM_198828 GAAACUCUGUCGGAGGAAGTT CUUCCUCCGACAGAGUUUCTG 24-B4 LY6G5B NM_021221 GUGCUGUCAGUACGAUUAUTT AUAAUCGUACUGACAGCACTG 24-B5 MADD NM_003682 GCGAGUAGACAUCGAGGUCTT GACCUCGAUGUCUACUCGCTT 24-B6 MAGI-3 NM_020965 GGCGAUGUUAUUGUAGACATT UGUCUACAAUAACAUCGCCTG 24-B7 MAGI1 NM_173515 GGAAGUUCACUUACCAAACTT GUUUGGUAAGUGAACUUCCTC 24-B8 MAP2K1IP1 NM_021970 GCUCCCAUGGAGUUAGUCUTT AGACUAACUCCAUGGGAGCTG 24-B9 MAP3K1 XM_042066 GGAAACAAACCGCCGUGUUTT AACACGGCGGUUUGUUUCCTC 24-B10 MAP3K7IP1 NM_006116 GGUUAAAUAUGGCUACACGTT CGUGUAGCCAUAUUUAACCTT 24-B11 MAP3K7IP2 NM_015093 GCAAAGGAACAUCUAGCCUTT AGGCUAGAUGUUCCUUUGCTG 24-C1 MAP3K9 NM_033141 GGUCUAUCGUGCUUUCUGGTT CCAGAAAGCACGAUAGACCTT 24-C2 MAPK3 NM_002746 GGAACAUUCCUUAGUCUCATT UGAGACUAAGGAAUGUUCCTT 24-C3 MAPK8IP1 NM_005456 GGCAAAUGCAGUUUAUUGUTT ACAAUAAACUGCAUUUGCCTC 24-C4 MAPK8IP2 NM_012324 GGAGUUUGAGAUGAUCGAUTT AUCGAUCAUCUCAAACUCCTG 24-C5 MAPK8IP3 NM_015133 GCUAUCUCUGUACAGAAUCTT GAUUCUGUACAGAGAUAGCTG 24-C6 MAPKAP1 NM_024117 GCUCAAAGAUUAGAACGACTT GUCGUUCUAAUCUUUGAGCTG 24-C7 MARCKS NM_002356 CCACCAUUCCAACAGGUCGTT CGACCUGUUGGAAUGGUGGTT 24-C8 MAST3 XM_038150 GCGUGACAUUCUCACCUUUTT AAAGGUGAGAAUGUCACGCTC 24-C9 MLL2 NM_003482 GGAACAAAACACCGUAGACTT GUCUACGGUGUUUUGUUCCTT 24-C10 NEK1 NM_012224 GGUUUUAUAGCCAAACGCATT UGCGUUUGGCUAUAAAACCTT 24-C11 NIPA NM_016478 GCCCUUUGAGCUGUCUCCATT UGGAGACAGCUCAAAGGGCTT 24-D1 NJMU-R1 NM_022344 GGAUGUUGUAUGCCCAAUCTT GAUUGGGCAUACAACAUCCTC 24-D2 NME5 NM_003551 GGCUGUACUUCUACUGUUUTT AAACAGUAGAAGUACAGCCTT 24-D3 NRGN NM_006176 GGGUUGUUUUGGUUUCGGATT UCCGAAACCAAAACAACCCTC 24-D4 OSRF NM_012382 GGUGCUAAUGUCUCUUCAUTT AUGAAGAGACAUUAGCACCTG 24-D5 P101-PI3K NM_014308 GGUCUGUACCUCCGUGAACTT GUUCACGGAGGUACAGACCTT 24-D6 PACSIN2 NM_007229 GGCUGUGUACAGUUAUACUTT AGUAUAACUGUACACAGCCTT 24-D7 PACSIN3 NM_016223 GGAUAUGCUGCUCACCUUATT UAAGGUGAGCAGCAUAUCCTT 24-D8 PANK2 NM_024960 GCGACUUUGAUCACCAUCATT UGAUGGUGAUCAAAGUCGCTC 24-D9 PANK3 NM_024594 GGACCUGCCUACUUUUAUCTT GAUAAAAGUAGGCAGGUCCTG 24-D10 PANK4 NM_018216 GCUGGCCUACUAUUCAACGTT CGUUGAAUAGUAGGCCAGCTT 24-D11 PCM1 NM_006197 GCUUAGUGAAAACCGAAAGTT CUUUCGGUUUUCACUAAGCTG 24-E1 PCTK3 NM_002596 CGGAUGACAGAAUCAAGGCTT GCCUUGAUUCUGUCAUCCGTG 24-E2 PHKA1 NM_002637 GGAUCCCAAUCGUCUGUACTT GUACAGACGAUUGGGAUCCTC 24-E3 PHKA2 NM_000292 GGACGCAAGUCAGUGAUUCTT GAAUCACUGACUUGCGUCCTC 24-E4 PHKB NM_000293 GCUACAUUCGAGCUCGGUUTT AACCGAGCUCGAAUGUAGCTC 24-E5 PIK3AP1 NM_152309 GGUUAUAGUGUGAGACUUGTT CAAGUCUCACACUAUAACCTC 24-E6 PIK3C2A NM_002645 GCUUACCUAUCUAUCGUCCTT GGACGAUAGAUAGGUAAGCTT 24-E7 PIK3C2B NM_002646 GCAUCGGAUCCUAGAAGAGTT CUCUUCUAGGAUCCGAUGCTC 24-E8 PIK3C2G NM_004570 GGAAAGCUAUCUCGAAAGCTT GCUUUCGAGAUAGCUUUCCTG 24-E9 PIK3C3 NM_002647 GCUGUUAUCCUCUCAUUACTT GUAAUGAGAGGAUAACAGCTC 24-E10 PIK3CA NM_006218 GCAUGCCAAUUGGUCUGUATT UACAGACCAAUUGGCAUGCTC 24-E11 PIK3CB NM_006219 GGGAUCCCUUGUCUCAACUTT AGUUGAGACAAGGGAUCCCTG 24-F1 PIK3CD NM_005026 GCGUGGGCAUCAUCUUUAATT UUAAAGAUGAUGCCCACGCTG 24-F2 PIK3CG NM_002649 GCAAUUGGAGGCGAUCAUATT UAUGAUCGCCUCCAAUUGCTT 24-F3 PIK3R1 NM_181504 GCGGUACAGCAAAGAAUACTT GUAUUCUUUGCUGUACCGCTC 24-F4 PIK3R2 NM_005027 GGUUUUGUACGGUACGUUGTT CAACGUACCGUACAAAACCTG 24-F5 PIK3R3 NM_003629 GGAGAUUGAACGAAUUAUGTT CAUAAUUCGUUCAAUCUCCTT 24-F6 PIP5K1B NM_003558 GGAGGCAUUAAUAUCAGGATT UCCUGAUAUUAAUGCCUCCTG 24-F7 PIP5K1C NM_012398 GGAGAAAUGUGCAAACCUCTT GAGGUUUGCACAUUUCUCCTG 24-F8 PIP5KL1 NM_173492 GGGACUGAUCAAGAUCAUUTT AAUGAUCUUGAUCAGUCCCTT 24-F9 PKIA NM_006823 GCCCUCUCAGAUCCAAAUGTT CAUUUGGAUCUGAGAGGGCTG 24-F10 PKIB NM_032471 GGUAAGCUAUUAAAAGGCATT UGCCUUUUAAUAGCUUACCTT 24-F11 PKIG NM_007066 GGAAAUAAAUGCCGAUGAUTT AUCAUCGGCAUUUAUUUCCTG 24-G1 PNCK NM_198452 GAACUUUGCUCGGACACACTT GUGUGUCCGAGCAAAGUUCTT 24-G2 PRKAB1 NM_006253 GGUUUGUCCAGGCAGAGCATT UGCUCUGCCUGGACAAACCTT 24-G3 PRKAB2 NM_005399 GGCAGAAUGCAGAACACCUTT AGGUGUUCUGCAUUCUGCCTT 24-G4 PRKAG1 NM_002733 GGACUCCUUUAAACCGCUUTT AAGCGGUUUAAAGGAGUCCTG 24-G5 PRKAG2 NM_016203 GCAGAUAGUAUUGUGGGUATT UACCCACAAUACUAUCUGCTT 24-G6 PRKAG3 NM_017431 GGAACAAUCCAUGAACUUATT UAAGUUCAUGGAUUGUUCCTT 24-G7 PRKAR1A NM_002734 GAAGCAUAACAUUCAAGCGTT CGCUUGAAUGUUAUGCUUCTG 24-G8 PRKAR1B NM_002735 GGAGUGACAUAUUCGAUGCTT GCAUCGAAUAUGUCACUCCTC 24-G9 PRKAR2B NM_002736 GGAACAUCGCUACCUAUGATT UCAUAGGUAGCGAUGUUCCTT 24-G10 PRKCABP NM_012407 CCUCCUACGGGCCUUUUAUTT AUAAAAGGCCCGUAGGAGGTT 24-G11 PRKCBP1 NM_012408 GGUCGAUGCCCGAUUCUUUTT AAAGAAUCGGGCAUCGACCTG 24-H1 PRKCDBP NM_145040 CGCCCUAAUAAGGAGCGAATT UUCGCUCCUUAUUAGGGCGTG 24-H2 PRKCSH NM_001001 GGAGAAGCAGAAAAAGCUCTT GAGCUUUUUCUGCUUCUCCTC 24-H3 PRKRIR NM_004705 GGCUUACAUUGUCUCUAGUTT ACUAGAGACAAUGUAAGCCTG

24-H4 SCAP1 NM_003726 GGUCUCUUCUACUACUAUGTT CAUAGUAGUAGAAGAGACCTC

66 Materials and Methods

24-H5 SH3KBP1 NM_031892 GGUGUUCUCAACGGGAAGATT UCUUCCCGUUGAGAACACCTT 24-H6 SIK2 NM_015191 GGAAGAUUCCGGAUUCCGUTT ACGGAAUCCGGAAUCUUCCTT 24-H7 SKIV2L NM_006929 GGUUCACUAUAUCAACGAUTT AUCGUUGAUAUAGUGAACCTC 24-H8 SKP2 NM_005983 GCACAUGGACCUAUCGAACTT GUUCGAUAGGUCCAUGUGCTG 24-H9 STK11IP NM_052902 GGUCUUGUCACUGACAGAUTT AUCUGUCAGUGACAAGACCTT 24-H10 T3JAM NM_025228 GCUAUACACCUGUACCCAGTT CUGGGUACAGGUGUAUAGCTG 24-H11 TSKS NM_021733 GGAAAAGACCAACCGGGUUTT AACCCGGUUGGUCUUUUCCTT 25-A1 AK2 NM_001625 CCCUUGAUCCGUCGAUCAGTT CUGAUCGACGGAUCAAGGGTT 25-A2 AK7 NM_152327 GCUAUAUGACAUGACUAUGTT CAUAGUCAUGUCAUAUAGCTT 25-A3 AKAP1 NM_003488 GCCUGGUUGUAAUUUGUAATT UUACAAAUUACAACCAGGCTT 25-A4 AKAP10 NM_007202 GGAUGCAGUGAAUAUCUUATT UAAGAUAUUCACUGCAUCCTC 25-A5 AKAP11 NM_016248 GCAUUUCAUGUGAAGUACUTT AGUACUUCACAUGAAAUGCTT 25-A6 AKAP12 NM_005100 CCAAAGACGUACCAUUUUUTT AAAAAUGGUACGUCUUUGGTT 25-A7 AKAP2 NM_001004 GAUCACAAAAACAUGGAAATT UUUCCAUGUUUUUGUGAUCTG 25-A8 AKAP28 NM_178813 GUGGAUGACUCACGGUGAATT UUCACCGUGAGUCAUCCACTT 25-A9 AKAP3 NM_006422 CGAAUCUAGUCAUAGCCAUTT AUGGCUAUGACUAGAUUCGTG 25-A10 AKAP4 NM_003886 CCAACAAUAAUCAAAGUCCTT GGACUUUGAUUAUUGUUGGTG 25-A11 AKAP5 NM_004857 GCCAGUAACAGUACCCUAGTT CUAGGGUACUGUUACUGGCTT 25-B1 AKAP6 NM_004274 GGGCAGUAAAGAUAUAAGUTT ACUUAUAUCUUUACUGCCCTT 25-B2 AKAP7 NM_004842 CGAUGGGUCUGAAGUCCAATT UUGGACUUCAGACCCAUCGTT 25-B3 AKAP8 NM_005858 CCUAUACCUUUGUAGUGCATT UGCACUACAAAGGUAUAGGTT 25-B4 AKAP8L NM_014371 GGAUCCAGUUUGUGUGUUCTT GAACACACAAACUGGAUCCTT 25-B5 AKAP9 NM_005751 GCUUCUAUUUAGUCACGAATT UUCGUGACUAAAUAGAAGCTG 25-B6 AKIP NM_017900 CGCAAGCAGAUCAAGUUCGTT CGAACUUGAUCUGCUUGCGTC 25-B7 ASK NM_006716 CCAGUAUCAAGUUGUUGAUTT AUCAACAACUUGAUACUGGTT 25-B8 CALM1 NM_006888 CCUAUUCAAAUGGGUUCUATT UAGAACCCAUUUGAAUAGGTT 25-B9 CALM2 NM_001743 CGGGUGUAUUAUCCAGGUATT UACCUGGAUAAUACACCCGTT 25-B10 CDC42SE2 NM_020240 GGAGACCUGUUCAGUGGAATT UUCCACUGAACAGGUCUCCTG 25-B11 CDKN1B NM_004064 CGUAAACAGCUCGAAUUAATT UUAAUUCGAGCUGUUUACGTT 25-C1 CDKN1C NM_000076 CCGUUCAUGUAGCAGCAACTT GUUGCUGCUACAUGAACGGTC 25-C2 CDKN2A NM_000077 GCUUUUAAAAAUGUCCUGCTT GCAGGACAUUUUUAAAAGCTC 25-C3 CDKN2B NM_004936 GCAAUUGUAACGGUUAACUTT AGUUAACCGUUACAAUUGCTC 25-C4 CDKN2C NM_001262 GCAGGUUUCCUGGACACUUTT AAGUGUCCAGGAAACCUGCTC 25-C5 CDKN2D NM_001800 CGGUUCAGUUUCUUCUGCGTT CGCAGAAGAAACUGAACCGTT 25-C6 CHKB NM_005198 GUACAGCAGUUAUAACUAUTT AUAGUUAUAACUGCUGUACTC 25-C7 CIB2 NM_006383 GGGUUUUAACGAGCUAUGCTT GCAUAGCUCGUUAAAACCCTG 25-C8 CIB3 NM_054113 CGACUACAUUUGUGCGUGGTT CCACGCACAAAUGUAGUCGTC 25-C9 CNKSR1 NM_006314 CCAGGUCUGUACAGGUGUUTT AACACCUGUACAGACCUGGTT 25-C10 CRIM1 NM_016441 CCAACCAAUAUACCCAUUGTT CAAUGGGUAUAUUGGUUGGTT 25-C11 CSNK2B NM_001320 GGAACCCUGUAUGGUUUUUTT AAAAACCAUACAGGGUUCCTG 25-D1 CaMKIINalpha NM_018584 GCGGGUUGUUAUUGAAGAUTT AUCUUCAAUAACAACCCGCTT 25-D2 DGKH NM_152910 GGUGGAGUAUAAUGACAUATT UAUGUCAUUAUACUCCACCTG 25-D3 DLG1 NM_004087 GGAGAUCGUAUUAUAUCGGTT CCGAUAUAAUACGAUCUCCTT 25-D4 DLG3 NM_021120 CGGCAAGAAAUUCACUAUGTT CAUAGUGAAUUUCUUGCCGTG 25-D5 DLG4 NM_001365 GCGGGAGUAUGAGAUAGAUTT AUCUAUCUCAUACUCCCGCTT 25-D6 DOK1 NM_001381 GGAUGCAUGGUGGUGCCAATT UUGGCACCACCAUGCAUCCTT 25-D7 ERF NM_006494 CGGUUCACCUACAAGUUCATT UGAACUUGUAGGUGAACCGTT 25-D8 FLT3LG NM_001459 CGGAGAUACACUUUGUCACTT GUGACAAAGUGUAUCUCCGTG 25-D9 GCKR NM_001486 CGGAAAUCGAUACUGUGGUTT ACCACAGUAUCGAUUUCCGTG 25-D10 HGS NM_004712 CGGUAUCUCAACCGGAACUTT AGUUCCGGUUGAGAUACCGTG 25-D11 IHPK2 NM_016291 GCUUAAACACUAUAACCCUTT AGGGUUAUAGUGUUUAAGCTG 25-E1 IKBKAP NM_003640 CGUAAAGCCUUCCAUUUUATT UAAAAUGGAAGGCUUUACGTT 25-E2 IKBKB NM_001556 GGUGGUGAGCUUAAUGAAUTT AUUCAUUAAGCUCACCACCTC 25-E3 IKBKG NM_003639 CCGAUGUGUAUUUAACCAGTT CUGGUUAAAUACACAUCGGTC 25-E4 IPMK NM_152230 GCUACGAAAAUAUUUGCCATT UGGCAAAUAUUUUCGUAGCTC 25-E5 KIAA0551 XM_039796 CGAUUCAACUCAGAAAUACTT GUAUUUCUGAGUUGAAUCGTT 25-E6 KIAA1446 NM_020836 CCGUCUUACUCUACCUCAGTT CUGAGGUAGAGUAAGACGGTG 25-E7 KIAA1765 XM_047355 GCAAGACAUAUCCUAAUAUTT AUAUUAGGAUAUGUCUUGCTG 25-E8 LIM NM_006457 GCCCAUUCGGAACAAUGUUTT AACAUUGUUCCGAAUGGGCTT 25-E9 LOC283846 NM_199284 GGACUUACCCAAGUUAUUUTT AAAUAACUUGGGUAAGUCCTG 25-E10 LOC375133 NM_199345 GUGGAGAUCUUCUCCAGCCTT GGCUGGAGAAGAUCUCCACTT 25-E11 LOC375449 NM_198828 AAUGCAGCAACCCAGAUGUTT ACAUCUGGGUUGCUGCAUUTC 25-F1 LY6G5B NM_021221 GAUUCAGUGGUUCUACCAGTT CUGGUAGAACCACUGAAUCTG 25-F2 MADD NM_003682 GCGAAUUCACAACAAUAUGTT CAUAUUGUUGUGAAUUCGCTG 25-F3 MAGI-3 NM_020965 CCUGACCAGUCUAUAUAUATT UAUAUAUAGACUGGUCAGGTC 25-F4 MAGI1 NM_173515 GCUAUAAGAUUCGCUGACATT UGUCAGCGAAUCUUAUAGCTT 25-F5 MAP2K1IP1 NM_021970 GCCGUUAAGUGCUGCCAAUTT AUUGGCAGCACUUAACGGCTC 25-F6 MAP3K1 XM_042066 GCAAUUUUAACCUUACUCATT UGAGUAAGGUUAAAAUUGCTC 25-F7 MAP3K7IP1 NM_006116 CCGUGCACUUUUAUGCAAATT UUUGCAUAAAAGUGCACGGTT 25-F8 MAP3K7IP2 NM_015093 CCACCAACCAUUCAUUCAUTT AUGAAUGAAUGGUUGGUGGTT 25-F9 MAP3K9 NM_033141 GCAACAUAUUGAUCCUCCATT UGGAGGAUCAAUAUGUUGCTG 25-F10 MAPK3 NM_002746 GCAGCUGAGCAAUGACCAUTT AUGGUCAUUGCUCAGCUGCTG 25-F11 MAPK8IP1 NM_005456 CCACCGCAUGAACACAUCUTT AGAUGUGUUCAUGCGGUGGTG 25-G1 MAPK8IP2 NM_012324 GGGAUCAUCCCAUCCUUGATT UCAAGGAUGGGAUGAUCCCTT 25-G2 MAPK8IP3 NM_015133 CCCACAUAUCUGCUCUGUATT UACAGAGCAGAUAUGUGGGTT 25-G3 MAPKAP1 NM_024117 GCAGUCGAUAUUAUCUGUATT UACAGAUAAUAUCGACUGCTT 25-G4 MARCKS NM_002356 GGUGUUCUAAUUUUCUGUGTT CACAGAAAAUUAGAACACCTT 25-G5 MAST3 XM_038150 GCUUUGAUGGAUGAAUAGGTT CCUAUUCAUCCAUCAAAGCTC 25-G6 MLL2 NM_003482 CCUAUGACUAUCAGUUUGATT UCAAACUGAUAGUCAUAGGTT 25-G7 NEK1 NM_012224 GCGAGAAAUACUUCGUAGATT UCUACGAAGUAUUUCUCGCTT 25-G8 NIPA NM_016478 CCGAACUGAUGAGAGAAAATT UUUUCUCUCAUCAGUUCGGTG

25-G9 NJMU-R1 NM_022344 CCGGUUUUGUGAGGAUUGGTT CCAAUCCUCACAAAACCGGTT

67 Materials and Methods

25-G10 NME5 NM_003551 GGGCAAUUUAUGGCACAGATT UCUGUGCCAUAAAUUGCCCTC 25-G11 NRGN NM_006176 GCACACUCACUUAAAGAAATT UUUCUUUAAGUGAGUGUGCTT 25-H1 OSRF NM_012382 CGUGCUCAACUUGGUUUAGTT CUAAACCAAGUUGAGCACGTC 25-H2 P101-PI3K NM_014308 GGAUGGGUAAAUUGACCUCTT GAGGUCAAUUUACCCAUCCTT 25-H3 PACSIN2 NM_007229 GCACUCGUCUACUGUUUUATT UAAAACAGUAGACGAGUGCTT 25-H4 PACSIN3 NM_016223 GCUCAGUAUGAGCAGACGCTT GCGUCUGCUCAUACUGAGCTT 25-H5 PANK2 NM_024960 GGCUACUAUGCGUUAUAAUTT AUUAUAACGCAUAGUAGCCTG 25-H6 PANK3 NM_024594 GGAAUUAGUCAAUACAUGUTT ACAUGUAUUGACUAAUUCCTG 25-H7 PANK4 NM_018216 GGAAUGUGUUUUUACCACCTT GGUGGUAAAAACACAUUCCTG 25-H8 PCM1 NM_006197 GCUAAGAGAAUUAGUUCAUTT AUGAACUAAUUCUCUUAGCTC 25-H9 PCTK3 NM_002596 CCCAGAUAUGCAGAGUCACTT GUGACUCUGCAUAUCUGGGTG 25-H10 PHKA1 NM_002637 GCUGUACAGUGAAGAUUAUTT AUAAUCUUCACUGUACAGCTT 25-H11 PHKA2 NM_000292 GGACUUAUUGAGGAAACACTT GUGUUUCCUCAAUAAGUCCTT 26-A1 PHKB NM_000293 GCCAUUAAUAUACGAACUGTT CAGUUCGUAUAUUAAUGGCTG 26-A2 PIK3AP1 NM_152309 GCAUUUAGAUCGUAACAUGTT CAUGUUACGAUCUAAAUGCTT 26-A3 PIK3C2A NM_002645 GCAACAUAACUUAGAAACATT UGUUUCUAAGUUAUGUUGCTG 26-A4 PIK3C2B NM_002646 GGAAUUACACAAUAAGUUGTT CAACUUAUUGUGUAAUUCCTG 26-A5 PIK3C2G NM_004570 GGACAUGUCUUAAUGCUUATT UAAGCAUUAAGACAUGUCCTT 26-A6 PIK3C3 NM_002647 GCAUUGUUGAAGGGUGAUATT UAUCACCCUUCAACAAUGCTT 26-A7 PIK3CA NM_006218 GCAUUGACUAAUCAAAGGATT UCCUUUGAUUAGUCAAUGCTT 26-A8 PIK3CB NM_006219 GGAAUUUACACUGUCCUGUTT ACAGGACAGUGUAAAUUCCTC 26-A9 PIK3CD NM_005026 CGAGAUGCUGUGCAAGACGTT CGUCUUGCACAGCAUCUCGTT 26-A10 PIK3CG NM_002649 GCUUUAGAGUUCCAUAUGATT UCAUAUGGAACUCUAAAGCTT 26-A11 PIK3R1 NM_181504 CGUAUGAACAGCAUUAAACTT GUUUAAUGCUGUUCAUACGTT 26-B1 PIK3R2 NM_005027 CCCUGAUUUUUAAGCCAUATT UAUGGCUUAAAAAUCAGGGTG 26-B2 PIK3R3 NM_003629 GGAUAUCAAUCGAGUACAATT UUGUACUCGAUUGAUAUCCTC 26-B3 PIP5K1B NM_003558 GGCAUUAAUAUCAGGAUUGTT CAAUCCUGAUAUUAAUGCCTC 26-B4 PIP5K1C NM_012398 GGACGGCAAGACCUAUUUATT UAAAUAGGUCUUGCCGUCCTC 26-B5 PIP5KL1 NM_173492 GAUGGAACUGGAUACCACCTT GGUGGUAUCCAGUUCCAUCTG 26-B6 PKIA NM_006823 CCAGUAAUAUGCCAAUGGUTT ACCAUUGGCAUAUUACUGGTT 26-B7 PKIB NM_032471 GGCAGGUUACUUCCAAAUCTT GAUUUGGAAGUAACCUGCCTT 26-B8 PKIG NM_007066 GGAUAAGUGACACCUAGGATT UCCUAGGUGUCACUUAUCCTG 26-B9 PNCK NM_198452 GGACUCGAAGAUCAUGGUCTT GACCAUGAUCUUCGAGUCCTC 26-B10 PRKAB1 NM_006253 CCUACAUUCUCGAUUUUUCTT GAAAAAUCGAGAAUGUAGGTT 26-B11 PRKAB2 NM_005399 GCCUUAUCUCAGUCUAUUUTT AAAUAGACUGAGAUAAGGCTT 26-C1 PRKAG1 NM_002733 GGAAAGAGAAUGGAUUUAUTT AUAAAUCCAUUCUCUUUCCTC 26-C2 PRKAG2 NM_016203 CGGUUAGAGAAUCGCAUCUTT AGAUGCGAUUCUCUAACCGTT 26-C3 PRKAG3 NM_017431 GCCGUUAUUUAUAGAACUGTT CAGUUCUAUAAAUAACGGCTG 26-C4 PRKAR1A NM_002734 GAUUCUAUUGUGCAGUUGUTT ACAACUGCACAAUAGAAUCTT 26-C5 PRKAR1B NM_002735 CGACUUUUACAUCAUCACGTT CGUGAUGAUGUAAAAGUCGTC 26-C6 PRKAR2B NM_002736 GGAUGUUAUCAUAGGCUAUTT AUAGCCUAUGAUAACAUCCTT 26-C7 PRKCABP NM_012407 CGUACCUCAACAAAGCCAUTT AUGGCUUUGUUGAGGUACGTG 26-C8 PRKCBP1 NM_012408 CGUUUUUAUGUAAGCAUGATT UCAUGCUUACAUAAAAACGTG 26-C9 PRKCDBP NM_145040 GCGAAUCCUACAUCCACCATT UGGUGGAUGUAGGAUUCGCTC 26-C10 PRKCSH NM_001001 GGAGCUGGAUGAUGACAUGTT CAUGUCAUCAUCCAGCUCCTT 26-C11 PRKRIR NM_004705 CCUUUGACUAAUAGGAGUUTT AACUCCUAUUAGUCAAAGGTC 26-D1 SCAP1 NM_003726 GGAGUAGACUAUGCCAGUUTT AACUGGCAUAGUCUACUCCTT 26-D2 SH3KBP1 NM_031892 GCCAAUCAAACUAAGACCATT UGGUCUUAGUUUGAUUGGCTT 26-D3 SIK2 NM_015191 GGACAUCAUGUUAGCCAAUTT AUUGGCUAACAUGAUGUCCTT 26-D4 SKIV2L NM_006929 CCCACCAUAACAGAUCUGATT UCAGAUCUGUUAUGGUGGGTT 26-D5 SKP2 NM_005983 CCUAUCGAACUCAGUUAUATT UAUAACUGAGUUCGAUAGGTC 26-D6 STK11IP NM_052902 CCUAAGCCACAAUCAAGUCTT GACUUGAUUGUGGCUUAGGTT 26-D7 T3JAM NM_025228 GCAACUCAGCGAAAAACUCTT GAGUUUUUCGCUGAGUUGCTT 26-D8 TSKS NM_021733 CGCAGACAUCACGGAAAUCTT GAUUUCCGUGAUGUCUGCGTC

68 Materials and Methods

3.3 Experimental setup

Experiments were generally set up by transfection of HEK293 cells, SHSY5Y cells or CGCs with APP-construct and addition of inhibitors or co-transfection with APP- construct and siRNA. Transfections were conducted either using the Amaxa electroporation method using the Amaxa Nucleofector II (Amaxa, Köln, Germany) or with Lipofectamine2000 (Invitrogen, San Diego, CA, USA). Usually, samples of the supernatant were taken for later analysis and immediately frozen. HEK293 cells were transfected using Amaxa Nucleofector II with program Q-001, after mixing a single cell suspension (from trypsinization) of 106 cells in 100 µl Opti- MEM ( Gibco) and 5 µg of plasmid with or without 4 µM (4 µl of 100 µM) siRNA and after transfer to a transfection cuvette. Immediately after transfection 950 µl of DMEM with 5% FBS and 1% P/S were added and 100 µl (105 cells) plated (at a density of 3 x 105/cm2) per 96-well. 24 h after cotransfection with APP-construct and siRNA the supernatants and the cells were frozen separately. For inhibitor experiments the medium was changed 24 h post-transfection with the concomitant addition of different concentrations of the inhibitor. Inhibitor stock solutions were prepared by dissolution of the inhibitor salt to a final concentration of 10 mM in DMSO. The stock solution was further diluted with DMSO to different concentrations and of these 1 µl was added to 99 µl cell medium to achieve the desired final concentration. Samples of the supernatant were taken 24 h after addition of the inhibitor. For viability measurements the cells were incubated with MTT or Resazurin for 30 min. Using Lipofectamine2000, 5 x 104, 7.5 x 104 or 105 HEK293 cells were seeded per 96-well overnight before transfection to end up in a monolayer on the next day. Between 5 ng and 100 ng DNA of the different APP-constructs were transfected with or without 50 nM siRNA. For the screen 7.5 x 104 cells (106 cells/ml) per 96-well were seeded overnight in 75 µl DMEM supplemented with 1% FBS and 1% P/S. Only the inner 60 wells of the plate were used for cells; the outer wells were filled with PBS. For one kinome siRNA library plate six 96-well-plates with 60 cell-containing wells were prepared. The transfection mix consisting of Opti-MEM, Lipofectamine, APP- constructs and siRNA was prepared in several steps from predilutions of Opti-MEM with the other reagents. For each plate of the kinome library, 90 µl Lipofectamine were prediluted with 3510 µl Opti-MEM to 2.5% v/v. For cotransfection 1.5 µg of SEAP-APPswe-EpoR and 12 µg of APPswe were added to 2500 µl Opti-MEM and

69 Materials and Methods

1.5 µg of SEAP-APPswe to 1250 µl Opti-MEM, also per library plate. 1 µl of the kinome library siRNA was added to 31 µl Opti-MEM in a 96-well diluting the siRNA from 20 µM to 625 nM. Then after short mixing on a microtube-incubator, 10 µl of this siRNA predilution were transferred to an empty 96-well. To the remaining siRNA predilution 20 µl of the cotransfection mix were added, and to the 10 µl siRNA predilution 10 µl of SEAP-APPswe mix. Only after combination of siRNA and APP- construct, the Lipofectamine was added, 10 µl of the predilution in each well, reducing the siRNA concentration to 208 nM. The 30 µl of the transfection mix contained at this point 208 nM siRNA, 6 ng SEAP-APPswe-EpoR and 48 ng APPswe or 12 ng SEAP-APPswe, and 0.3 µl Lipofectamine. Addition of 25 µl of this mix to the cells with 75 µl cell medium thus resulted in 52 nM siRNA, 5 ng SEAP-APPswe-EpoR and 40 ng APPswe or 10 ng SEAP-APPswe and 0.25 µl Lipofectamine per well. The transfection mixes in the 96-well-plates were distributed (halved) on the plates with 60 cell-containing wells, thus ending up with four cell-plates for the SEAP-APPswe- EpoR/APPswe-mix and two for the SEAP-APPswe-mix. 72 h after addition of the transfection mix, the supernatants and the cells were frozen separately.

Transfection mix scheme:

1: 31 µl Optimem added to empty 96-well-plate

2: 1 µl siRNA added; 20 µM  625 nM kinome library plate

3: after mixing 10 µl transferred on other empty 96-well-plate

4: 10 µl of SEAP-APPswe added 20 µl of Cotransfection mix added 5: 10 µl of Lipofectamine mix added to all wells; siRNA conc. 625 nM  208 nM 6: 25 µl of transfection mix added to cells; siRNA conc. 208 nM  52 nM

70 Materials and Methods

SHSY5Y

For experiments the procedure was the same as for HEK293 cells, except for 10% FBS instead of 5%.

CGCs

For transfection of CGC with Amaxa Nucleofector II, 6 x 106 cells in single cell suspension obtained directly after cell isolation were nucleofected using 100 µl of the mouse neuron nucleofection (Amaxa) and program G-13. 5 µg of APP-constructs were transfected either separately or in co-transfection with or without 4 µM siRNA. After transfection either 0.2 Mio or 0.5 Mio CGC were seeded on 50 µg/ml PLK- coated 96-well-plates. For inhibitor experiments, Neurobasal medium (Gibco 21103) supplemented with 2% B27 (Gibco 17504-044), 20 mM KCl, 2 mM glutamine and 1% P/S was used for the medium change 45 min after plating instead of BME. Then, 24 h post-transfection the medium was changed again with the concomitant addition of different concentrations of the inhibitor. Inhibitor stock solutions were prepared by dissolution of the inhibitor salt to a final concentration of 10 mM in DMSO. The stock solution was further diluted with DMSO to different concentrations and of these was 1 µl added to 99 µl cell medium to achieve the desired final concentration. 24 h or 72 h after addition of the inhibitor supernatant samples were frozen. For viability measurements the cells were incubated with MTT or Resazurin for 2 h.

3.4 Data collection and analysis

Data were mostly generated from frozen supernatants and frozen cells. Samples of frozen supernatants were subjected to measurements with a SEAP activity assay and Aβ-ELISA and frozen cells to measurements of mRNA content by qPCR. Viability measurements were conducted directly after freezing the supernatants. The data were analyzed using MS Office Excel and processed data arranged in Graph Pad Prism 4.0 for presentation.

71 Materials and Methods

3.4.1 SEAP activity measurement

The SEAP assay is based on the hydrolytic activity of the reporter enzyme SEAP [439]. The colorless substrate para-nitrophenylphosphate (pNPP, Sigma N2765) is hydrolyzed by SEAP to produce phosphate and the yellow chromophore para- nitrophenolate (King-Armstrong-method) [440]. For each measurement, 10 µl samples were added to 100 µl sample buffer (2.4 mM pNPP (1 mg/ml), 0.1 M glycine,

1 mM MgCl2, 1 mM ZnCl2, pH 10.4). The absorption change was measured at 405 nm at ambient temperature with the 96-well-plate-reader EL-X808 (Biotek). HEK293 samples from the screen were measured twice, at 0 min and at 30 min after addition of the sample buffer and in between stored in the refrigerator to minimize spontaneous hydrolysis of pNPP. CGC samples were measured after 0 min, 30 min, 60 min and sometimes longer periods when the SEAP activity was very low, and also stored in the refrigerator in between. Data were initially recorded with the reader software Gen5 and for analysis exported as Excel-files.

3.4.2 A40 quantification

Aβ40 was determined with sandwich ELISA-Kits (BioSource, Camarillo, CA, USA) according to the manufacturers‟ instructions. Usually, HEK293 samples were diluted with sample incubation buffer 1 + 3 and CGC samples 1 + 1. Data from measurement at 450 nm were initially recorded with the reader software Gen5 and for analysis exported as Excel-files.

3.4.3 Cell viability measurements

For detection of cell viability, the percentage of viable cells was quantified by their capacity to reduce MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrasodium bromide, Calbiochem 475989) or Resazurin [441]. HEK293 cells and CGCs were incubated for 30 min and 2 h, respectively, with 0.5 mg/ml MTT or 10 µg/ml Resazurin. Yellow MTT is reduced to blue formazan forming crystals inside mitochondria which are then lysed with 5% formic acid and 95% isopropanol to solubilize the formazan in order to measure its absorbance at 550 nm (with absorbance at 690 nm as reference to subtract contribution of cell debris). Blue Resazurin is reduced inside mitochondria to pink Resorufin which returns to the supernatant and can be measured by absorbance at 550 nm or fluorescence at 590 nm after excitation at 570 nm. Viability was routinely measured after all

72 Materials and Methods pharmacological experiments for all conditions and revealed significant effects on the viability of CGCs at ≥10 µM IPAD or ≥2 µM AQD, respectively. HEK293 cells were nearly unaffected by the inhibitors.

3.4.4 Western blot analysis

APP and APP-cleavage products either from cell lysate or medium were separated with 4-12% gradient SDS-PAGE and transferred onto PVDF membranes (Amersham, Buckinghamshire, UK) by semidry blotting. The membranes were blocked by 4% milk powder-PBS for 2 h, incubated with the primary antibody at 4°C over night, washed and incubated with the secondary peroxidase-conjugated antibody. Protein bands were detected by film exposure (Biorad, Hercules, CA, USA) to the chemiluminescent product of the enhanced ECL substrate (Pierce, Rockford, IL, USA). Cell lysate was prepared by collecting cells in harvest buffer (130 mM NaCl, 3 mM KCl, 10 mM

Na2HPO4, 2 mM KH2PO4, 10 mM EDTA) and centrifugation at 2000xg for 10 min at 4ºC. The cell pellet was lysed with RIPA-buffer (50 mM Tris, pH 7.4, 1% NP-40 (Igepal), 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EGTA, 1% PMSF, 1% protease inhibitor mix). Debris was separated by 15000xg for 15 min at 4°C and the protein concentration of the resulting supernatant was measured. 20 µg of total protein was loaded onto the gel respectively. Medium was mixed with sample buffer

5:1, heated at 95°C for 10 min and the proteins separated and detected as above.

3.4.5 Immunocytochemistry

CGCs transfected with APP-constructs were subjected to immunofluorescence microscopy for estimation of the transfection efficiency. Cells were washed once in PBS and fixed in 4% paraformaldehyde followed by permeabilizing in 0.1% Triton X- 100. After washing with PBS/Tween and blocking for 30 min with 10% horse serum in PBS the cells were incubated overnight with the primary antibody anti-APP, in PBS with 1% FBS, for APPswe- and SEAP-APPswe-transfected CGCs and with anti- SEAP-antibody for SEAP-APPswe-, SEAP-APPswe-AβK16V- and SEAP-APPswe- EpoR-transfected cells. Following two washing steps, incubation with the secondary fluorescently labeled antibodies and addition of 250 ng/ml Höchst 33342 (Fluka 14533), fluorescent images were taken and analyzed using an Olympus microscope equipped with a Hitachi CCD camera or with a Leica microscope with the usual digital camera Canon PowerShot A640 with 10 megapixels. Images were processed with Adobe Photoshop CS2.

73 Materials and Methods

3.4.6 Determination of mRNA knockdown

Knockdown of mRNA targeted with siRNA was determined by comparison of levels of the mRNA of interest in siRNA-treated and control-siRNA-treated cells relative to GAPDH.

RNA isolation was done according to the TRIzol© Invitrogen 2006 protocol. Briefly, adherent cells were lysed for 3 min with one volume equivalent (100 µl per 96-well) of TRIzol reagent (Invitrogen 15596018) at RT. The TRIzol solution was then frozen and stored at -20°C. To extract RNA from the TRIzol solution, 0.2 volume equivalents of chloroform were added. After thorough vortexing, the solution was incubated for 3 min at RT and then centrifuged at 12.000 x g for 15 min for phase separation. The upper (aqueous) phase, containing RNA, was carefully removed and transferred to another vial for RNA extraction. RNA was precipitated from the watery phase by addition of one volume equivalent of isopropanol and subsequent centrifugation at 12.000 x g for 15 min. The RNA pellet was washed once with 75 % ice-cold ethanol and centrifuged again at 10.000 x g for 5 min. After removal of the ethanol and air-drying the pellet, the RNA was resuspended in 10 μl RNAase-free water (Invitrogen 10977035). The concentration of RNA was measured in a Nanodrop device (ND-1000) or an Eppendorf BioPhotometer from Prof. Hartig‟s group, aliquoted and stored at -80 °C. The purity of the samples was determined through the quotient of the absorptions at 260 and 280 nm (A260/A280). A quotient between 1.7 and 2.0 indicated a sufficient purity. For the synthesis of cDNA, 2 μg mRNA, 1 μl 0.5 μg/μl random hexamers (GE Healthcare 27-2166-01) or 1 µl 10 µM specific reverse primers (MWG Eurofins) were mixed and the volume was adjusted to 12.5 μl with sterile, RNAse-free water. The mixture was incubated at 70 °C for 10 min and quickly chilled on ice. 4 μl 4x first strand buffer (250 mM Tris*Cl pH 8.3, 375 mM KCl, 15 mM MgCl2), 2 μl 0.1 M DTT and 1 μl 10 mM dNTP mix (Fermentas) were added and incubated at 42 °C for 2 min. In the following, 0.5 μl Superscript II Reverse Transcriptase (200 U) (Invitrogen 18064) were added and cDNA-synthesis was performed for 50 min at 42 °C. Then, the reaction was inactivated by heating at 70 °C for 15 min. For the q-PCR the cDNA template was diluted 1:5 in RNAse free water. Semi-quanitative Real-Time PCR was performed in a MyiQ cycler (BioRad) using the Platinum® SYBR® Green qPCR Supermix kit (Invitrogen 11722-046) according to

74 Materials and Methods the manufacturer‟s instructions. Briefly, 1 μl cDNA was added to a mixture of 10 μl Supermix and 0.2 μl of the respective forward and reverse primers (10 μM each). The volume was adjusted to 20 μl with milliQ water. Program: 95 °C 3 min, 40x (95 °C 30 sec, 60 °C 30 sec, 72 °C 30 sec), 95 °C 1 min. The specificity of the amplification was controlled by melting point curve analysis.

The CT-values (Cycle of Threshold) of the housekeeping gene GAPDH and the gene of interest were determined by averaging three replicate wells. Data from measurement were initially recorded with the cycler software iQ5 V2.0.148.060623 and for analysis exported as Excel-files.

For data analysis, the difference (ΔCT) in CT-value of the sample- and GAPDH/ActinB-cDNA was calculated:

ΔCT = CT(sample) – CT(GAPDH/ActinB)

Then, the ΔCT-value of the control was subtracted from the ΔCT-value of the sample, yielding the ΔΔCT-value:

ΔΔCT = ΔCT (sample)– ΔCT(control)

Because the initial molecules are doubled within every cycle, the ΔΔCT-value was logarithmized: -ΔΔCT 2 The obtained value was equal to the relative gene expression.

3.4.7 Statistics

All experiments are displayed as means of triplicates ± standard deviation (SD). One-way analysis of variance (ANOVA) and post-hoc Student's t-tests were employed for statistical evaluation using the statistical software GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA).

IC50 values were calculated by performing sigmoid dose-response curve fitting (GraphPad Prism).

3.4.8 Screen data analysis

Data were analyzed with Excel. SEAP activity raw data for each half plate were converted to fraction(=%)-values (=normalized) by dividing the SEAP activity from each well through the average SEAP activity from all wells of the half plate. The average SEAP activity was taken as control value because, firstly, concomitant transfection of GFP siRNA, from a different company and with a different stock concentration than the library, resulted in very different SEAP activities although the

75 Materials and Methods same final concentration was applied. Secondly, the assumption that most of the siRNAs do not affect cleavage of APP by BACE1 is most likely true. To account for slight differences due to position of wells on the plates, each well was also normalized to the surrounding wells, however differences in globally (whole plate) and locally (surrounding wells) normalized SEAP activities were usually not large. Then, for the SEAP-APPswe-EpoR/APPswe-transfected cells, the two globally normalized SEAP activities for each siRNA from the two identically transfected half plates were averaged. First-level-hits were defined with 75% or less of normalized SEAP activity. Normalized SEAP activities from the SEAP-APPswe-transfected cells were calculated as above (except for averaging of duplicates due to a single value only) and looked up for the first-level hit siRNAs. Here, first-level-hits with 80% or less of normalized SEAP activity were removed from the hit list. 80% was taken as threshold because SEAP activities from SEAP-APPswe-transfected decreased by 20% with complete inhibition of BACE1. Supernatant samples of the remaining second-level-hits, and of three neighboring wells, were used in the Aβ40-ELISA. The values in absorbance at 450 nm minus absorbance of empty wells of the hits were divided by the average value of the neighboring wells and here 90% or less was required for third-level-hits.

3.5 Laboratory equipment

Laboratory equipment was mainly purchased from VWR (VWR International GmbH, Bruchsal, Germany) and Integra Biosciences (IBS GmbH, Fernwald, Germany). Pipettes (Eppendorf Research), adjustable 0,5-10μl, 2,0-20μl, 10-100μl, 20-200μl, 100-1000μl, were purchased from VWR (613-0479, 613-0480), also multipipettes with 8 channels 0,5-10μL (613-3622) and 12 channels 20-200μl (613-0146). Pipettors (Pipetboy acu) for serological pipettes were purchased from Integra. Laminar flow sterile benches (class 2) from Nu-Aire (NU-437-500E, NU-437-400E) with rack (NU-400-144) were purchased from Integra. Another class 2 bench from Heraeus (HS18) and a horizontal flow bench (class 1) from Kojair (KH-115 Safety) for the isolation of CGCs were taken over from the group of Prof. Hartung. Incubators from NuAire (NU-5500E) with small inner doors (5500I16) and from Heraeus (390-1024) with small inner doors (390-4429) were purchased from Integra and VWR, respectively.

76 Materials and Methods

Absorbance and fluorescence was measured with a Biotek Reader ELX-808, equipped with filters for 340, 405, 450, 562, 630 and 690 nm ± 10 nm, and a Tecan infinite M200 with wavelengths adjustable 200-900 nm (from Prof. Hartig‟s group), respectively. Gel electrophoresis chambers, the power pack and a semidry blotter were purchased from Biorad. A vacuum pump (181-1521), the analytical balance SARTORIUS TALENT TE124S (611-1109), the balance SARTORIUS TALENT TE3102S (611-1117), the magnetic stirrer with contact thermometer RCT BASIC+VT5 (442-2398), magnetic stirrers VMS-A (442-0185), the pH/mV/C Meter with Cal Check HI 221 (662-0086), a waterbath (462-6719), vortexers VV3 (444-0007), the ultrasound bath USC200T (142-6046) with lid and basket (142-6049), the ultrasound homogenizer Sonoplus HD 2200 (431-5504), the liquid nitrogen tank ARPEGE 140 (478-3255) with rack (478- 3282), the Eppendorf microtube mixer THERMOMIXER COMFORT, TYP 5355 (460- 1112) with tube holder 24X2,0 ML (460-1116), and the trolly MSW 8X5/2 (139-9940) were purchased from VWR. Centrifuges Kendro FRESCO 21 WITH SEALED ROTOR 24X1,5/2 ML (521-1098), Eppendorf CENTRIFUGE 5702 (521-0020) with rotor A-4-38 (521-0032) and adaptors 50ml-conical tube (521-0038) and 15ml-conical tube (521-0037), Kendro MULTIFUGE® 1 S-R (521-0170) with rotor TTH-400 (521-0153) and beakers (521- 0155) and adaptors 15ml-conical tubes (521-0177) and 50ml-conical tubes (521- 1879) and multiwell-rotor MP 3300 (521-0160), and table centrifuges Galaxy 7D (521-2830) and Galaxy 16DH (521-2839) were purchased from VWR. Forceps (14-0322, 14-0422, 14-0203, 14-0100, Z03-102-3C, ßßSI-14-1546) and scissors (12-2104, 12-2021) for the isolation of CGCs were purchased from Schreiber_GmbH_(Fridingen,_Germany).

77 Materials and Methods

4 Sequences of C3orf52 and orthologs

Used sequences The following sequences were obtained from the National Institute of Health (NIH) (USA) database at the NIH Consortium for Bioinformatics (NCBI) and localized in the genome using the NCBI MapViewer. species gene genomic mRNA human APLP1 NC_000019 NP_001019978.1 human APLP2 NC_000011 NP_001633.1 human APP NG_007376 NM_000484 mouse APP NM_007471 fruitfly APPL AE014298 AAF45520 human TTMP NM_024616 NP_078892.2 chimpanzee TTMP NC_006490 XM_001154447 mouse TTMP NC_000082 NM_145389 rabbit TTMP NC_013682 XM_002716844 horse TTMP NC_009162 XM_001501326 cattle TTMP AC_000158 XM_865244 chicken TTMP NW_001471529.1|Gga1_WGA29_2 XM_416636 dog, not yet reported dog TTMP: NW_876299.1|Cfa33_WGA78_2 exon 1: in 4401019-4401760 bp, 444 “n”(undefined) instead of bases exon 2: 4407300-4407429 bp, appr. 6000 bp after assumed location of exon 1 (in human 6814 bp) exon 3: 4415493-4415620 bp exon 4: 4421367-4421443 bp exon 5: 4425125-4425306 bp

Sequences used for the examination of the regions containing the APP family members are found in the ENSEMBL genome browser (www.ensembl.org) under the names as in the results chapter. If not otherwise stated, the longest isoforms were used.

78 Materials and Methods

Sequences were translated using the DNAprotein translation program at the proteomics server ExPasY (Switzerland) or Microsoft Excel. Alignments were done by CLUSTALW as on-line version on the server “mobyle portal” run by the Pasteur Institute (Paris, France). Trees were visualized from tree data with newicktops 1.0 and drawtree also at “mobyle portal”.

Primers for the detection of TTMP/ANNE expression Primers were designed with AiO and purchased from Eurofins/MWG Operon. TTMP forward: 5‟-AACACGCCTCTCAATGGTGC-3‟ TTMP reverse: 5‟-CTTTCGGTGAGCAGGTGAGG-3‟ ANNE1 forward: 5‟-AGCACTGCTTCAGCTGGGTC-3‟ ANNE1 reverse: 5‟-GCAGTGCCATGATCACGCCA-3‟ ANNE2 forward: 5‟-AGAGGCAGGCAGCCAGGTTA-3‟ ANNE2 reverse: 5‟-GTGTTCCTAAGGCTGGCTGGC-3‟ ANNE3 forward: 5‟-CTCTTGGAAGCTTTTGCCAGC-3‟ ANNE3 reverse: 5‟-AGCTGCCCTTCACACTGACTT-3'

79 Results

5 Characterization of cells and APP-constructs

The choice of the cell types used in this project, Cerebellar Granule Cells (CGCs) and HEK293, was based on criteria to be met and trade-offs to be made. Criteria of prime importance were endogenous BACE1-activities which are sufficient for detection without overexpression and a human or at least rodent origin with neuronal or at least neural traits. Trade-offs in cell type physiology and relevance had to be made for superior logistics in cell culture and availability. The APP-constructs which have been engineered at Lundbeck A/S achieved reduction in costs by around 99% and readout assay duration by around 90% in comparison to the Aβ-ELISA. The trade-off was the substitution of most of APP, the N-terminal 84%, for Secreted Alkaline Phosphatase (SEAP) and the modification of the C-terminus shortly after the BACE1-cleavage site such that cleavage by α- and γ- secretases is prevented [442]. Then, instead of sAPPβ SEAP is liberated and secreted into the culture medium. Measurement of the activity of SEAP by a colorimetric assay allows quantifying indirectly BACE1-activity. The BACE1-specific inhibitors IPAD and AQD were used to verify BACE1-dependent processing of the APP-constructs in HEK293 and CGCs, in the case of HEK293 additionally with BACE1-siRNA.

5.1 Characterization of CGCs, HEK293 and SHSY5Y cells CGCs

CGC-cultures consist to approximately 95% of primary murine neurons, which express sufficient measurable endogenous BACE1-activity. Although of cerebellar origin and therefore, as human counterpart, only with limited relevance in AD and only affected in its end stage, murine CGCs possess the advantages of a reasonably pure and easy isolation and a reasonably high transfection potential (Fig. 8A) which is rarely found for neurons. The morphology of this cell type with the long neurites and the distribution of proteins within the different compartments cell body, axon and dendrites provide information which needs to be captured by imaging of (many) single cells. Therefore CGCs were mainly characterized by imaging under different conditions.

80 Results

CGCs developed a dense network of neurites after several days in culture (Fig. 8B) and the proliferation of still dividing cell types like astrocytes (Fig. 8C/D) can be suppressed with cytosine-arabinoside to keep their fraction in the initial lower percentage range.

A B C D

Fig. 8: CGC-culture. A. CGCs could easily be transfected with GFP (live imaging with longpass 525 nm filter). B. CGCs developed a dense network of neurites (live imaging with phase contrast). C/D. Overview and close-up of a CGC culture with around 5% astrocytes stained for the astrocyte marker GFAP. Scale bars: 50 µm.

The cytoskeleton of CGCs was visualized with red fluorescent actin (Fig. 9A/B) or staining of β-III Tubulin (Fig. 9C) which were found all the way along the neurites.

A B C

Fig. 9: Cytoskeleton of CGCs. A/B. CGCs were transfected with red fluorescent actin (life imaging with longpass 590 nm filter). B. CGCs were isolated by S. Penzkofer and stained by B. Zimmer for β-III Tubulin. From [443]. Scale bars: 50 µm.

81 Results

CGCs were subjected to excitotoxicity experiments with glutamate with or without the NMDA receptor blocker MK-801 which rescued the neurons from cell death (Fig. 10). Without MK-801, the CGCs completely desintegrated, leaving only vesicles from cell bodies and neurites which still contain flourescent calcein. A B C D

E F G H

Fig.E 10: Excitotoxicity of FCGCs. G H A-D: 400 µM glutamate. E-H: 400 µM glutamate + 1 µM MK-801. A/E. Calcein-containing cytosol (LP 525 nm). B/F. Overlay of A&C or E&G. C/G. Höchst33342-stained nuclei (LP 425 nm). D/H. Phase-contrast. Scale bars: 50 µm.

CGCs were exposed for 72 h to aspartame to test its toxic potential. As positive control, glutamate with or without MK-801 was applied. The viability assay based on reduction of MTT resulted in 50% decreased reduction with glutamate (Fig. 11). It was assumed that the remaining 50% stem from the larger and metabolically more active astrocytes which are not strongly affected by glutamate (Fig. 10A/B). Aspartame did not exert toxic effects up to 3 mM, as measured by MTT reduction.

125

SD] Fig. 11: Aspartame on CGCs.  CGC cultures were exposed for 72 100 h to aspartame concentrations of 10 nM, 100 nM, 300 nM, 1 µM, 10 µM, 30 µM, 100 µM, 300 µM, 1 mM 75 and 3 mM with or without the NMDA receptor inhibitor MK-801. 100 µM glutamate was used as 50 positive control for toxicity and killed the neurons which could be Aspartame rescued with 1 µM MK-801. 25 Aspartame + 1 µM MK801 100 µM Glutamate 100 µM Glutamate + 1 µM MK801

0 MTT MTT reduction [% of MK801-treated -8 -7 -6 -5 -4 -3 -2 Aspartame concentration [log M] 82 Results

The impact of botulinum neurotoxin type C (BoNT/C) on CGCs was tested with 2.5, 5, 10 and 100 ng per 24-well and 0.7 Mio CGCs. After a few hours with 100 ng, they demonstrated the typical morphology of dead CGCs with neurites completely disintegrated into small vesicles and small cell bodies without spherical appearance. Dose-dependently more CGCs survived longer with lower doses of BoNT/C, the last ones up to 24 h with 2.5 ng (no images available, dead CGCs looked the same as in Fig. 10A-D). CGCs were transfected with a dominant-negative mutant of Reggie fused to GFP (Fig. 12) to test the impact of disturbed Reggie-function on neurite outgrowth. In over ten transfections neurite outgrowth was abolished only once (transfected dead cells).

A B C

Fig. 12: CGCs transfected with dominant-negative mutant of Reggie fused to GFP. A. GFP-fluorescence (LP 525 nm). B. Overlay of A and C. C. Höchst33342-stained nuclei (LP 425 nm). Scale bars: 50 µm.

Murine BACE1 in CGCs was stained with human-BACE1-antibodies (Fig. 13), showing BACE1 in cell bodies and neurites.

A B C D

E F G

Fig. 13: BACE1 in CGCs. A. CGCs were stained with anti-human BACE1 ectodomain antibody. B. Overlay of A and C. C. Höchst33342-stained nuclei. D. Secondary antibody control. E. CGCs were stained with anti-human BACE1 N-terminal antibody. F. Overlay of E and G. G. Höchst33342-stained nuclei. Scale bars: 20 µm. 83 Results

HEK293 cells express sufficient endogenous BACE1-activity as measurable by detection of (overexpressed) Aβ, are of human origin and exhibit neural features like the expression of neurofilaments [444]. The lack of relevance in AD of this kidney- derived cell type is the trade-off for its uncomplicated cell culture with the potential for high transfection efficiency and a fast production of large cell numbers necessary in (high-throughput) screens. The impact of different compounds on BACE1 activity was tested in this project with HEK293 and always controlled by general cell viability with the reduction potential for MTT. HEK293 underwent basic characterization with different compounds (Fig. 14) which were also used for characterization of an APP-construct. SHSY5Y cells also express sufficient endogenous BACE1 activity and are of human neuroblastoma origin. They were characterized (Fig. 14) as counterpart for stably transfected SHSY5Y. Generally SHSY5Y were more sensitive to the compounds tested than HEK293, although the viability curves were left-shifted not more than half of a magnitude.

A B C

SD] SD]

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Fig. 14: Different compounds on HEK293 and SHSY5Y. A-F. The compounds were administered at the concentrations indicated with a medium change 24 h after seeding of the cells and after additional 24 h MTT reduction potential was measured. 84 Results

5.2 Optimization of transfection for APP-construct characterization

The different constructs could either be stably or transiently transfected into the HEK293, whereas CGCs could only be transfected transiently. It was decided to transfect also the HEK293 with plasmids transiently because, firstly, the cells had to be transfected transiently with siRNA anyway, which is usually not 100% efficient and also variable to some extent. Depending on their overall percentage, stably transfected cells without siRNA would therefore distort results. Secondly, with stable transfection the plasmids integrates usually somewhere in the genome with the potential to induce unpredictable effects like destruction of a gene or insertion of the plasmid after APP-unrelated transcription enhancer/repressor binding elements. For the screen of specifically BACE1-affecting kinases, production of the BACE1- substrate as equal as possible is advantageous, so that if transcription factors are affected by a kinase, the likelihood to act somehow on one of the two major determinants, BACE1 and substrate, is much higher for BACE1. Actually, this should be solved with the CMV-promoter on the plasmids. Additionally, the mandatory usage of the resistance antibiotic, here G418, to maintain stable transfection could exert unspecific cell-culture side effects in the rather high concentrations employed, even if the antibiotic is not used in the kinase-siRNA-experiments. For characterization of the APP-constructs with transient transfection, the efficiency of transfection was determined and optimized using GFP, more specifically enhanced GFP with a C-terminal linker region (eGFP-C1). Transfected cells were imaged and the transfection efficiency determined by calculating the percentage of GFP- transfected cells.

85 Results

Transfection of CGCs was conducted with electroporation. The efficiency of electroporation is first of all dependent on strength, duration and shape of the electric pulse. These parameters are predefined by usage of a cell type-specific electroporation program with the Amaxa electroporator. For CGCs was already at Lundbeck A/S the program G-013 (chicken neurons) found to be most suitable, which could be confirmed (data not shown). Other tested programs resulted either in lower efficiency, although higher viability like O-005 for murine neurons and G-004 for SHSY5Y, or higher efficiency along with dramatic decrease in viability like Q-001 for HEK293 (data not shown). With usage of the provided nucleofection solution and the recommendation to keep added volume as low as possible only the two parameters cell number and plasmid concentration were left for optimization. Different numbers of CGCs, but same amount of plasmid, per transfection had as outcome different transfection efficiencies (Fig. 15) and 5 Mio CGCs were from then on used. Amounts of plasmid exceeding 5 µg were not found to increase the efficiency (data not shown) and therefore were chosen 5 µg for transfection of CGCs.

A B C

E D cell transf. nuclei Total % transf. number 5 Mio 146 273 419 35 10 Mio 97 367 464 21 15 Mio 73 206 279 26

Fig. 15: Transfection efficiencies for different numbers of CGCs. A-C. 5, 10 or 15 Mio CGCs were electroporated (overlay of GFP fluorescence and stained nuclei). D. labeled transfected cells and nuclei (for counting in A). E. Transfected cells and nuclei from non-transfected cells counted from A, B and C.

86 Results

HEK293 were either transfected with electroporation or lipofection. Electroporation was conducted using Opti-MEM, having 304 mOsm without any supplements in comparison with 329 mOsm for neuron nucleofection solution, program Q-001 (HEK293 ATCC), an optimal cell number of around 1 Mio, confirming the manufacturer‟s instruction for HEK293, and an optimal amount of also 5 µg plasmid (Fig. 16A). In cotransfection with siRNA a concentration of 4 µM was chosen to account for both a reasonable knockdown and only a small volume added (4 µl of 100 µM stock solution) to obtain a high transfection efficiency.

A HEK293 after 24 h C HEK293 after 24 h 100 1 Mio cells 0.5 Mio cells B 100 3 µg eGFP-C1

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D E F G H

- 0.5 µM 1 µM 2 µM 4 µM GFP siRNA

Fig. 16: Optimization of electroporation of HEK293 with GFP plasmid and GFP siRNA. A-C. Transfection efficiencies for different numbers of HEK293 and different amounts of GFP plasmid. B. 95% transfection efficiency (overlay of GFP and nuclei). D-H. Cotransfection of GFP plasmid and increasing concentrations of GFP siRNA.

Transfection efficiency with Lipofectamine is strongly dependent on the volume of Lipofectamine used per well. Especially in cotransfection with siRNA the volume of Lipofectamine, the amount of plasmid and the concentration of siRNA have to be 87 Results adjusted because siRNA and plasmids seem to compete for Lipofectamine and insufficient volume of Lipofectamine leads to decreased transfection efficiencies in addition to a strong non-specific “knockdown” from control siRNA which reduces strongly the window for test siRNAs. When mixing the reagents it was important to combine firstly siRNA and plasmid and add Lipofectamine later. For characterization of the APP-constructs, 0.25 µl Lipofectamine and 0.1 µg plasmid per well with 0.1 µM siRNA was found to be optimal (Fig. 17G/H). 0.2 µg eGFP-C1

A no GFP siRNA B 0.2 µM GFP siRNA C 0.8 µM GFP siRNA

52 % 27 % apparent 20 % apparent

transfection efficiency transfection efficiency transfection efficiency

D E F

0.1 µg eGFP-C1 0.04 µg eGFP-C1 74% transfection efficiency 60% transfection efficiency (434 of 587) (376 of 624) 0.1 µg eGFP-C1 0.04 µg eGFP-C1 0.1 µM GFP siRNA 0.05 µM GFPsiRNA

G H I J

Fig. 17: Optimization of lipofection of HEK293 with GFP plasmid and GFP siRNA. A-C. Transfection efficiency and apparent transfection efficiencies for 0.2 µg GFP plasmid per well without or with GFP siRNA. D-F. LP 525 nm of A-C. G/H. Transfection efficiency with 0.1 µg GFP plasmid per well and effect of 0.1 µM GFP siRNA. I/J. Transfection efficiency with 0.04 µg GFP plasmid per well and effect of 0.05 µM GFP siRNA.

88 Results

5.3 Characterization of the APP-constructs

Plasmids containing APPswe (K595N/M596L) or APP (Fig. 18A) served as basis for the other APP-constructs engineered by Lundbeck A/S, Valby, Denmark, and were briefly characterized using microscopy (APPswe only) and specific secretase inhibitors (both APPswe and APP). In HEK293 they were additionally characterized with siRNA. The impact of inhibitors and siRNAs was estimated by comparison of the Aβ-production of treated and control cells.

In order to link BACE1 activity to SEAP activity, as practical readout substitute for Aβ- production, APPswe and APP were modified subsequently in two steps. The first modification of APPswe and APP resulted in the intermediate constructs SEAP- APPswe and SEAP-APP where the reporter enzyme SEAP was fused 13 amino acids N-terminal to the BACE1 cleavage site. SEAP is liberated both by α-secretases and BACE1 as SEAPα and SEAPβ in analogy to sAPPα and sAPPβ (Fig. 18B). SEAP-APPswe transfected CGCs were imaged and both constructs were tested with secretase inhibitors and siRNAs. Unfused SEAP from the source plasmid was briefly characterized. A second modification, a lysine to valine point-mutation at the α-cleavage site (K16V) of SEAP-APPswe or SEAP-APP, prevents cleavage by α-secretases and should result in liberation of SEAPβ only (Fig. 18C). These constructs were called SEAP- APPswe-AβK16V and SEAP-APP-AβK16V and also briefly characterized. For another approach to prevent cleavage by α- and γ-secretases, a second modification was applied. The SEAP-APPswe- or SEAP-APP-C-Terminus was substituted 9 amino acids after the BACE1 cleavage site by 52 amino acids of the (EpoR) [427] containing the transmembrane domain, which should also result in liberation of SEAPβ only (Fig. 18D). In analogy to the other constructs these were called SEAP-APPswe-EpoR and SEAP-APP-EpoR and characterized similarly. Generally the constructs with the Swedish mutation were characterized more thoroughly because most experiments were conducted with them.

89 Results

Fig. 18: Overview of APP-constructs and secretase cleavage sites. A. APPswe and APP differ from each other by the Swedish double mutation KMNL. B. SEAP was fused to APPswe and APP 13 amino acids before the BACE1-cleavage site. C. AβK16V in the two SEAP-constructs abolishes the α-cleavage site. D. EpoR was fused to the SEAP-constructs 9 amino acids after the BACE1-cleavage site.

90 Results

A completely unrelated plasmid with APP fused to fluorescent citrin (GFP derivative), APP-Citrin, was also imaged in transfected CGCs (Fig. 19) which demonstrated good overlap of fluorescence due to citrin (Fig. 19A) and staining with antibodies against APP (Fig. 16C) and thus made staining of the other constructs reliable.

A B C D

Fig. 19: CGCs transfected with APP-Citrin. A. Fluorescence from Citrin (LP 525 nm). B. Overlay of A and C and stained nuclei. C. Fluorescence from A594-conjugated sec. antibody recognizing 6E10 (LP 590 nm). D. Secondary antibody control. Scale bars: 20 µm.

APPswe in transfected CGCs could be visualized (Fig. 20) with the same antibodies as APP in APP-Citrin-transfected CGCs.

A B C

Fig. 20: CGCs transfected with APPswe. A. Fluorescence from A594-conjugated sec. antibody recognizing 6E10 (LP 590 nm). B. Overlay of A and C. C. Höchst33342-stained nuclei (LP 425 nm). Scale bars: 20 µm.

91 Results

The pharmacological characterization of APPswe with specific secretase inhibitors demonstrated the dependency of Aβ-production on BACE1 and γ-secretase. Both in CGCs (Fig. 21A) and HEK293 (Fig. 21B) the dynamic range of the BACE1-inhibitor Amino-Quinazoline-Derivative (AQD) spanned the three magnitudes from 1 nM to 1µM and at this concentration Aβ-production is completely abolished. At higher concentrations, AQD exerts toxic effects (Fig. 21B). The γ-secretase inhibitor LY450139, currently in phase 3 trial as Semagacestat, also abolished Aβ-production at 1 µM (Fig. 21C). The α-secretase inhibitor GM6001 in contrast had no effect on Aβ-production in the concentrations tested (Fig. 21D). transfected CGC after 24h

A 175 B 125 transfected HEK293 after 24h

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Fig. 21: Pharmacological characterization of APPswe in GCGs and HEK293. A. CGCs were electroporated with 5 µg APPswe, the BACE1 inhibitor AQD was administered with a medium change after 24h and supernatants sampled after additional 72 h. B. HEK293 were electroporated with 5 µg APPswe, AQD was administered with a medium change after 24 h and supernatants sampled after additional 24 h. MTT reduction was measured directly after sampling of the supernatants and addition of fresh medium. C. Treatment as in B but with the γ-secretase inhibitor LY450139. D. Treatment as in B but with the α-secretase inhibitor GM6001. 92 Results

siRNAs in combination with APPswe were used in HEK293 to show proof-of-principle of the technology. Firstly, siRNAs, similarly to inhibitors, exerted their effects in a concentration-dependent manner (Fig. 22A) like an siRNA directed against APPswe (APPswe siRNA) which decreased Aβ-production in co-electroporated HEK293 stronger with 2 µM than with 1 or 0.5 µM. Secondly, siRNAs possess a high specificity which is displayed by the failure of a non-specific control siRNA (Con) and an siRNA against SEAP to substantially decrease Aβ-production (Fig. 22B). A siRNA against BACE1 in contrast dramatically decreases it, again proving its dependency on BACE1 activity (Fig. 22B).

transfected HEK293 after 24h APPswe A 600 B 1000 4 µM siRNA 500 8 µM siRNA

] 750

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1-40 A  A 250 100

0 0 no siRNA -7.0 -6.5 -6.0 -5.5 -5 5 µg plasmid: APPswe siRNA [log M] siRNA: Con SEAP APPswe BACE1

Fig. 22: Characterization of APPswe in HEK293 with siRNAs. A. HEK293 were coelectroporated with 5 µg APPswe and 0, 0.5, 1 and 2 µM of an siRNA against APPswe, the medium was changed after 24 h and supernatants sampled after additional 24 h. B. Treatment as in A but with 4 and 8 µM of different siRNAs.

93 Results

APP in transfected CGCs and HEK293 gave rise to Aβ-production (Fig. 23A/B), which in the case of HEK293 was confirmed to be dependent on BACE1 and γ- secretase by use of IPAD and LY450139 (Fig. 23B). The Con siRNA and the SEAP siRNA did not interfere with Aβ-production, whereas the APPswe siRNA exerted a measurable effect, although by far not as strong as with the APPswe-construct (Fig. 23C). BACE1 siRNA also did not decrease Aβ as strong as in combination with the APPswe-construct, at least not at 4 µM.

A APP transf. CGCs B APP transf. HEK293 after 24 h 125 350

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Fig. 23: Characterization of APP in CGCs and HEK293. A. CGCs were electroporated with 5 µg APP and the supernatant sampled after 72 h. B. HEK293 were electroporated with 5 µg APP, the BACE1 inhibitor IPAD or the γ-secretase inhibitor LY450139 were administered with a medium change after 24 h and supernatants sampled after additional 24 h. C. HEK293 were coelectroporated with 5 µg APP and 4 or 8 µM of different siRNAs, the medium was changed after 24 h and supernatants were sampled after additional 24 h.

94 Results

Aβ was measured with an ELISA kit with a sigmoid standard curve, thus enhancing sensitivity in the lower concentration range by sacrificing it at higher concentrations (Fig. 24A).

The alternative readout SEAP shows a more linear dependency of measured absorption on concentration as displayed by undiluted and diluted solutions of a supernatant from pCI-neo-SEAP-transfected HEK293 (Fig. 24B). SEAP in the supernatant dephosphorylates chromogenic para-Nitro-Phenyl-Phosphate (pNPP) to colorless nitrophenol which deprotonates to nitrophenolate with its extended conjugated π-electron system and is therefore a chromophore, with a peak of absorbance close to 405 nm. pNPP is added to the supernatant in an alkaline buffer in order to prevent dephosphorylation by other phosphatases.

The reaction rate of SEAP, which was the actual parameter measured, differed between several conditions (Fig. 24C). The reaction was temperature-dependent because it was faster at 37°C than at room temperature (RT) and absorption values of around over 4 could not be measured because the upper limit of the reader was reached. At 37°C with 5% CO2, however, the measured absorption was strongly decreased, probably by decreasing the pH of the reaction solution away from the optimum of SEAP and/or by decreasing deprotonation of the colorless nitrophenol. As additional control HEK293 were transfected with SEAP under a non-functional promoter (pSEAP-basic).

Purified Shrimp Alkaline Phosphatase was incubated with pNPP for comparison with SEAP (Fig. 24D). Reaction solutions containing 0.02 and 0.1 mU/µl Shrimp AP resulted in reaction rates of 20 and 70 mAU/min, which was the range typically measured with the SEAP-modified APP-constructs.

95 Results

Curve A 4,000

3,500

3,000 U]

2,500

2,000 450 1,500

absorption[A 1,000

0,500

0,000 0 100 200 300 400 500 600 Aβ1-40 concentration [pg/ml]

B C time dependency of reaction reaction rate 37°C -CO2 5 37°C, -CO , pCI-neo-SEAP 1.25 undiluted 2 RT, -CO2, pCI-neo-SEAP 3/4 37°C, +CO2, pCI-neo-SEAP 1.00 4 1/2 1/4

0.75 3

[AU] [AU] 0.50 2

0.25 1 37°C, -CO2, pSEAP-basic

0.00 0 absorption from nitrophenolate at 405 at nm nitrophenolate from absorption absorptionnitrophenolatefrom 405 at nm 0.0 0.5 1.0 1.5 2.0 0 1 2 3 4 5 6

D reaction time [h] reaction time [h]

]

min

U/

A

[m

activity

hrimpAP S ShrimpAP [mU/µl]

Fig. 24: Aβ1-40-ELISA and characterization of SEAP in HEK293. A. Standard curve of the Aβ1-40-ELISA (axis units with decimal commas). B. HEK293 were electroporated with 5 µg SEAP (on plasmid pCI-neo-SEAP), the medium changed after 24 h and sampled after additional 24 h. For the following SEAP activity assay with the SEAP substrate pNPP the sample was used either undiluted or diluted to ¾, ½ or ¼ with medium. C. HEK293 were electroporated with 5 µg of either pCI-neo-SEAP or pSEAP-basic (SEAP without functional promoter), the medium was changed after 24 h and sampled after additional 24 h. For the following SEAP activity assay the samples were either incubated at 37°C, at room temperature (RT) or in an incubator (37°C, +CO2). D. Shrimp Alkaline Phosphatase (AP) in different amounts was incubated at 37°C with pNPP (axis units with decimal commas). 96 Results

SEAP-APPswe in transfected CGCs was stained with an antibody directed against the SEAP-moiety (Fig. 25A), partly because no endogenous CGC protein should give false-positive staining and partly because the APP-antibody 6E10 (used for APP- Citrin and APPswe) was only available anymore for extremely high prices. Typically most transfected cells had the SEAP staining in or on the cell body and some were stained also along the neurites. SEAP-APPswe-transfected HEK293 produced Aβ and SEAP time- and cell number- dependently and the time courses of Aβ and SEAP from a given cell number resemble each other largely (Fig. 25D).

A B C

D 1.00 SEAP-APPswe 60 [mAU/min SEAPactivity Fig. 25: Characterization of SEAP-APPswe in CGCs and 100,000 HEK293. 75,000 0.75 50,000 45 opensymbols A-C. CGCs were electroporated 25,000 with 5 µg SEAP-APPswe, the cells per well cells stained after 72 h with a 0.50 30 SEAP antibody (clone 8B6) and a A594-conjugated sec. ab (A,

[ng/ml]conc. LP 590 nm), the nuclei visualized filled symbols 1-40 with Höchst33342 (C, LP 425

 0.25 15 A  nm) and the images overlayed SD] (B). Scale bars: 50 µm. D. HEK293 were electroporated 0.00 0 with 5 µg SEAP-APPswe, seeded in different numbers per well, 0 6 12 18 24 the medium changed after 24 h time [h] and sampled after add. 24 h.

97 Results

The BACE1-inhibitors IPAD and AQD nearly abolished Aβ-production at 1 µM, as with APPswe, in CGCs (Fig. 26A/B) and HEK293 (Fig. 26C/D). At this concentration of IPAD and AQD, SEAP activity remained approximately at 80% in HEK293 and at 50% in CGCs, probably due to SEAPα from α-cleavage, and decreased even further at 5 µM. However, from the MTT and Resazurin measurements this concentration exerts a toxic effect on CGCs with both inhibitors (Fig. 26B). Also morphologically CGCs looked dead at 5 µM AQD (image not shown). In HEK293, toxicity was repeatedly measured at 10 µM AQD by reduced MTT reduction, while IPAD at 10 µM seemed to exert only a slight toxic effect.

SD]

A B SD]   transfected CGC 125 transfected CGC 125

100 100

75 75

50 50 IPAD, A AQD, A IPAD, SEAP AQD, SEAP 25 25 IPAD, MTT AQD, MTT

IPAD, Resazurin AQD, Resazurin conc., SEAP act., MTT red., red.Resazurin

0 conc., SEAP act., MTT red., red. Resazurin 0

1-40

1-40 

-10 -9 -8 -7 -6 -5  A -10 -9 -8 -7 -6 -5

A [% of pg/ml,154 0.6 mAU/min, 0.51 AU, kFU 38

DMSO [% ofpg/ml, 117 mAU/min,0.46 AU,0.49 kFU 37 inhibitor [log M] DMSO no DMSO inhibitor [log M] no DMSO

transfected HEK293 C D transfected HEK293 125 125

100 100

SD]

SD]

  75 75

50 SEAP-APPswe 50 SEAP-APPswe

[% of control [% of control AQD A 25 IPAD A1-40 25 1-40 IPAD SEAP AQD SEAP AQD MTT

IPAD MTT conc., SEAP activity MTT and reduction conc., SEAP activity MTT and reduction

0 0

1-40

1-40

 A A con. -10 -9 -8 -7 -6 -5 con. -10 -9 -8 -7 -6 -5 inhibitor [log M] inhibitor [log M]

Fig. 26:_Characterization of SEAP-APPswe with BACE1 inhibitors in CGCs and HEK293. A/B. CGCs were electroporated with 5 µg SEAP-APPswe, IPAD (A) or AQD (B) administered with a medium change after 24 h and the supernatants sampled after additional 72 h. MTT reduction was measured directly after supernatant sampling and addition of fresh medium. C/D. HEK293 were treated with IPAD (C) or AQD (D) as above but the supernatants sampled after 24 h. 98 Results

In HEK293, the Aβ concentration was strongly reduced at 1 µM LY450139 (Fig. 27A) as with APPswe whereas SEAP activity was unaffected by the γ-secretase inhibitor. The 80% remaining SEAP activity at the highest concentrations of the BACE1 inhibitors (Fig. 27C/D) was probably SEAPα from α-cleavage. The α-secretase inhibitor GM6001 decreased SEAP activity by 40% (Fig. 27B). Aβ-production was unaffected by GM6001.

A B

transfected HEK293 after 24h transfected HEK293 after 24h SD]

SD] 125 125  

100 100

75 75

50 50 -sec.-inh. LY450139, A -sec. inh. GM6001, A

25 -sec.-inh. LY450139, SEAP 25 -sec. inh. GM6001, SEAP

-sec.-inh. LY450139, MTT -sec. inh. GM6001, MTT conc., SEAP activity, MTT reduction

conc., SEAP activity, MTT reduction 1-40

1-40 0 0

  A

A

[% ofpg/ml, 785 mAU/min,62 AU 0.39 [% ofpg/ml, 822 mAU/min,65 AU 0.33 -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5 inhibitor [log M] inhibitor [log M] Fig. 27:_Characterization of SEAP-APPswe with γ- and α-secretase inhibitors in HEK293. A/B. HEK293 were electroporated with 5 µg SEAP-APPswe, γ-secretase inhibitor LY450139 (A) or α-secretase inhibitor GM6001 (B) administered with a medium change after 24 h and the supernatants sampled after additional 24 h. MTT reduction was measured directly after supernatant sampling and addition of fresh medium.

In co-electroporation with siRNA, both the SEAP siRNA and the APPswe siRNA achieved an effective knockdown of the construct, measurable by both readouts, while with the BACE1 siRNA around 40% of Aβ and, in similarity with the inhibitors, 80% of SEAP activity remained, probably mostly SEAPα (Fig. 28).

125

SD] SEAP-APPswe

 100 SEAP activity A 1-40 conc.

75 Fig. 28: Characterization of SEAP-APPswe in HEK293 with siRNAs. and SEAP activitySEAP and 50 HEK293 were coelectroporated with 5 µg SEAP-APPswe and 4 µM of different

conc. siRNAs, the medium was changed after 24 25

1-40 h and supernatants were sampled after 

A additional 24 h. ofpg/ml 548 mAU/min 38 and

[% 0 5 µg plasmid: - SEAP-APPswe 4 µM siRNA: - - SEAP APPswe BACE1 99 Results

In order to test for differences originating in the two transfection methods, HEK293 were either electroporated (Amaxa) with 5 µg SEAP-APPswe or lipofected (Lipofectamine) with 0.2 µg SEAP-APPswe and then the supernatant of 100000 cells per well sampled 24 h after addition of compounds to measure LDH activity, SEAP activity and Aβ concentration while the cells were used for an MTT assay. Generally the values of compound-treated HEK293 relative to non-treated HEK293 were quite similar with both transfection methods (Fig. 29), whereas SEAP activity and Aβ concentration were higher with lipofection and also the ratio of SEAP activity to Aβ concentration was higher with lipofection. The antibiotic Actinomycin D forms a stable complex with DNA and blocks the movement of the RNA . It was not toxic at 0.78 µM but decreased SEAP activity and Aβ concentration by more than 50% (Fig. 29A/B). The anti-malaria drug Chloroquine intercalates into DNA and enriches in lysosomes (=lysosomotropic) and nearly completely killed HEK293 at 2 mM (Fig. 29C/D). Cycloheximide interferes with the peptidyl transferase activity of the 60S ribosome, blocking translation, and had at 7 µM only a minor effect on SEAP activity and Aβ concentration with lipofection (Fig. 29E/F).

SEAPactivity

SEAPactivity 0.78 µM Actinomycin D A 0.78 µM Actinomycin D 100 B 100 100 100 Lipofectamine 167 of mAU/min [% Amaxa 17mAU/min of [%

and1337 pg/ml]

and388 pg/ml] 75 75 75 75

and

and 50 50

50 50

and LDH activityand and LDH activityand

A

A

40conc. 25 25 40conc. 25 25

MTT MTT assay 0 0

MTT MTT assay 0 0 MTT LDH SEAP A

MTT LDH SEAP A [% of -59 AU mAU/min]1.06 and [% of AU -57 1.57 and mAU/min]

2 mM Chloroquine 2 mM Chloroquine SEAPactivity

SEAPactivity Amaxa 100 Lipofectamine 100 C 100 100 D

[% of 159 mAU/min of [%

and1156 pg/ml]

[% of 19mAU/min of [%

and365 pg/ml] 75 75 75 75

and

and 50 50

50 50 and LDH activityand

A and LDH activityand

A

 25 25 40conc. 25 25 40conc.

MTT MTT assay 0 0

MTT MTT assay 0 0 MTT LDH SEAP A

MTT LDH SEAP A [% of -59 AU 1.06 mAU/min]and [% of -57 AU 1.57 mAU/min]and

SEAPactivity E 7 µM Cycloheximide F 7 µM Cycloheximide SEAPactivity 100 Amaxa 100 100 Lipofectamine 100

[% of 19mAU/min of [%

[% of 159 mAU/min of [%

and365 pg/ml]

and1156 pg/ml] 75 75 75 75

and 50 50 and

50 50 and LDH activityLDH and

A and LDH activityLDH and

A

 25 25 40conc. 25 25 40conc.

MTT assay MTT 0 0

MTT LDH SEAP A assay MTT 0 0

[% of-57 mAU/min]AU and 1.57 MTT LDH SEAP A [% of -59 AU mAU/min]1.06 and Fig. 29: Characterization of SEAP-APPswe in HEK293 with different compounds. A-F. HEK293 were electroporated with 5 µg SEAP-APPswe, the compounds added with a medium change after 24 h and supernatants were sampled after additional 24 h. 100 Results

SEAP-APP was characterized with electroporated HEK293. BACE1-inhibitor IPAD and γ-secretase-inhibitor LY450139 strongly reduced Aβ-production at 1 µM (Fig. 30 A), with the curve from IPAD being left-shifted in comparison with SEAP-APPswe. In co-transfection with SEAP siRNA again a strong reduction of both Aβ and SEAP activity was measurable while APPswe was by far not as efficient as with the SEAP- APPswe-construct (Fig. 30B). In this particular experiment 8 µM BACE1 siRNA decreased Aβ-production only around 50%, whereas remaining SEAP activity was closer to the expected value of around 75%. However, in another experiment only around 15% Aβ were left with 5 and 10 µM BACE1 siRNA with SEAP activity remaining quite high (Fig. 30C). SEAP siRNA at 5 and 10 µM completely abolished Aβ-production and SEAP activity (Fig. 30D). Even 10 µM of siRNA was not generally toxic. A B

125 SD]

transfected HEK293 after 24h SD] 

125  A 1-40 conc. SEAP activity 100 100

75

75 and SEAP activitySEAP and 50 50 IPAD, A conc. IPAD, SEAP 25

25 1-40 

IPAD, MTT A conc., SEAP activity, MTT reduction

% pg/mlofmAU/min 106 1269 and 0 1-40

0 [  LY450139, Aβ

A 5 µg plasmid: SEAP-APP [% of pg/ml,964 mAU/min,110 0.25 AU DMSO -10 -9 -8 -7 -6 -5 8 µM siRNA: con SEAP APPswe BACE1 inhibitor [log M] C D

transfected HEK293 after 24h transfected HEK293 after 24h SD] SD] 125

125  

100 100

75 75

50 50

A A 25

25 SEAP SEAP conc., SEAP activity,SEAP conc., reduction MTT

conc., activity,SEAP MTT reduction MTT MTT 1-40 1-40 0

0 

A

A

[% pg/ml, of mAU/min,661 104 0.6 AU [% ofpg/ml, 661 mAU/min, 104 0.6 AU -8.5 -7.5 -6.5 -5.5 -5 -8.5 -7.5 -6.5 -5.5 -5

BACE1 siRNA [log M] SEAP siRNA [log M] no siRNA5 µM Con no siRNA5 µM Con 10 µM Con 10 µM Con Fig. 30: Characterization of SEAP-APP in HEK293 with inhibitors and siRNAs. A. HEK293 were electroporated with 5 µg SEAP-APP, the inhibitors added with a medium change after 24 h and supernatants were sampled after additional 24 h. B-D. HEK293 were coelectroporated with 5 µg SEAP-APP and different siRNAs in the concentrations indicated, the medium changed after 24 h and sampled after additional 24 h. 101 Results

SEAP-APPswe-AβK16V could also be stained in transfected CGCs with the SEAP antibody (Fig. 31A). Again, most transfected cells were stained at the cell body while some were stained all over. A B C D

Fig. 31: Characterization of SEAP-APPswe-AβK16V in CGCs. A-C. CGCs were electroporated with 5 µg SEAP-APPswe-AβK16V, the cells stained after 72 h with a SEAP antibody (clone 8B6) and an A594-conjugated sec. ab (A, LP 590 nm), the nuclei visualized with Höchst33342 (C, LP 425 nm) and the images overlayed (B). D. Secondary antibody control. Scale bars: 20 µm. SEAP from SEAP-APPswe-AβK16V and BACE1 colocalized most strongly in a juxtanuclear compartment, probably the Golgi-apparatus (Fig. 32B). The raft protein Thy1 also colocalized in some neurons with SEAP (Fig. 32F). A B C D

E F G H

Fig. 32: Characterization of SEAP-APPswe-AβK16V in CGCs. A-D. CGCs were electroporated with 5 µg SEAP-APPswe-AβK16V. The cells were stained after 72 h with the SEAP antibody (A) and the human BACE1 ectodomain antibody (C). The nuclei were visualized with Höchst33342 (D) and the images overlayed (B). E-H. CGCs were electroporated with 5 µg SEAP-APPswe-AβK16V. The cells were stained after 72 h with the SEAP antibody (E) and a Thy1 antibody (G). The nuclei were visualized with Höchst33342 (H) and the images overlayed (F). Scale bars: 20 µm.

102 Results

The BACE1 inhibitors IPAD and AQD strongly reduced both Aβ- and SEAP-secretion in CGCs and HEK293 at 1 µM (Fig. 33), thus linking SEAP activity in form of SEAPβ to BACE1 activity. Compared to SEAP-APPswe-transfected HEK293, only 20% of SEAP activity is measurable from SEAP-APPswe-AβK16V-transfected HEK293 (SEAP-APPswe: 65 mAU/min; SEAP-APPswe-AβK16V: 18 mAU/min) when normalized to the Aβ-concentrations (SEAP-APPswe: 822 pg/ml; SEAP-APPswe- AβK16V: 1120 pg/ml), which equals the 20% of SEAP activity reduction with 1 µM BACE1 inhibitors in SEAP-APPswe-transfected HEK293.

A SEAP-APPswe-AK16V in CGC

125 SD]

 100

75

50

IPAD, A

25 IPAD, SEAP

40 conc. and SEAP activitySEAP and conc. 40  A

[% of pg/ml57 0.1 mAU/minand 0

DMSO -11 -10 -9 -8 -7 -6 inhibitor [log M] B C SEAP-APPswe-AK16V in HEK293 SEAP-APPswe-AK16V in HEK293

125 125

SD]

SD]   100 100

75 75

50 50 IPAD, A AQD, A 25 AQD, SEAP

25 IPAD, SEAP activitySEAP and conc. 40

40 conc. and SEAP activitySEAP and conc. 40

 A A

0 0 [% pg/mlof mAU/min 17 1141 and [% pg/mlof mAU/min 18 1120 and con. -11 -10 -9 -8 -7 -6 con. -11 -10 -9 -8 -7 -6 inhibitor [log M] inhibitor [log M] Fig. 33: Characterization of SEAP-APPswe-AβK16V with BACE1 inhibitors in CGCs and HEK293. A. CGCs were electroporated with 5 µg SEAP-APPswe-AβK16V. IPAD was administered with a medium change after 24 h and the supernatants sampled after additional 72 h. B/C. HEK293 were electroporated and treated with IPAD (B) or AQD (C) as above but the supernatants sampled 24 h after the medium change. 103 Results

SEAP-APP-AβK16V was characterized in HEK293, firstly with IPAD and AQD (Fig. 34A/B), which had left-shifted inhibitory curves compared to SEAP-APPswe-AβK16V, paralleling the left-shift with SEAP-APP in comparison with SEAP-APPswe. Secondly, SEAP-APP-AβK16V was coelectroporated with siRNAs resulting in Aβ/SEAP-reduction by SEAP siRNA and BACE1 siRNA but hardly by APPswe siRNA (Fig. 34C). Thirdly, western blotting demonstrated that SEAP-APP-AβK16V was not cleaved anymore at the mutated site by α-secretases because no band from SEAPα was found in the supernatant (S) (Fig. 34D). No band from SEAPβ could be found because it does not contain the Aβ-epitope for the 6E10 antibody in contrast to uncleaved SEAP-APP-AβK16V in cells (C) which was recognized by the antibody. In comparison with SEAP-APPswe-AβK16V, the Aβ-concentrations were extremely low in contrast to similar SEAP activities.

A transfected HEK293 after 24h B transfected HEK293 after 24h SD]

125 SD] 125  

100 100

75 75

50 50

IPAD, A AQD, A 25 25

IPAD, SEAP AQD, SEAP conc., SEAP activity, MTT reduction

IPAD, MTT conc., SEAP activity, MTT reduction AQD, MTT 1-40 0 1-40

 0

[% of pg/ml,73 mAU/min,13 0.93 AU

A [% of pg/ml,84 mAU/min,13 AU 0.99 A DMSO -10 -9 -8 -7 -6 -5 DMSO -10 -9 -8 -7 -6 -5 inhibitor [log M] inhibitor [log M] C 125

SD] A 1-40 conc. D  SEAP- SEAP activity non- SEAP- SEAP- 100 APP- transf. AβK16V APPswe APP

75 C S C S kD C S C S

50 and SEAP activityand 50 SEAP- APP- 37 conc. SEAPα

25 AβK16V

1-40 

A

50 % of pg/ml61 9.3 and mAU/min [ 0 GAPDH 5 µg plasmid: SEAP-APP-AK16V 37 8 µM siRNA: con SEAP APPswe BACE1

Fig. 34: Characterization of SEAP-APP-AβK16V with BACE1 inhibitors and siRNAs in HEK293. A/B. HEK293 were electroporated with 5 µg SEAP-APP-AβK16V. IPAD (A) or AQD (B) was administered with a medium change after 24 h and the supernatants sampled after additional 24 h. C. HEK293 were coelectroporated with SEAP-APP-AβK16V and siRNA. D. Electroporated HEK293 and supernatant were subjected to western blotting and the same blot was first incubated with APP antibody 6E10 and then with GAPDH antibody. 104 Results

SEAP-APPswe-EpoR was visualized in CGCs with the SEAP antibody (Fig. 35A). In similarity with the other SEAP-constructs, mainly the cell bodies were stained. Interesting to experimenters might be the large difference in fluorescence intensity of replicates due to different plastic ware (Fig. 35 D/E), which was reproduced by nine more (=all) comparisons in the dataset, thus confirming an initial observation.

A B C

D E

F G H

Fig. 35: Characterization of SEAP-APPswe-EpoR in CGCs. A -C. CGCs were electroporated with 5 µg SEAP-APPswe-EpoR, the cells stained after 72 h with the SEAP antibody (A), the nuclei visualized with Höchst33342 (C) and the images overlayed (B.). D. The original fluorescence intensity from A in a Falcon 96-well-plate. E. The original fluorescence intensity of the duplicate from A in a Nunc 96-well-plate. F -H. Same as A-C but with higher transfection efficiency (40%). Scale bars: 20 µm.

105 Results

In addition to IPAD and AQD, the BACE1 inhibitors “Amgen” and “Eli Lilly” were applied on SEAP-APPswe-EpoR-transfected CGCs (Fig. 36A-C.) and all four inhibitors strongly reduced SEAP activity, more specifically the SEAPβ activity fraction. IPAD and AQD achieved BACE1 inhibition at 1 µM and generally reproduced the curves obtained with the other constructs. AQD again killed all cells at 5 µM.

A B transfected CGC transfected CGC

125 125

SD]

SD]

 

100 100

75 75

50 50

IPAD, SEAP AQD, SEAP 25 25 IPAD, MTT AQD, MTT

IPAD, Resazurin AQD, Resazurin SEAP SEAP act., MTT red., red.Resazurin

0 SEAP act., MTT red., red.Resazurin 0 [% of 0.8 mAU/min, 0.42 AU, kFU 52 [% of 0.6 mAU/min, 0.44 AU, kFU 54 -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5

DMSO inhibitor [log M] DMSO inhibitor [log M] no DMSO no DMSO

C

125 transfected CGC

SD]  100

75

of controls  50 Amgen Eli Lilly 25

SEAP SEAP activity [ 0

con. -10 -9 -8 -7 -6 -5

inhibitor [log M]

Fig. 36: Characterization of SEAP-APPswe-EpoR with BACE1 inhibitors in CGCs. A-C. CGCs were electroporated with 5 µg SEAP-APPswe-EpoR. IPAD (A), AQD (B), “Amgen” or “Eli Lilly” (both C) was administered with a medium change after 24 h and the supernatants sampled after additional 72 h. Reduction potential for MTT or Resazurin was measured directly after supernatant sampling and addition of fresh medium.

106 Results

SEAP-APPswe-EpoR-transfected HEK293 produced SEAP time- and cell number- dependently (Fig. 37A) and in these cells the four BACE1 inhibitors IPAD, AQD, “Amgen” and “Eli Lilly” decreased SEAP activity by between 50% and 75% (the SEAPβ fraction) (Fig. 34B). The ranking of their potencies was preserved with IPAD being most potent (in vitro: 15 nM), followed by AQD (in vitro: 30 nM), “Eli Lilly” (in vitro: 105 nM) and “Amgen” (in vitro: 1.5 µM). Around 25% of SEAP activity was not inhibitable and AQD decreased the MTT reduction potential by 20% at 10 µM. The α- secretase inhibitor GM6001 and the γ-secretase inhibitor LY450139 did not affect SEAP activity in the concentrations applied to transfected HEK293, thus proving monospecificity of SEAP-APPswe-EpoR for BACE1 cleavage (Fig. 37C).

A 60 SEAP-APPswe-EpoR

SD]

 100,000 45 75,000 50,000 25,000 cells per well 30

15

activitySEAP [mAU/min 0

0 6 12 18 24 time [h]

B C transfected HEK293 after 24h 125 transfected HEK293 after 24 h 150

125 100

SD] 100  75

75 IPAD 50 AQD 50 -sec.-inh. GM6001, SEAP

Amgen [% of control [% of control ±SD] -sec.-inh. GM6001, MTT 25 Eli Lilly 25 -sec.-inh. LY450139, SEAP

AQD MTT SEAP activity MTT reductionand -sec.-inh. LY450139, MTT SEAP activitySEAP MTT reductionand 0 0

con. -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5 inhibitor [log M] inhibitor [log M] Fig. 37: Characterization of SEAP-APPswe-EpoR with time-course and secretase inhibitors in HEK293. A. HEK293 were electroporated with 5 µg SEAP-APPswe-EpoR, seeded in different numbers per well, the medium was changed after 24 h and sampled after additional 24 h. B/C. HEK293 were electroporated with 5 µg SEAP-APPswe-EpoR. IPAD , AQD , “Amgen” or “Eli Lilly” (all B), LY450139 or GM6001 (both C) was administered with a medium change after 24 h and the supernatants were sampled after additional 24 h. Reduction potential for MTT was measured directly after supernatant sampling and addition of fresh medium. 107 Results

In co-electroporation both the SEAP siRNA and the APPswe siRNA achieved a measurable or rather good knockdown of the construct in CGCs (Fig. 38), depending on the control taken as reference value. This was especially true for BACE1 siRNA with either around 35% decrease in SEAP activity in comparison with a non-specific siRNA (Con) or nearly 50% in comparison with GFP siRNA (GFP).

125

100

SD] 

75 Fig. 38: Characterization of SEAP- APPswe-EpoR with siRNAs in CGCs. 50 CGCs were coelectroporated with SEAP activitySEAP 5 µg SEAP-APPswe-EpoR and 4

of mAU/min27 µM of different siRNAs, the

[% 25 medium was changed after 24 h and sampled after additional 72 h.

0

5 µg plasmid: SEAP-APPswe-EpoR 4 µM siRNA: GFP Con SEAP APPswe BACE1

In transfected HEK293, the decrease of SEAP activity due to SEAP siRNA, APPswe siRNA and BACE1 siRNA was much more efficient (Fig. 39A), as with the other constructs, and here a direct comparison of the control siRNAs revealed for the GFP siRNA an MTT reduction more similar to those from the other siRNAs than the MTT reduction from the Con siRNA and a SEAP activity more similar to siRNA-non-treated cells than the SEAP activity from the Con siRNA (Fig. 39B). The knockdown of the construct and of BACE1 was confirmed by semi-quantitative qPCR (Fig. 39C). In the case of BACE1 siRNA the level of the qPCR product, relative to GAPDH qPCR product and GFP siRNA-treated cells, was lower than the remaining level of SEAP activity. Lipofection with the SEAP siRNA and the APPswe siRNA achieved not a decrease in SEAP activity as strong as with electroporation but it was retained for the BACE1 siRNA (Fig. 39D). Also, transfection with PSEN1 siRNA did not change SEAP activity relative to the controls and thus corroborated the non-dependency of construct-cleavage on γ-secretase (Fig. 39D).

108 Results

125 MTT reduction

A 125 MTT reduction B SD]

SEAP activity  SEAP activity SD]  100 100

mAU/min 75

SEAP activitySEAP 75 107

50

50 AU and 25

25 1.25

MTT MTT reduction activitySEAP and

MTT MTT reduction and

% of mAU/min AU 64 0.47 and [ 0 [% of 0 5 µg plasmid: SEAP-APPswe-EpoR 5 µg plasmid: SEAP-APPswe-EpoR 4 µM siRNA: - GFP Con SEAP BACE1 4 µM siRNA: Con SEAP APPswe BACE1

C 125 SD: D 175 SEAP BACE1

150 0.1 µg SEAP-APPswe-EpoR

SD] SD]

 100 100 nM siRNA SEAP activity  t level 125 uc 75 100

GAPDH)ct qPCR product

 SEAP BACE1 75 50

mAU/min259 and

SEAP SEAP activity 50 SEAP SEAP activity and

GFP siRNA) 25

[% of 25



-

[% of mAU/min101 2 relative qPCR prod 0 0 5 µg plasmid: SEAP-APPswe-EpoR GFP Con SEAP BACE1 PSEN1 4 µM siRNA: GFP SEAP BACE1 APPswe no siRNA Fig. 39: Characterization of SEAP-APPswe-EpoR with siRNAs in HEK293. A-C. HEK293 were coelectroporated with 5 µg SEAP-APPswe-EpoR and 4 µM of different siRNAs, the medium changed after 24 h and sampled after additional 24 h. D. HEK293 were colipofected with 0.1 µg SEAP-APPswe-EpoR per well and 100 nM of different siRNAs, the medium changed after 24 h and sampled after additional 24 h. Detection of SEAP by western blotting using the same SEAP antibody as for immunocytochemistry was not possible (Fig. 40A). However, western blotting was not listed as application for this antibody by the manufacturer. A siRNA B siRNA APPswe GFP BACE1 SEAP - kDa APPswe GFP BACE1 SEAP -

SEAP ab 1/200 dilution GAPDH ab 1/5000 dilution

Fig. 40: Characterization of SEAP-APPswe-EpoR with siRNAs in HEK293. A/B. HEK293 were coelectroporated with 5 µg SEAP-APPswe-EpoR and 4 µM of different siRNAs, the cells lysed after 48 h and the same western blot firstly incubated with the SEAP antibody (A) and then with a GAPDH antibody (B). 109 Results

Transfected HEK293 were also administered the NF-κB-inhibitor Celastrol (Fig. 41), which was demonstrated to reduce Aβ-production in SHSY5Y cells to less than 10% remaining at 5 µM but not at 0.5 µM [445]. The effect at 5 µM was reproducible with the constructs APPswe (Fig. 41A), APP (Fig. 41B) and SEAP-APPswe-EpoR (Fig. 41C) in HEK293 and with SEAP-APPswe both Aβ concentration and SEAP activity decreased to around 50% (Fig. 41D). At 5 and 10 µM however, MTT reduction declined by exactly the same value in SEAP-APPswe- and SEAP-APPswe-EpoR- transfected HEK293. At lower concentrations on the other hand, Celastrol did not exert measurable general toxicity but rather seemed to inhibit BACE1-dependent cleavage, up to 70% with SEAP-APPswe-EpoR.

A APPswe transf. HEK293 after 24 h B APP transf. HEK293 after 24 h 1250 100

1000 75

750 50

500 conc. [pg/ml] [pg/ml]conc.

1-40 25

1-40 

 250

A A 0 0 Celastrol: - 0.5 µM 5 µM 20 µM Celastrol: - 0.5 µM 5 µM 20 µM C D transfected HEK293 after 24h 125 SD] transfected HEK293 after 24h

 125

SD]  100 100

75 75

50 50 NF-B-inh. Celastrol, A NF-B-inh. Celastrol, SEAP 25 NF-B-inh. Celastrol, SEAP NF-B-inh. Celastrol, MTT 25

SEAP SEAP activity, MTT reduction NF-B-inh. Celastrol, MTT

conc., activity,SEAP MTT reduction [% of mAU/min,90 AU 0.26

0 1-40 0

 A

DMSO -11 -10 -9 -8 -7 -6 -5 [% pg/ml,of mAU/min,2288 158 AU 0.39 DMSO -11 -10 -9 -8 -7 -6 -5 inhibitor [log M] inhibitor [log M]

Fig. 41: Characterization of different APP-constructs with the NF-κB inhibitor Celastrol in HEK293. A-D. HEK293 were electroporated with 5 µg of either APPswe (A), APP (B), SEAP-APPswe- EpoR (C) or SEAP-APPswe (D). Celastrol was administered in the concentrations indicated with a medium change 24 h after electroporation and after additional 24 h the supernatants were sampled. 110 Results

SEAP-APP-EpoR was briefly characterized in HEK293 with IPAD and AQD (Fig. 42). The effect of both inhibitors on SEAP activity was by far not as strong as with SEAP- APPswe-EpoR, more similar to the SEAP-APP-construct. However, sequencing of the plasmid confirmed the presence of the EpoR-part.

A 40

30

20

10 SEAP-APPswe-EpoR

SEAP activity [mAU/min ±SD] SEAP-APP-EpoR 0 con. -10 -9 -8 -7 -6 -5 B inhibitor IPAD [log M] 125 control

1 µM AQD SD]

 100

75 mAU/min [ 50

25 SEAP activitySEAP

0 0.2 µg plasmid: SEAP-APPswe SEAP-APP SEAP-APPswe-EpoR SEAP-APP-EpoR Fig. 42: Characterization of SEAP-APP-EpoR with BACE1 inhibitors in HEK293. A. HEK293 were electroporated with 5 µg of either SEAP-APP-EpoR or SEAP-APPswe- EpoR. IPAD was administered in the concentrations indicated with a medium change 24 h after electroporation and after additional 24 h the supernatants were sampled. B. HEK293 were lipofected with 0.2 µg per well of either SEAP-APPswe, SEAP-APP, SEAP-APPswe-EpoR or SEAP-APP-EpoR. AQD was administered in the concentrations indicated with a medium change 24 h after lipofection and after additional 24 h the supernatants were sampled.

Finally, SEAP activities normalized to Aβ concentrations from different APP- constructs yielded 256, 14, 9, 570, 65, 6 pg/mAU for SEAP-APPswe in CGCs, SEAP- APPswe in HEK293, SEAP-APP in HEK293, SEAP-APPswe-AβK16V in CGCs, SEAP-APPswe-AβK16V in HEK293 and SEAP-APP-AβK16V in HEK293, respectively.

111 Results

Stably transfected cell lines were also generated at Lundbeck. They were characterized with the BACE1 inhibitors IPAD and AQD and the γ-secretase inhibitor LY450139. APPswe-HEK293 produced different amounts of Aβ dependent on cell number and time-point of sampling, which was inhibitable with 1 µM IPAD and 1 µM LY450139 (Fig. 43A). APPswe-HEK293 developed long processes after some days in culture (Fig. 43B).

APPswe-HEK293 A 500 20,000 400 200 10,000 SD]  2,500 150 cells per well 100

50 25 [pg/ml conc. 20

1-40 1µM LY450139

 1µM IPAD

A 10

0

0 6 12 18 24 B time [h]

Fig. 43: Characterization of HEK293 stably transfected with APPswe. A. HEK293 stably transfected with APPswe were seeded in different numbers per well. IPAD or LY450139 was administered at 1 µM with a medium change 24 h after seeding and after additional 6 h and 24 h the supernatants were sampled. B. HEK293 stably transfected with APPswe after 4 d in culture with 2% FBS. 112 Results

SHSY5Y stably transfected with APPswe, SEAP-APPswe (#379), SEAP-APP (#395) and SEAP-APPswe-EpoR (#396) were administered IPAD at 2 µM and LY450139 at 1 µM. IPAD reduced Aβ and SEAP in #379 not as much as in #395, while SEAP from #396 was similarly reduced as in HEK293 transiently transfected with SEAP- APPswe-EpoR (Fig. 44A-D). LY450139 abolished Aβ-production in #379 and #395 but did not reduce SEAP activity in #396, as expected.

A APPswe-SHSY5Y B 130 20,000 150 SEAP-APPswe-SHSY5Y 10,000 (#379) after 24 h

SD] 125 2,500 A 1-40 conc.

 80 cells per well SEAP activity 100 30 30 75

conc. [pg/ml conc. 20 50

1-40 1µM IPAD conc. and SEAP activitySEAP and conc.

 10 A

1-40 25 1µM LY450139  0 A [% mAU/min] pg/ml of145 and 1050 0 0 6 12 18 24 no inh. 2 µM IPAD 1 µM LY450139 time [h]

C D 150 150 SEAP-APP-SHSY5Y SEAP-APPswe-EpoR-SHSY5Y (#395) after 24 h (#396) after 24 h 125 A conc. 125 1-40 A 1-40 conc. SEAP activity SEAP activity 100 100

75 75

50 SEAP activity 50

conc. and SEAP activitySEAP and conc. [% of mAU/min]328

1-40 25

 25 A

[% pg/mlof mAU/min] 53 294 and 0 0 no inh. 2 µM IPAD 1 µM LY450139 no inh. 2 µM IPAD 1 µM LY450139

Fig. 44: Characterization of SHSY5Y stably transfected with APP-constructs. A. SHSY5Y stably transfected with APPswe were seeded in different numbers per well. IPAD or LY450139 was administered at 1 µM with a medium change 24 h after seeding and after additional 6 h and 24 h the supernatants were sampled. B-D. SHSY5Y stably transfected with SEAP-APPswe (B), SEAP-APP (C) or SEAP-APPswe- EpoR (D) were seeded. 2 µM IPAD or 1 µM LY450139 was administered with a medium change 24 h after seeding and after additional 24 h the supernatants were sampled.

113 Results

6 Kinome screen for BACE1-affecting kinases

Before conducting the large kinome screen for BACE1-affecting kinases cotransfection experiments with APP-construct and siRNAs, especially against kinases, were necessary as preparatory test-runs to find both a suitable and economical condition. The APP-construct of choice was SEAP-APPswe-EpoR because SEAP activity was dependent on BACE1, much more than with SEAP-APP- EpoR, and SEAP activity was much higher than with SEAP-APPswe-AβK16V or SEAP-APP-AβK16V, where SEAP activity was often close to detection limit which resulted in high well-to-well-variability.

6.1 Optimization of cotransfection with SEAP-APPswe-EpoR and siRNAs

The condition to be found should account for a sufficient effect of kinase siRNAs, a parsimonious use of plasmid and the large number of siRNAs in the kinome library. For a sufficient effect of kinase siRNAs several concentrations and experiment durations were compared. Lipofectamine allowed simultaneous transfection with 12 siRNAs in contrast to the one-by-one electroporation method and was optimized by comparison of different amounts of plasmid, siRNA and lipofectamine.

114 Results

Since most of the construct-characterization was carried out with electroporation, the effect of kinase siRNAs was firstly tested with this method. siRNAs against Casein Kinase 1 isoforms δ and ε (CSNK1), Mitogen-Activated-Protein 3 Kinase Kinase Kinase (MAP3K3), and MAP2K1 Interacting Protein 1 (MAP2K1IP1) were chosen because CSNK1 was demonstrated to phosphorylate BACE1 and the other two were hits in a previous screen with APPswe and SEAP-APPswe at Lundbeck. At 4 µM siRNA had CSNK1 no effect on SEAP activity while MAP3K3 and MAP2K1IP1 strongly decreased it (Fig. 45A). Knockdowns between 25% and 60% were achieved (Fig. 45B).

125 125 SD: MAP2K1IP1

A B SD] CSNK1  SEAP

SD] 100 100 MAP3K3 

75 GAPDH)ct 75 

50 50 SEAPactivity

GFP siRNA)  25 - 25

[%of 259 mAU/min relative qPCR product level

0 [% of 2 0

GFP GFP SEAP SEAP CSNK1 CSNK1 4 µM siRNA: MAP3K3 4 µM siRNA: MAP3K3 MAP2K1IP1 MAP2K1IP1

Fig. 45: Effect of kinase siRNAs on SEAP activity and kinase mRNAs in HEK293. A/B. HEK293 were coelectroporated with 5 µg SEAP-APPswe-EpoR and 4 µM of siRNAs. 24 h after electroporation was the medium changed and sampled after additional 24 h to measure SEAP activities (A) whereas the cells were subjected to mRNA purification and the obtained cDNA to qPCR (B).

115 Results

The dependency of SEAP activity from SEAP-APPswe-EpoR-transfected HEK293 on Lipofectamine and plasmid amounts, experimental duration and siRNA concentration was examined in 96-well-plates, the format which was also to be used in the kinome screen. In a kinome screen with the same kinome siRNA library to investigate neurite outgrowth, the siRNAs were left for 72 h with the cells and used at a concentration of 50 nM [446]. A prolonged use of siRNA like this is maybe necessary to account for protein half lifes when they are unknown and which can last up to 30 h and longer. For lipofection with GFP, 0.25 µl Lipofectamine per 96-well were sufficient, which was also enough to generate conveniently high SEAP activities after 72 h and provided more buffer capacity than 0.1 µl with regards to future co-transfection with siRNA which also binds to Lipofectamine, while 0.05 µl was unsuitable (Fig. 46A). Up to 0.25 µl per well, Lipofectamine seemed to be the limiting factor because more than 40 ng plasmid per well did not result in a corresponding increase of SEAP activity. In comparison of experiment durations, samples from 0-72 h contained over twice as much SEAP activity as samples from 0-48 h (Fig. 46B).

A B

SD] 350 SD]

 350

0.25 µl Lipofectamine per well  0-72 h 300 0.1 µl " 300 0-48 h 250 0.05 µl " 250

200 200 [mAU/min [mAU/min 150 150 100 100 50 50 0 0

SEAP activity SEAP SEAP activity 0 0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 100 100 plasmid per well [ng] plasmid per well [ng]

Fig. 46: Effect of transfection conditions and duration of experiment on SEAP activity. A. HEK293 were lipofected with different amounts of Lipofectamine and SEAP-APPswe- EpoR-plasmid. Supernatants were sampled without medium change after 72 h. B. HEK293 were lipofected with 0.25 µl Lipofectamine and different amounts of SEAP- APPswe-EpoR-plasmid per well. Supernatants were sampled without medium change after 48 h or 72 h.

116 Results

A cotransfection experiment with siRNA to determine a suitable experiment duration and setup was conducted either with 100 ng plasmid and 100 nM siRNA (Fig. 47A/C) or 40 ng plasmid and 50 nM siRNA (Fig. 47B/D). Supernatants were sampled either from 24-48 h (Fig. 47A/B) or 0-72 h (Fig. 47C/D) experiments. The experiment with 24-48 h required a medium change after 24 h and was the standard scheme with transfection by electroporation. For experiments with the library, however, this experimental scheme with only 48 h total time in addition to removal of the siRNAs by the medium change also seemed not suitable, mostly because of the unknown half lifes of the kinases. Moreover, the medium change was not successful with some wells in the sense that a large fraction of the cells was accidently removed together with the medium which was not too bad with the usually ten replicates from electroporation. With duplicates at most, however, the medium change beared too much of a risk, especially for the generation of false positives. Also, hardly any difference is noticeable in comparison of 24-48 h and 0-72 h.

A 175 SEAP activity from 24-48 h B 175 SEAP activity from 24-48 h SD]

SD] 150 100 ng SEAP-APPswe-EpoR 150 40 ng SEAP-APPswe-EpoR   100 nM siRNA 50 nM siRNA 125 125

100 100 75 75

50 50 SEAP activitySEAP activitySEAP 25 25

[% ofmAU/min 101 0 [% of mAU/min48.5 0

GFP Con GFP Con SEAP SEAP BACE1PSEN1CSNK1 BACE1PSEN1CSNK1 APPswe MAP3K3 APPswe no siRNA no siRNA MAP3K3 MAP2K1IP1 MAP2K1IP1

C 175 D 175 SEAP activity from 0-72 h

SEAP activity from 0-72 h SD]

SD] 150 100 ng SEAP-APPswe-EpoR 150 40 ng SEAP-APPswe-EpoR   100 nM siRNA 50 nM siRNA 125 125 100 100 75 75 50

50 activitySEAP activitySEAP

25 25 [% ofmAU/min 117 [% ofmAU/min 151 0 0

GFP Con GFP Con SEAP SEAP BACE1PSEN1CSNK1 BACE1PSEN1CSNK1 APPswe MAP3K3 APPswe MAP3K3 no siRNA no siRNA MAP2K1IP1 MAP2K1IP1 Fig. 47: Effect of transfection conditions and duration of experiment with siRNAs. A-D. HEK293 were colipofected either with 100 ng SEAP-APPswe-EpoR and 100 nM siRNA (A/C) or 40 ng SEAP-APPswe-EpoR and 50 nM siRNA (B/D). Supernatants were either sampled 24 h after a medium change (24 h after lipofection) (A/B) or without a medium change after 72 h (C/D).

117 Results

As alternative to Lipofectamine2000, HiPerfect was tested which was developed primarily for transfection of siRNA and not for plasmids which explains the low SEAP activities (Fig. 48), especially when not co-transfected with siRNA. The quite similar SEAP activities from the different siRNAs clearly precluded use of the transfection reagent in cotransfection with the plasmids.

A B

SD]

SD]  SEAP activity from 24-48 h  SEAP activity from 0-48 h 150 150

100 100

50 [% of mAU/min28 50 [% of mAU/min12.7

0 0

GFP GFP SEAP SEAP activity SEAP PSEN1 BACE1 PSEN1 SEAP activitySEAP BACE1 no siRNA no siRNA MAP2K1IP1 MAP2K1IP1 50 ng SEAP-APPswe-EpoR 100 ng SEAP-APPswe-EpoR 100 nM siRNA 100 nM siRNA

Fig. 48: Transfection with HiPerfect instead of Lipofectamine. A/B. HEK293 were colipofected either with 50 ng SEAP-APPswe-EpoR and 100 nM siRNA (A) or 100 ng SEAP-APPswe-EpoR and 100 nM siRNA (B). Supernatants were either sampled 24 h after a medium change (24 h after lipofection) (A) or without a medium change after 48 h (B).

118 Results

Further experiments to try less plasmid amounts and siRNA concentrations were carried out in order to minimize potential future off-target effects from siRNAs. Comparisons with 30 or 10 ng plasmid in combination with 50 or 10 nM siRNA and 100 or 160 µl final volume favored the condition with 30 ng plasmid, 50 nM siRNA and 100 µl final volume because it achieved the third highest Z-factor (0.88) and had much more total SEAP activity than the condition with the second highest Z-factor (0.93) and a higher ratio of GFP siRNA-SEAP activity / SEAP siRNA-SEAP activity than the condition with the highest Z-factor (0.99) (Fig. 49).

100 Condition plasmid [ng] siRNA [nM] final vol. [µL] Z factor

1 30 50 100 0.88

2 10 50 100 0.77 3 30 50 160 -0.06 75 4 10 50 160 0.93

SD] 5 30 10 100 0.99

 6 10 10 100 0.82 7 10 10 160 0.85

50

SEAP activitySEAP [mAU/min 25

0

1-no 2-no 3-no 4-no 5-no 6-no 7-no 1-GFP 2-GFP 3-GFP 4-GFP 5-GFP 6-GFP 7-GFP 1-SEAP 2-SEAP 3-SEAP 4-SEAP 5-SEAP 6-SEAP 7-SEAP

Fig. 49: Colipofection with different conditions. HEK293 were colipofected under different conditions (table insert) without siRNA or with GFP siRNA or SEAP siRNA (left, middle and right bars). Supernatants were sampled after 72 h without a medium change.

119 Results

An experiment with more kinase siRNAs demonstrated clearly a stronger effect on SEAP activity with 50 nM than with 5 or 10 nM for 8 out of a total of 19 siRNAs (Fig. 50A/B), among them also APPswe siRNA (Fig. 50A).

A 125

SD] left: 5 nM  middle: 10 nM 100 right: 50 nM

75

50

activity SEAP 25

0 mAU/min 84 and 104 115, [%of GFP Con SEAP BACE1 PSEN1 APPswe BACE1Kao3 BACE1Singer6

B

125 left: 5 nM SD]

 middle: 10 nM 100 right: 50 nM

75

50

activity SEAP 25

0 mAU/min 84 and 104 115, [% of BCR PFKM CSNK1 IRAK3 STK10 MAP3K3 PRKCA MAP2K1IP1 MAP3K3#2 MAP2K1IP1#2MAP2K1IP1#3

Fig. 50: Colipofection with 5, 10 or 50 nM siRNA. A/B. HEK293 were colipofected with 30 ng SEAP-APPswe-EpoR and either 5, 10 or 50 nM siRNA (left, middle and right bars). Supernatants were sampled after 72 h without a medium change. 115, 104 and 84 mAU/min refer to 5, 10 and 50 nM, respectively.

120 Results

Four of the kinase siRNAs, two with a large difference (MAP2K1IP1#2, PFKM) and two without (IRAK3, MAP2K1), were used along with GFP siRNA and Con siRNA to generate a dose-effect-curve (Fig. 51). The large difference in SEAP activity from 10 to 50 nM was reproduced for MAP2K1IP1#2 and PFKM, and was also measurable for IRAK3, while MAP2K1IP1 displayed a quite uniform slope with the equidistant plotting applied on the data points. Since a decrease in SEAP activity of around 50% was sufficient to raise interest, 100 or 200 nM was not necessary and was assumed to raise merely the risk of off-target effects. 125

SD] 100 

75

[mAU/min 50

25 GFP PFKM

SEAP SEAP activity Con MAP2K1IP1#2

0 IRAK3 MAP2K1IP1

1 5 10 50 0.1 0.5 100 200 siRNA [nM] Fig. 51: Colipofection with different concentrations of siRNA. HEK293 were colipofected with 30 ng SEAP-APPswe -EpoR and the siRNA concentrations as indicated. Supernatants were sampled after 72 h without a medium change.

The search for kinase affecting BACE1-dependent cleavage of APP was planned to start with the remaining SEAP activity from SEAP-APPswe-EpoR and then to examine the remaining Aβ concentration. Since some well-to-well-variability in transfection efficiency existed, identical conditions for transfection of both constructs should be realized by co-transfection with kinase-siRNA and both SEAP-APPswe- EpoR and APPswe. At equal amounts of 20 ng per plasmid, Aβ was only measurable from samples where cells were cotransfected with SEAP siRNA (data not shown). At 5 ng SEAP-APPswe-EpoR and 40 ng APPswe both readouts were satisfactory and used in the kinome screen.

121 Results

Another factor which was examined was the dependency of SEAP activity on the position in the 96-well-plate. It is known that differences exist between the outermost wells, especially the corner wells, and the inner wells with regards to evaporation. This is probably due to the conductance of heat via the plastic from the walls which stand on the metal plate to the outer and then to the inner wells. This effect was seen by thawing samples in a 96-well-plate in a 37°C incubator and resulted in completely thawed 36 outermost and still frozen 60 inner samples. When a 96-well-plate filled with 100 µl PBS in each well was placed on an additional metal plate of the right size such that all wells were in direct contact with the metal but the plastic walls of the plate not anymore, evaporation was equal and about 40 µl per day in a 37°C- incubator. Also, HEK293 reached confluency always fastest along the ridges on which the culture flask stands on the metal plate. In order to test the effect of well position on SEAP activity HEK293 in differently positioned wells were transfected with the same amounts of SEAP-APPswe-EpoR- plasmid. This was carried out with the same experiment which demonstrated dependency of SEAP activity on plasmid amounts (Fig. 46A). Of the four replicates of each plasmid the outer wells had the highest SEAP activity (Fig. 52). With four of the eight amounts (10, 20, 30, 50 ng) the difference between the second and the third outermost row was not large, so that the 60 inner wells could be used for the kinome screen. replicates 1-4 of 5 ng plasmid per well

plas. [ng] 5 10 20 30 40 50 75 100

SEAP act. 71 176 268 286 361 326 368 382 A

31 137 200 250 321 261 333 346 B

32 143 223 237 288 266 291 267 C

69 153 190 237 257 236 210 262 D

1 2 3 4 5 6 7 8 9 10 11 12

...

Fig. 52: Dependency of SEAP activity on plasmid amount and well position in a 96-well-plate. HEK293 were lipofected with different amounts of SEAP-APPswe-EpoR. Supernatants were sampled after 72 h without a medium change.

For one 96-well-plate of the library, two 96-well-plates with cells were therefore used, more specifically, two times two for duplicates in co-transfection of siRNA, SEAP- APPswe-EpoR and APPswe and two for co-transfection of siRNA and SEAP- APPswe as described in detail in the material and methods section.

122 Results

6.2 Analysis and validation of screen results

The results acquired during the optimization experiments led to the following screening strategy which was applied to 1357 siRNAs of the kinome siRNA library. The screen was conducted as siRNA-plasmid cotransfection. Due to some well-to- well-variability in transfection efficiency, which demanded identical conditions for the primary SEAP-activity readout and the secondary but decisive Aβ-readout, HEK293 were co-transfected with kinase-siRNA and both SEAP-APPswe-EpoR and APPswe to search for kinase hits (samples: hit search), and this in duplicate wells for reduction of random hits. The control-well was co-transfected with kinase-siRNA and SEAP-APPswe (samples: hit control). SEAP-APPswe was used in the screen as control to exclude general effects of the kinase-siRNAs on viability, protein synthesis, trafficking and others, thus removing false-positive hits. SEAP-activities from samples of hit search and hit control were measured simultaneously with the same pNPP substrate solution. Kinase hits identified and validated in the HEK293 screen would then further be validated and characterized in CGCs. Analysis of the HEK293 screen for the identification of kinase hits was divided in three levels with different filters in order to increase the likelihood for true hits and to decrease it for false hits to end up in the dataset with identified hits (Fig. 53). O SEAP-APPswe-EpoR, 5 ng O SEAP-APPswe, 20 ng O APPswe, 40 ng + +

= siRNA 50 nM, Kinase X = siRNA 50 nM, Kinase X = siRNA 50 nM, Kinase Y = siRNA 50 nM, Kinase Y 2x = siRNA 50 nM, Kinase Z 1x = siRNA 50 nM, Kinase Z for duplicates

96-well-plate 96 - well -plate 75000 HEK293 / well 75000 HEK293 / well

Supernatants sampled and frozen after 72 h Hit identification ───────────────────────────────────────── Measurement 1: SEAPβ activity SEAPα activity Data analysis 1: activity ≤ 81% of average?

Data analysis 2: 1. level hit activity ≥ 65% of average? Measurement 2: Aβ concentration 2. level hit Data analysis 3: conc. ≤ 90% of neighboring wells? Hit validation of: 3. level hits ─────────────────────────────── Repetition with validated siRNAs and measurement of remaining mRNA-level

Fig. 53: Screening strategy. Circles represent plasmids, parallel lines siRNAs. 123 Results

The hits from the kinome siRNA library added up to 190 first level hits (14% from 1357 siRNAs), 93 second level hits (6.9%) and 38 third level hits (2.8%) (Tab. 4 below).

hit search: Hit control: hit search: plate no. SEAP activity: SEAP activity: Aβ40: <81% >65% <90% 4 11 7 4 5 18 6 6 6 14 8 1 8 14 7 0 9 16 11 1 12 19 14 5 14 11 3 0 15 25 9 5 17 13 4 3 18 15 7 7 21 8 4 2 22 6 3 1 23 3 1 0 24 6 3 1 25 6 3 0 26 5 3 2 siRNAs: 1357 190 93 38

Third-level-hits were no longer handled as mere siRNA-identifier but as targeted kinase. One reason was to compare them with hits from a previous screen by Lundbeck using SEAP-APPswe. The hit siRNAs against IRAK3 and MAP2K1IP1 were the same hit siRNAs as in the previous screen by Lundbeck, therefore not new hits, and the same siRNAs which were used for screen optimization. However, the hit siRNA against MAP3K3 in the Lundbeck screen and used for screen optimization achieved 84% and 90% remaining SEAP activities from SEAP-APPswe-EpoR and SEAP- APPswe and was therefore not a hit. Another reason was to check whether some hit siRNAs targeted the same kinase. Against each kinase usually three siRNAs were used in the screen and the occurrence of two or more hit siRNAs against the same kinase would be a meaningful hint. Actually, two siRNAs indeed targeted PI3K. However, one was against regulatory subunit 2 and one against catalytic subunit 2α and in total 37

124 Results siRNAs targeted PI3K. The random chance for two hits from this group of 37 was calculated by (37/1357)∙(36/1357) to 0.072% which is 218fold higher than the random chance double hit for most other kinases with (3/1357)∙(2/1357)=0.00033% but still 70times lower than their actual contribution (2/39=5%) to the hit list. The only other double hit, and targeted by the usual three kinases, was PRKAA2: one from this screen and one from the parallel screen by Dr. Yana Chernyshova with the remaining 885 siRNAs of the library. Further, the relevance of the kinases in AD or neurons was looked up in the literature. Third-level-hits had average remaining values of 59.5%, 90.0% and 54.3% for SEAP activity from SEAP-APPswe-EpoR, SEAP activity from SEAP-APPswe and Aβ concentration from APPswe, respectively (Tab. 5 below).

name plate well hit search: SEAP hit control: SEAP hit search: Aβ40 PRKAA2 4 4c 70.8 82.5 55.3 CHEK2 4 7f 62.2 91.5 54.2 MKNK1 4 7h 45.5 64.8 64.6 STK29 4 8d 59.9 65.6 39.8 TRAD 5 1c 62.2 78.4 88.3 VRK3 5 4d 51.9 100.4 95.5 PRPF4B 5 6h 38.2 74.6 47.2 VRK2 5 8d 41.6 66.7 95.1 TSSK1B 5 9b 45.2 71.2 22.5 CSNK1D 5 10c 65.1 91.6 45.3 MAPK6 6 8b 68.3 86.7 70.6 EPHA4 9 3c 71.9 86.5 81.8 ZAK 12 3h 31.7 90.1 30.2 RIPK4 12 4e 43.5 109.8 25.4 IRAK3 12 7b 72.0 94.5 70.6 KIAA1804 12 9b 73.5 100.0 72.8 RIPK3 12 9g 41.7 104.3 25.0 MOS 15 b1 39.5 70.2 51.2 ERN2 15 3g 80.7 95.0 31.7 PRKWNK1 15 7c 73.0 98.2 58.0 TEX14 15 11d 67.9 81.2 46.3 FN3KRP 17 3f 40.0 78.1 24.7 TLK1 17 4b 59.4 119.6 33.2 FN3K 17 8f 47.1 67.7 32.3 SEPHS1 18 3e 73.6 105.4 63.6 AK3 18 5f 74.8 100.4 53.9 PIK4CB 18 6c 36.4 66.0 50.5 PRKRA 18 6d 59.3 102.6 82.8 CDADC1 18 8a 71.8 108.9 69.3 UGP2 18 9e 57.4 88.6 70.2 CSF1R 18 9g 61.5 126.0 85.8 MPP5 21 1g 71.3 95.5 49.4 PKLR 21 3b 46.8 76.4 20.1 SPHK1 21 3c 67.2 87.2 52.8 MPP7 22 2a 71.1 134.1 53.3 MAP2K1IP1 22 11f 55.8 67.6 n.a. PI3KR2 24 4f 73.2 92.4 50.5 PHKB 26 1a 65.3 91.5 59.2 PI3KC2A 26 3a 80.8 99.3 41.1 average 59.5 90.0 54.3

125 Results

In order to test reproducibility and validity of these results three hits were selected for repetition of the experiments with examination of the knockdown on mRNA level. The selected hits were STK29, TRAD and CSNK1D. All three were interesting with regards to relevance in neurons or AD and their hit profiles were different. STK29 had a SEAP activity from SEAP-APPswe-EpoR of 59.9%, a not much higher SEAP activity from SEAP-APPswe of 65.6% and a remaining Aβ concentration of 39.8%. On the other hand, the SAD kinase STK29 was interesting because it is required in forebrain neurons to acquire polarity and endow axon and dendrites with distinct properties which is promoted by phosphorylation of Tau [447]. The hit profile from TRAD with 62.2%, 78.4% and 88.3% had a higher SEAP activity from SEAP-APPswe (78.4%) but also a very high remaining Aβ-level (88.3%). It was interesting because it was most consistently underexpressed in AD-hippocampus [448]. CSNK1D had a hit profile more or less as expected (65.1%, 91.6%, 45.3%) and was interesting because CSNK1 was demonstrated to phosphorylate BACE1 [92] and to be upregulated in AD hippocampus, 30fold on protein level [299] and 24fold on mRNA level [300]. For the repetition experiments (Fig. 51A) siRNAs with the same sequences were reordered, so that the library was not used unequally. Remaining SEAP activity and Aβ concentration with CSNK1D siRNA were similar (47.5%, 42.2%) to the screen results (65.1%, 45.3%) whereas with STK29 siRNA repetition results (96.3%, 112.8%) differed strongly from screen results (59.9%, 39.8%), even more than with TRAD siRNA (75.9%, 106.4% vs. 62.2%, 88.3%). qPCR product levels from cDNA generated from mRNA samples were analyzed according to the ΔΔct method where the difference in ct (cycle of threshold) between gene of interest and housekeeping gene (here GAPDH) was taken first and then from this difference the difference in ct of the housekeeping gene of kinase siRNA sample minus GFP siRNA sample was subtracted. The CSNK1D siRNA did not knockdown the related CSNK1E but also not CSNK1D (Fig. 51B), assuming that qPCR product levels and mRNA levels correlate to each other. Strangely, the qPCR product levels obtained with a primer for SEAP and one for APP were much higher (80fold and 20fold) in the CSNK1D siRNA sample than in the GFP siRNA sample. The most likely explanation is contamination of the purified mRNA from CSNK1D siRNA treated cells with SEAP-APPswe-EpoR- and APPswe-plasmid. The difference in ct-values of APP and SEAP was 2.93 which is the same as 7.62fold more APP than SEAP,

126 Results hinting on the amounts of plasmids cotransfected: 40 ng APPswe and 5 ng SEAP- APPswe-EpoR. The TRAD siRNA did not knockdown TRAD and, as with CSNK1D, also not BACE1 (Fig. 54C). SEAP- and APP-qPCR product levels are similar as with GFP siRNA and the difference of their ct-values was 2.78 which is the same as 6.87fold more APP than SEAP. The qPCR product levels of Actin B, another housekeeping gene, were also measured and its ct-value from the GFP siRNA sample was 27 in comparison with 20 for GAPDH and also 27 for CSNK1D.

A 125 black bar: APPswe white bar: SEAP-APPswe-EpoR

100

75

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conc. and SEAP activity SEAP and conc.

25 1-40

 A

[% of 15 pg/ml and 81 mAU/min±SD] 81 and pg/ml 15 [%of 0 GFP CSNK1D STK29 TRAD

qPCR data from CSNK1D-siRNA B C qPCR data from TRAD-siRNA

ct] 8200

ct] 200  2250 

SD, 300 150 SD,

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100 50 qPCR product levels qPCR

product levels qPCR

of GFP-siRNA of GFP-siRNA 0 0 [% Actin B SEAP APP BACE1 CSNK1D CSNK1E [% Actin B SEAP APP BACE1 TRAD primer primer

Fig. 54: Effects of hit siRNAs in repetition experiment. A. HEK293 were cotransfected with 40 ng APPswe, 5 ng SEAP-APPswe-EpoR and 50 nM of siRNAs and the medium sampled after 72 h. B/C. The CSNK1D siRNA and TRAD siRNA transfected cells from A were subjected to mRNA purification and the obtained cDNA to qPCR. The same ct-value for an abundant housekeeping gene as for a kinase seemed strange, like the “upregulation” of CSNK1D in CSNK1D siRNA-treated cells. Therefore, cDNA synthesis and qPCR were repeated, however, under differing conditions. The usual condition with the usage of random hexamer primers for cDNA synthesis was compared with one where only a specific reverse primer was used, thus excluding random binding efficiencies to the different mRNAs. In order to have

127 Results identical conditions during cDNA synthesis for all mRNAs of interest, all the specific reverse primers were mixed to result in same final concentrations. As another modification to the usual cDNA synthesis the usual duration of 50 min was compared with shortened reaction durations of 15 and 5 min. Reverse transcriptase from Avian Myoblastosis Virus (AMV) [449] is known to possess not only 5‟-3‟ RNA-directed DNA polymerase activity but also 5‟-3‟ DNA-directed DNA polymerase activity in addition to RNase H activity [450]. The actual reverse transcriptase used in this project, Invitrogen Superscript II, is also RNA- and DNA- directed but is engineered to possess 106fold reduced RNase H activity (product description). The DNA-directed DNA polymerase activity would lead to additional cDNA synthesis, once cDNA is present. It would maybe result in a PCR in which each synthesized cDNA is immediately used as template. This might distort the correlation from e.g. CSNK1D mRNA to CSNK1D cDNA when those of the random hexamer primers which bind to the sequence are close to being used up after 50 min of PCR with the GFP siRNA sample but not yet with the CSNK1D siRNA sample and thus gives CSNK1D cDNA from the CSNK1D siRNA sample a chance to “catch up” with CSNK1D cDNA from the GFP siRNA sample. The use of the specific reverse primer only without the specific forward primer prevents a PCR. In comparison of random hexamer primers and specific reverse primer only with the same mRNA sample, the ct-values with specific reverse primer were 3-5 cycles lower (Fig. 55A/B). For Actin B this difference was nearly 6 cycles and importantly, the ct- values of Actin B were lower than those of CSNK1D whereas they were again higher with random hexamer primers at 50 min of cDNA synthesis. After 5 min of cDNA synthesis, Actin B and CSNK1D from GFP siRNA and CSNK1D siRNA samples had a very similar ct-value of around 30.5 with random hexamer primers in contrast to distinct ct-values already after 5 min with specific reverse primer. When the ΔΔct-calculation was applied to assess knockdown of CSNK1D, the results were not dramatically different between the different types of primers (Fig. 55C/D). However, whereas with random hexamer primers the levels from CSNK1D siRNA- treated cells seemed to catch up with the levels from GFP siRNA-treated cells with longer cDNA synthesis duration, the remaining levels decreased slightly with specific reverse primer and longer cDNA synthesis duration. The knockdown for CSNK1D was at best between 50% and 75%.

128 Results

A cDNA synthesis with random hexamer primers 35 left bars: 50 min cDNA synthesis 34 middle bars: 15 min " 33 right bars: 5 min " 31.0 GFP siRNA CSNK1D siRNA 30.5

30.0

29.5 29.0

28.5 cycle of threshold- #]threshold- cycleof

[

productqPCR of Detection 28.0 27.5 ActB CSNK1E CSNK1D CSNK1D

cDNA synthesis with specific reverse primer only B 32.0 left bars: 50 min cDNA synthesis 30.5 middle bars: 15 min " 29.0 right bars: 5 min " 27.0 GFP siRNA CSNK1D siRNA 26.5 26.0 25.5 25.0

24.5 cycle of threshold-#] cycleof

[ 24.0

productqPCR of Detection 23.5 23.0 ActB CSNK1E CSNK1D CSNK1D C D random hexamer primers specific reverse primer 400 800 left bars: 50 min cDNA s. left bars: 50 min cDNA s.

middle bars: 15 min " middle bars: 15 min " ct] ct] 300

400 right bars: 5 min " right bars: 5 min "   300 200

SD,

SD, 150 120   100 100 75 75 50 50 X

25 25

[% of GFP-siRNA [% of GFP-siRNA 0 0

CSNK1E CSNK1D CSNK1E CSNK1D

qPCR qPCR product levels of CSNK1D siRNA treated qPCR product qPCR oflevels siRNA CSNK1D treated

Fig. 55: Different conditions for cDNA synthesis. A/B. Purified mRNA from Fig. was used for 5, 15 and 50 min of cDNA synthesis with random hexamer primers (A) or specific reverse primers only (B). C/D. qPCR product levels from CSNK1E cDNA and CSNK1D cDNA of CSNK1D siRNA transfected HEK293 in comparison with GFP siRNA transfected HEK293.

129 Results

In a first attempt to prioritize the hits they were ranked according to their deviation of their screen results from the hypothetical ideal hit profile with 0% remaining SEAP activity from SEAP-APPswe-EpoR, 80% remaining SEAP activity from SEAP- APPswe and 0% remaining Aβ concentration. So, for each kinase hit the difference of each value (converted from % to fraction of 1) minus the ideal value (as fraction of 1) was calculated and the three differences used to calculate the distance from the ideal hit as length of a vector in a space with the three dimensions SEAP from SEAP- APPswe-EpoR, SEAP from SEAP-APPswe and Aβ from APPswe by taking the square root of the added squares of the differences (Tab. 6 below). ranking formula: ______√(SEAP -APPswe-EpoR%/100 - 0)2 + (SEAP-APPswe%/100 - 0.8)2 + (APPswe%/100 - 0)2 Rank Kinase Rank Kinase 1 ZAK 20 PHKB 2 FN3KRP 21 PIK3R2 3 PKLR 22 PRKAA2 4 TSSK1B 23 UGP2 5 RIPK3 24 PIK3C2A 6 FN3K 25 AK3 7 RIPK4 26 PRKWNK1 8 PRPF4B 27 MAPK6 9 PIK4CB 28 SEPHS1 10 MOS 29 IRAK3 11 STK29 30 CDADC1 12 TLK1 31 MPP7 13 CSNK1D 32 PRKRA 14 MKNK1 33 VRK2 15 TEX14 34 KIAA1804 16 CHEK2 35 TRAD 17 SPHK1 36 EPHA4 18 ERN2 37 VRK3 19 MPP5 38 CSF1R

130 Results

However, by this system STK29 was ranked higher than CSNK1D whereas by remaining SEAP activity and Aβ concentration in the repetition experiment, CSNK1D was closer to a true hit than STK29 and also TRAD. In comparison of the SEAP and Aβ values from the screen, the profile of CSNK1D siRNA had a higher remaining SEAP activity with the control SEAP-APPswe than the other two profiles or rather a higher difference between SEAP activity from SEAP- APPswe and the other two values. Also, in the end, decrease of Aβ was assumed to be more important than decrease of SEAP from SEAP-APPswe-EpoR. With the ranking formula changed accordingly, the rank of CSNK1D was much higher than that of STK29 while most of the high ranking kinases stayed (Tab. 7 below). ranking formula: - (SEAP-APPswe-EpoR% - 0%) + (SEAP-APPswe% - 80%) - (APPswe% - 0%) +(SEAP-APPswe% - SEAP-APPswe-EpoR%) + (SEAP-APPswe% - APPswe%) +(SEAP -APPswe-EpoR% - APPswe%) Rank Kinase Rank Kinase

1 RIPK4 20 CDADC1 2 TLK1 21 PRPF4B 3 RIPK3 22 TEX14 4 MPP7 23 SPHK1 5 ZAK 24 PHKB 6 PKLR 25 MOS 7 FN3KRP 26 STK29 8 ERN2 27 PRKAA2 9 TSSK1B 28 PIK4CB 10 PIK3C2A 29 KIAA1804 11 CSNK1D 30 PRKRA 12 MPP5 31 IRAK3 13 AK3 32 UGP2 14 FN3K 33 MAPK6 15 CSF1R 34 VRK3 16 PIK3R2 35 MKNK1 17 SEPHS1 36 EPHA4 18 CHEK2 37 TRAD 19 PRKWNK1 38 VRK2

131 Results

In order to corroborate the effect of the kinases on reduction of Aβ concentration and SEAP activity APPswe, SEAP-APPswe or SEAP-APPswe-EpoR were cotransfected with siRNAs which have been demonstrated to achieve at 25 nM more than 70% knockdown on mRNA level in HEK293 cells [451]. The kinases chosen for this further test were in total 19 – roughly the upper half of the ranking and additionally some to substitute for higher ranking hits where no validated siRNA was available. Along with these siRNAs, GFP siRNA with the same sequence as in all other experiments was ordered to assure identical conditions regarding the stock solution, dilution steps and so on. The experiment with these siRNAs was conducted twice and hardly any reduction of Aβ concentration and SEAP activity was measured in comparison with GFP siRNA (Fig. 56). Also, the outcomes of the two independent experiments were very similar which is demonstrated by the quite low standard deviations from the pooled data. The Aβ concentration and SEAP activity from APPswe and SEAP- APPswe-EpoR was reduced by ZAK, PKLR and TSSK3, however paralleled by SEAP activity from SEAP-APPswe. ERN2, CSNK1D and PIK4CB on the other hand selectively reduced Aβ from APPswe, however only by 10-15% compared to GFP.

150

. 100

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0 siRNA: GFP PKLR FN3KRP ERN2 TSSK1B PIK3C2A

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conc. andSEAP activity

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- 1 50 Aβ

[%pg/ml of 200 and 51 mAU/min ±SD] 0 siRNA: GFP CHEK2 PRPF4B MOS PIK4CB TSSK3 Fig. 53: Validated siRNAs against hit kinases from the screen. HEK293 were colipofected with either 40 ng APPswe and 5 ng SEAP-APPswe-EpoR or 20 ng SEAP-APPswe and 50 nM of the siRNAs and the medium was sampled after 72 h. 132 Results

In the case of CSNK1D, PI3K, p38-MAPK and JNK, inhibitors were also used to test the effect of kinase-inhibition on β-cleavage. The highly specific CSNK1 inhibitor PF 670462 [430] with an IC50 of 14 nM for CSNK1D and an IC50 of 7.7 nM for CSNK1E was administered at 100 nM which resulted in 13% less SEAP activity from SEAP- APPswe-EpoR and 5% less Aβ40 from APPswe. Inhibitors for PI3K, p38-MAPK which was associated to AD [452] and amyloid deposition [453] and attenuated by IRAK3 [454], and, not linked to results of the screen, for JNK which phosphorylated APP [294] and JIP1 to activate the JNK module [325], were administered to SEAP-APPswe-EpoR transfected HEK293 and did not reduce SEAP activity up to 1 µM (Fig. 57). A. B 150 125

125 100 100 75 75 50 50

LY294002, SEAP 25 25 p38 inh., SEAP LY294002, MTT

p38 inh., MTT

SEAP SEAP activity MTT and reduction SEAP activitySEAP MTT reduction and

[% of mAU/min65 0.55 AU and ±SD] 0 [% of mAU/min82 0.4 AU and ±SD] 0 DMSO -11 -10 -9 -8 -7 -6 -10 -9 -8 -7 -6 -5 inhibitor [log M] inhibitor [log M] C 150

125

100 75 Fig. 57: Effect of inhibition of PI3K, p38- MAPK and JNK on SEAP activity. 50 HEK293 were electroporated with 5 µg SEAP-APPswe-EpoR. LY294002 (A), p38- 25 JNK inh. II, SEAP MAPK-inhibitor (B) or JNK-inhibitor II (C) JNK inh. II, MTT was administered at the concentrations

SEAP activity MTT and reduction 0 indicated with a medium change 24 h after [% of mAU/min90 0.42 AU and ±SD] electroporation and after additional 24 h DMSO -11 -10 -9 -8 -7 -6 the medium was sampled. inhibitor [log M]

Since all initial hits from this screen dropped out in the validation experiments and the previous hits from Lundbeck A/S seemed questionable, no mice were sacrificed for further experiments with CGCs.

133 Results

The validated RIPK4 siRNA seemed to upregulate SEAP activity from SEAP- APPswe (Fig. 56), maybe via upregulation of α-cleavage. In order to verify or falsify this effect, the same siRNA was ordered from another company together with a non- hit- and the hit-siRNA against RIPK4 used in the screen. The validated RIPK4 siRNA showed then no upregulation of SEAP activity from SEAP-APPswe or downregulation of SEAP activity from SEAP-APPswe-EpoR in comparison with the RIPK4 non-hit siRNA (Fig. 58). 125 left bars: SEAP-APPswe right bars: SEAP-APPswe-EpoR Fig. 58: Test for upregulation 100 of SEAP activity by validated RIPK4 siRNA. 75 HEK293 were colipofected with either 5 ng SEAP- 50 APPswe-EpoR or 20 ng activity SEAP SEAP-APPswe and 50 nM of 25 the siRNAs. The medium

was sampled after 72 h. % of 9.4 and 31 mAU/min ±SD] and31mAU/min 9.4 of % [ 0 siRNA: non-hit RIPK4 hit RIPK4 validated RIPK4 Kinase siRNAs upregulating SEAP from SEAP-APPswe in the screen are listed in Tab. 8 below. Kinase names upregulating SEAP to values higher than 170%, in the order of their appearance in Tab. 8, are: MGC4796, PRKCN, GSK3B, AMHR2, ZAK, CABC1, PXK, WEE1, RNASEL, CDKN1A, PI4KII, PNKP, PRKAR2A, AK5.

SEAP activity: plate no. SEAP activity: >140% total >170% 4 5a, 11f 4e, 6c 4 5 3g, 4g, 8c 3 6 11f 7f 2 8 2b 1 9 6c, 10c 2 12 4c, 4f, 7e, 9c 5a, 5d 6 14 3c, 3e, 4e 3d, 4b, 9h 6 15 3f, 5c 2 17 2b, 4e, 4f, 5b, 6e, 6g, 9g, 10f 1a, 11c 10 3e, 3f, 4e, 4f, 5c, 7d, 8c, 8e, 18 4c, 4d, 5d, 7f 15 9d, 10c, 10e 21 6c, 6e, 6f, 6g, 7h, 8d 6 22 1e, 9e, 10b 3 23 4a 1 24 2c, 3b, 11c 3 25 2g, 10c 2 26 0 siRNA: 1357 52 14 66

134 Results

Kinase siRNAs upregulating SEAP from SEAP-APPswe-EpoR in the screen are listed in Tab. 9 below. Kinase names upregulating SEAP to values higher than 150%, in the order of their appearance in Tab. 9, are: LOC91807, PCTK1, CDKL2, ERK8, MAPK7, BLK, ERBB3, LMTK2, STK39, FER, ALK, TNNI3K, TGFBR1, BMPR1A, TTK, ADK, NME7, MMAA, IKBKB.

plate no. SEAP activity: >130% SEAP activity: >150% total

1a, 4d, 8f, 8g, 9a, 10f, 4 8 11c, 11d 2f, 4g, 5g, 6a, 6e, 6g, 5 7c 10 7d, 7f, 8f 2d, 2g, 3b, 4c, 4d, 5a, 6 2c, 2e, 3a, 9b 11 11a 8 7h 1 2a, 3f, 4b, 4c, 8a, 10d, 9 2b, 2d, 2f, 3a, 4d, 11a 13 11c 12 5f, 6a, 6d, 6f, 7h 1c, 1h, 8a 8 14 4c 1 1c, 2a, 2d, 2e, 4d, 4g, 15 4e 10 9c, 10c, 11b 17 1c, 4a, 6a, 11a 2c 5 18 1c, 1h, 4a, 5g, 6a, 8g 7a 7 21 5h, 10h, 11a 8h 4 22 7d 1 23 4h 1 24 1b, 1c, 2g, 9a 5a 5 25 1h, 6f 2 26 2b, 7a 2 siRNA: 1357 70 19 89

135 Results

Kinase siRNAs downregulating SEAP from SEAP-APPswe in the screen are listed in Tab. 10 below. They were mostly located outermost of the inner 60 wells in 96-well- plates, indicated by column numbers 1, 5, 6 and 11 and hinting on an effect on SEAP production due to position in plate.

SEAP activity: plate no. SEAP activity: <75% total <50% 4 1a, 1b, 2d, 3a, 5h, 6a, 10b 6b 8 5 1a, 1b, 3h, 4b, 5c, 7h, 11d 9h, 11e 9 6 6c, 8c, 8e, 8g, 10g, 11a 6 8 1b, 2g, 6b, 9g 4 9 1c, 1g, 3h, 4e, 10g, 11g 6 1e, 1f, 1g, 1h, 2a, 2e, 2f, 3h, 4a, 4h, 5g, 12 2h, 8h, 10h 22 6h, 8e, 8g, 9h, 10g, 11d, 11g, 11h 14 1b, 1e, 1h, 3a, 5h, 6a, 6c, 8g, 11d, 11g 1ª 11 15 4b, 6h 6a, 6b 4 17 2c, 2g, 4c, 5h, 6f, 6h, 8h, 9a, 10h, 11f 6b, 6c, 6d 13 1d, 1e, 1g, 1h, 5e, 5h, 6a, 6e, 7e, 7h, 9f, 18 1a, 4h, 7a, 7b 18 9h, 10h, 11e 21 1b, 1d, 7g, 9b, 9f, 10b 6a 7 1b, 2b, 3g, 5e, 6b, 6d, 6f, 8b, 9a, 10d, 22 6a, 6c 15 11a, 11f, 11h 23 5f, 6b, 7g, 10e, 11a, 11d 2f, 11g 8 24 1a, 4a, 5g, 5h, 10b, 11f 6a 7 25 1c, 7e, 11f 3 26 1a, 1b, 2c, 4e, 7b 5 siRNA: 1357 122 22 144

136 Results

7 Identification of potential APLP-like pseudogenic fragments in C3orf52 7.1 Examination of potential APLP-like pseudogenic fragments In an unsuccessful bioinformatic attempt to identify a fourth member of the APP- family, potential APLP-like processed pseudogenic fragments were accidently discovered. A fourth member of the APP-family was searched because the APP-family contains 3 members and according to the widely accepted theory of two whole genome duplication events in early vertebrates, however, four members should have been derived from the ancestral gene. The selection of the chromosomal location to be examined followed an earlier assignment of the APP family to the chromosomes 3, 11, 19, 21 [455] and the logics of coparalogy: Coparalogy is the phenomenon of co- localization of members of two different gene families on same chromosomes. Coparalogy for the APP family and other gene families, e.g. the IgLON- and the PHLDB family was suspected based on the location of their known members on chromosomes 11q24, 19q13.1, 21q21.3 and chromosomes 3q13, 11q23, (19q13,) respectively [456]. So, chromosome 3q contains an IgLON member called LSAMP but so far no APP member. Cues on the exact location of an APP member on 3q might be gained from the coparalogous pair on 11q: The IgLON member OPCML on 11q is located 2.5 mbp downstream of APLP2 [NCBI: MapViewer], with their reading directions pointing to each other (Fig. 59). 4 million base pairs (mbp) upstream of LSAMP on 3q was found a gene labeled Chromosome 3 open reading frame 52 (C3orf52) – with both reading directions also pointing to each other (Fig. 59 below), and initially suspected to be remnants of the fourth member.

Chromosome 11 PHLDB1 APLP2 OPCML ↔ 2.5

mbp PHLDB APP IgLON family family? family

C3orf52? ↔ Chromosome 3 PHLDB2 4 mbp LSAMP

Fig. 59: Chromosomal locations of the APP-, the IgLON- and the PHLDB family and C3orf52. Chromosome images modified from ENSEMBL (www.ensembl.org). 137 Results

C3orf52 in human was detected in 2005 as a TPA-inducible transmembrane protein, therefore named TTMP, and characterized in silico [457]. TTMP exists in three isoforms from in total seven transcript variants of C3orf52. The isoforms have three N-terminal exons in common and differ by the C-terminal exons (Fig. 60 below). Isoform 2 corresponds to TTMP of chicken (red jungle fowl, Gallus gallus) and the teleost fish stickleback (Gasterosteus aculeatus). The long “exon 6”, in addition to its 5‟-region possesses potential APLP-like processed pseudogenic fragments (Fig. 60).

C3orf52, Hs chromosome 3 location 111,805,182-111,849,851 bp direction + TTMP isoform 1 3‟UTR TTMP isoform 3 transcript: spanning whole genomic region of C3orf52, non-coding

potential APLP-like processed pseudogenic fragments TTMP isoform 2

“exon 6”=3‟UTR

C3orf52, Gg location 91,943,876-91,952,441 bp direction + TTMP 3‟UTR

C3orf52, Ga group I location 14,454,279-14,459,159 bp direction + TTMP 3„UTR

Fig. 60: Human, chicken and stickleback C3orf52 and the location of the potential APLP-like processed pseudogenic fragments in human. Modified from the NCBI and ENSEMBL databases.

“Exon 6” and 923 bp of its 5‟-flanking region were translated and in the three amino acid reading frames, a signal peptide-like sequence, several smaller stretches and a potential transmembrane domain were reminiscent of APP family members, mostly of APLP2. The similarities with APLPs are reflected in the name APLP-Near Notes Extant (ANNE) (Fig. 61 p148). The program RepeatMasker [458] identified the complement of the 3‟-half of an AluJb (c3‟Alu) on DNA level, of which the first third is even translated in TTMP isoform 1. ANNE (698 amino acids without c3‟Alu) was compared to isoforms of APLP2 and APP with similar lengths from human and the fish fugu (Takifugu rubripes), and to APLP1 from the green anole lizard (Anolis carolinensis) and the fish medaka (Oryzias latipes). These APLP1s have lengths of 699 and 723 amino acids and are therefore longer than mammalian APLP1 with

138 Results usually 651 amino acids. In order to facilitate the comparison to ANNE, the translation of c3‟Alu was copied into the APP family members at the same position as in ANNE and a wild card (X) copied into the C-terminus of ANNE. For the APLP1s alone, this was sufficient to align their transmembrane domains and C-termini to this part of ANNE (not shown). For the APLP2- and APP-sequences to become aligned similarly, the C-termini of all sequences were elongated by a block of 10 tryptophanes (W). These sequences were aligned by CLUSTALW [459] and then the N-terminal part of ANNE was manually shifted 46 amino acids upstream to align the putative APLP-like fragments there. Finally, the first exon of human APP was manually shifted upstream for alignment with APLP2 and ANNE (Fig. 62 p149). Expected-values (e-value; similar to p-values) were calculated by BLASTP [460]. In direct pair wise alignments of ANNE to human APLP2 by BLASTP two fragments with similarity were assigned with an e-value (Tab. 11 p150), however even the lower one only at 0.02. By alignment, ANNE has in average 13.7% and 5.3% identity to the human APP family members on DNA and protein level, respectively (Tab. 12A p150). The genomic region around ANNE was examined for flanking direct repeats, which were not found, a poly-A-tail, which was not found up to 20000 bp downstream and the GC-content, which is low although higher than of the genomic region (Tab. 12B p150). C3orf52, actually TTMP, was predicted by NCBI in most mammals by automated analysis of the genomes (Tab. 13 p151). TTMP in dog was identified during this study located downstream of the Transmembrane Serine Protease 7 (TMPRSS7) as in the other mammals and probably missed by automated analysis because the first exon is located in a region with unknown DNA sequence. Regions orthologous to ANNE were identified by DNA alignment (Tab. 14 p151) with the program package AiO [462] and used to calculate a tree for mammalian divergence (Fig. 63 below) (and also by CLUSTALW which did not align a long insert in cattle correctly probably due to gap penalties (not shown)). Rabbit

Cattle Horse

Dog Chimpanzee

Human

Mouse Fig. 63: Tree for mammalian divergence calculated from alignment in Tab.14. 139 Results

Regions orthologous to ANNE in these mammals were translated and resulted generally in less similarity to APP family members, as demonstrated by the two most similar stretches, of which one contains the potential transmembrane domain (Fig. 64 p156). A more N-terminally CV-repeat (on DNA level: gt) is however best conserved in chimpanzee. The APLP-like pseudogenic appearance of ANNE and its poor conservation could be explained by an ancient insertion event of an APP family member mRNA into the genome. The time point of a potential insertion was roughly calculated as in [463], where DNA substitution rates of rodents with 4.6∙10-9 and slowed rates for humans with 1.2∙10-9 and 0.8∙10-9/site/year were used for the time points up to 40, 25 and 0 million years ago (mya), respectively. Alternatively, substitution rates from chicken with 3.6∙10-9/site/year were combined with the slowed human rates. From 2184 nucleotides of human ANNE, in average 66% around 100 are not substituted in the similar amino acid stretches. ANNE would thus reach an age of around 239 my or 294 my (calculations in Tab. 15 p156). The calculated ages are below the divergence of teleost fish and tetrapods 450 mya and the age of 294 my, calculated with chicken substitution rates, is close to the mammal-reptilian divergence [405]. In order to test whether ANNE is present in sauria and teleosts, the orthologous regions in predicted C3orf52 of chicken, the teleost fishes stickleback and Tetraodon nigroviridis had to be found. (In the clawed frog, C3orf52 and TMPRSS7 were not found.) As starting point, and as mentioned above, human TTMP isoform 2 corresponds to chicken and teleost TTMP (Tab. 16 p157). In human isoform 2 and chicken, the end of exon 5 marks in both species the end of the coding region (Fig. 60). The regions downstream of human and chicken exon 5 up to the ends of exon 6 (human without c3‟Alu) were aligned by CLUSTAL to the 3‟- untranslated region (3‟UTR) and the 3‟-flanking region up to the downstream gene GUCA1C in stickleback in order to test which 3‟-regions correspond to each other. As result, alignments started not far downstream of the beginning of ANNE in human, however, translations of chicken and stickleback DNA stretches aligned to the APLP- like stretches in ANNE did not exhibit similarity to APLPs (not shown). Also when searched more in detail, translations of the region downstream of chicken TTMP did not exhibit similarity to APP family members (not shown).

140 Results

In contrast to human and chicken C3orf52 between TMPRSS7 and GCET2, teleost C3orf52 is located between RCSD1 and GUCA1C. The different location of C3orf52 in tetrapods and teleosts with respect to neighboring genes leaves room for gene changes in tetrapods or teleosts including the loss of the intronic region in teleosts due to or following chromosomal rearrangements. In order to test whether C3orf52 in tetrapods or in teleosts is closer to the common chondrichthyan ancestor, C3orf52 and TMPRSS7 were blasted against the genome of the elephant shark (Callorhinchus milii) [464, 465] because sequences of identified genes, like TMPRSS7, are either hard to find or not annotated in the databases. TMPRSS7 or its immediate downstream duplication, also present in chicken, was identified in AAVX01345579.1 (e-value: 10-75, not shown), however no tetrapodal downstream gene like C3orf52, UPK1A or CD200 or teleost downstream gene like FLT1, so that the question for the chondrichthyan location of C3orf52 and its 3‟UTR had to be left unanswered. For an original constellation of the genes as in tetrapods speaks, that synteny in this region of the elephant shark genome seems to be closer to tetrapods than to teleosts because of more synteny clusters. While TAGLN3 and TMPRSS7, for example, are found as cluster in tetrapods, teleosts and the elephant shark [465, supplementary file HumanSyntenyClusters: #372], ATG3 and CCDC80 in contrast build a cluster in tetrapods and the elephant shark (cluster #373) but not in teleosts (Tab. 17 p157). Also, GCET2, in mammals and chicken immediately downstream of C3orf52, was not found at all in teleosts (but also not in elephant shark). In order to test whether maybe parts of other genes or pseudogenes might be found in the region downstream of human exon 5 up to the end of exon 6, the three 5‟- and the three 3‟-reading frames were translated and blasted against the human genome. Indeed 119 hits with e-values less than 10-25 and several of them nearly as low as 10-50 were found (not shown), however, they were Alu sequences as exemplified by a hit against isoform 3 of -Like Factor 2 (CRLF2) (Tab. 18 p158).

7.2 Gene expression data analysis of C3orf52 ANNE and C3orf52 are expressed in B-cells and fetal tissues (Tab. 19 p158). In order to test expression of parts of ANNE and TTMP, mRNA of 3d HESC9 (kindly provided by Matthias Weng) was subjected to qPCR and sequencing which resulted in detection of 5 amplicons (Fig. 65 p159) with ct-values of 32, 34, 33, 33 and 62 in

141 Results comparison to 21 for GAPDH (Tab. 20 p159). The first 4, named TTMP and ANNE1- 3, were found in transcript variant 1 of TTMP in the predicted lengths. Two antisense mRNAs (NCBI: AA210770, AA485191) from germinal center B-cells share parts of amplicons 3 and 4. Their possibly identified [466, 467] promoters are TFIIB- and RXR-sites (Tab. 21 p161). Amplicon 5 with around 500 bp (targeted 1800 bp) was found in the human genome at chromosome 1, 45.2 mbp, as ribosomal protein S15a (processed) pseudogene 11 (RPS15AP11), in an intron of 8642213, a predicted homolog to murine mCG114749 (Tab. 22 p161). The expression also of antisense transcripts in B-cells seemed interesting and C3orf52 gene expression data were compiled (Tab. 23A p163). Of 700 experimental array data, around 100 with immunological background have been roughly ordered in groups of cell types, leukemias, diseases, effectors and cellular activities (Tab. 23B p164). Taken together, around 40% of the array data are linked to the monocyte-macrophage lineage, for example as myeloid leukemia, around 20% each to lymphocyte lineages, leukemias, diseases and natural immunoreceptor interaction partners like PAMPs and interleukins, and 5% each to transcription factors and the cellular activities differentiation and response to low levels of oxygen (Tab. 23C p166). Entries linked to prostate cancer are listed in Tab. 23D p166. Small molecule effectors and linked disorders have been grouped into drugs, allergens, smoke components, receptor effectors, lipid-derived hormones and hormone cofactors (Tab. 23E p167). Regarding once more the initial suspicion of TTMP as APP family member, this hypothesis was also refuted by the location of C3orf52 upstream of GRAMD1C (Fig. 66 below) in comparison to APLP2 and APLP1 being downstream of GRAMD1B and GRAMD1A. C3orf52 has rather a similar relative location as the

Chromosomes 3 11 19

PHLDB2 111.6▼ PHLDB1 118.4▼

C3orf52 111.8▼ CXCR5 118.7▼

GRAMD1B 123.4▼ GRAMD1A 35.5▼

GRAMD1C 113.6▼ APLP2 129.9▼ APLP1 36.4▼

Fig. 66: Overview on paralogous regions of chromosomes 3 and 11 containing C3orf52 and CXCR5. Long arrows stand for the chromosomes and sequence directions as in the NCBI database. Numbers behind genes mark the location on the chromosome in million base pairs and arrowheads the reading direction. 142 Results

CXCR5. C3orf52 and CXCR5 possess also an overall length which is not very different (217/250aa vs. 372aa) and like C3orf52, CXCR5 is expressed in B-cells, however their sequences possess only 14% identity (not shown). With regards to a possible function of C3orf52 in monocyte-macrophage differentiation and its location close to GRAMD1C, it is interesting to note, that the primary response gene in myeloid differentiation, MyD88 [468], is located just 0.5 mbp downstream of GRAMD1C in the fish medaka (in comparison to 75 mbp upstream of GRAMD1C in human) and has a similar overall length (Tab. 24 p167). Of the vertebrates with completely sequenced genome, medaka had fewest chromosomal rearrangements as has been demonstrated [469, 470].

8 Second attempt to identify a fourth APP family member on chromosome 3 In a second approach to identify a fourth APP family member on chromosome 3, regions of interest were aligned to APP family members or whole regions of chromosomes were aligned in order to find an APP family-like gene sequence in genomic DNA. Alignments of APP fam. members to chromosome 3 between GRAMD1C and LSAMP did not result in sequences with sufficiently high identity. A challenge in this search might be discrimination between random similarity and similarity due to homology, which is virtually only possible above a certain level of sequence identity (approximated by equal random chance for all four nucleotides, Fig. 67 p168). Empirically, 40% identity seemed close to the upper limit for random similarity in gapless alignments of the 100 bp conserved APP family C-termini to 100 kbp regions of chromosome 3 (not shown). Further, so far no vertebrate APP family member other than APLP1, APLP2 or APP was reported and therefore the fourth member or parts of it might have been lost and thus be subjected to free mutation. By use of combined substitution rates, more than 40% remaining identity would date back to a loss not older than 175 million years ago (mya), which might be younger than the actual date of loss, given the teleost-tetrapodal divergence 450 mya, and the gene exons could thus not become discernible from random sequences with relatively high identity.

143 Results

The alignments of large chromosomal regions containing APP family members to the large chromosomal region of interest on chromosome 3 were due to the large amount of data thankfully conducted by Prof. Tancred Frickey from the bioinformatics chair at the University of Konstanz. However, this approach was also not successful. Below, the results of alignments of 111.4-116.3 mbp and 33.8-39.8 mbp on chromosome 3 and chromosome 19, thus covering the region around GRAMD1C and GRAMD1A with APLP1 (position in alignment file in Tab. 25 p169), demonstrate a pitfall with this method: Long alignment blocks of chromosome 3 aligning to the genomic sequence of APLP1 on chromosome 19 consist mostly of repetitive elements like Alu-copies found in APLP1 introns (Tab. 26/27 p169). Alu copies were spread beginning 80 mya so that they cannot have been shared due to the whole genome duplications over 450 mya and should be filtered before alignment. Further, as noticed only later, both in mouse and the green anole lizard, APP is found on the same chromosome as the region around GRAMD1C (Fig. 68 p171), which is a hint, that a fourth member might not be found on human chromosome 3 - this under the assumption that APP and GRAMD1C belonged formerly to one linkage group like APLP2 with GRAMD1B and also APLP1 with GRAMD1A.

9 Examination of the regions containing the APP family During the search for the fourth member, it was noticed that the phylogeny of the regions containing the APP family is complicated. To start with, in a publication on reconstruction of the ancestral vertebrate genome [470], APP family members were initially maybe not linked to the IgLON family (and others in these regions with two members like the BACE family) on one ancestral vertebrate protochromosome, as seen for human and chicken (Fig. 69 p172). Here also no hints exist whether to expect three or four regions of APP family members in the genome. The situation is slightly different in medaka. Here, from APP family members cues on four members and thus four regions to expect exist (Fig. 70 p173). In order to identify a “fourth region”, gene families with three members neighboring APP family members, and GRAMD1C, had to be selected. Families were found and examined and their paralogs mapped to an interesting region on chromosome 1 (Fig. 71 below). Importantly, they seem to hint mutually on an independent “fourth region” and one gene family, ARHGAP30-33, has supposedly a fourth member in it.

144 Results

Chromosome 19 APLP1 GRAMD1A

KIRREL2 ETV2:↔GABPA ERF:↔ETV3 ARHGAP33 APLP2 GRAMD1B Chromosome 11 PHLDB1 OPCML

KIRREL3 ETS1 TAGLN ARHGAP32

Chromosome 21 APP GABPA: ↔ETV2 ETS2

GRAMD1C Chromosome 3 PHLDB2 LSAMP

TAGLN3 ARHGAP31

Chromosome 1

KIRREL ETV3:↔ERF TAGLN2 ARHGAP30

Fig. 71: Distribution of the human APP, GRAMD1, PHLDB, IgLON, KIRREL, ETV, TAGLN and ARHGAP families on chromosomes 19, 11, 21+3, and 1. Chromosome images modified from ENSEMBL [www.ensembl.org].

Orthologs of these genes from mouse, green anole lizard, chicken, clawed frog and medaka were listed in groups A (APLP1, Tab. 28), B (APLP2, Tab. 29), C (APP, Tab. 30) and D (4. Region?, Tab. 31) (pp174-177) in order to determine the relationship among the family members and maybe to obtain hints on a possible location for a fourth APP family member. Several APP family entries in the ENSEMBL database are not yet labeled (Fig. 72A p178) but all can be classified as APLP1 by neighboring

145 Results genes (e.g. NFKBID and KIRREL2) and their consensus patterns (Fig. 72B p178). The two entries for platypus APLP1 are probably due to poor sequencing results (1/4 and 1/3 of nucleotides undefined). The longest APP family members of each branch, 699aa APLP1 of medaka, 787aa APLP2 of Takifugu rubripes and 787aa APP of stickleback were blasted against the genome of the elephant shark (because sequences of the reported genes APLP2 and APP were not found in the databases) and resulted for APLP2 in identification of an interesting APP family member in the entry AAVX01069756.1, although only one quarter of its expected length could be found here. It is interesting because it was found via an APLP2 and by alignment placed between APLP2 and APP, however like all known APLP1 members it possesses an arginine at a specific position, which is a glutamine in fruitfly appl and in all APLP2 and APP members identified so far (Tab. 32 below). Full-length appl has 887aa and apl-1 of Caenorhabditis elegans 686aa. However, this elephant shark APP family member could not be classified because no potentially neighboring genes in (by number) nearby entries were found. When asking the group leader of the elephant shark genome project for up- and downstream genes, he offered to send BACs including the sequence of interest but due to thesis deadline no advantage was taken of this option.

Tab. 32: CLUSTAL 2.0.12 alignment with APLP of elephant shark (Cm, yellow)

APLP2 Tr 447------RQTLIQHFQAMVESLEEETASEKQQLVETHLARVEAMLNDRRRLALENYLAALQ APLP Cm ?------PLVLQHFKAMVESLEQEAASEKQRLVETHLARVEALLNDRRRVSLENYLSALQ APP Ga 451------KKAVIQRFQEKVEALEQEAASERQQLVETHMARVEALLNDRRRLALESYLTALQ APLP1 Ol 355------RQALNEHFQSVLQTLEEQVAGERQRLVETHLARVEAILNNNRRVALENYLTAVQ apl-1 Ce 295KGAEKFKSQMNARFQKTVSSLEEEHKRMRKEIEAVHEERVQAMLNEKKRDATHDYRQALA appl Dm 451KAAQSFKQRMTARFQTSVQALEEEGNAEKHQLAAMHQQRVLAHINQRKREAMTCYTQALT : :*: :.:**:: ::.: * ** * :*:.:* : * *:

APLP2 Tr ADPP--RPHRILQALRRYVRAENKDRQHTIRHYQHVLAVDPE---KAAQMKSQ-VMTHLR APLP Cm ASPPPTQSHRILQALKRYVRAEQKDRQHTMRHYQHVMAVDPE---KAAQMKSQVVMTHLR APP Ga QDSP--RPRHVFSLLKKYVRAEQKDRQHTLKHFEHVRMVDPK---KAAQIRPQ-VLTHLR APLP1 Ol SDPP--QPERVLQTLKRYMAAEQKDRRHTLRHYQHIVAVDPQ---KAEQMKFQ-VYTHLH apl-1 Ce THVNKPNKHSVLQSLKAYIRAEEKDRMHTLNRYRHLLKADS----KEAAAYKPTVIHRLR appl Dm EQP--PNAHHVEKCLQKLLRALHKDRAHALAHYRHLLNSGGPGGLEAAASERPRTLERLI . . : . *: : * .*** *:: ::.*: . : . :*

APLP2 Tr VIEERMNQSLSLLYKVPYVADEIQDEIDELLQEQK------589 APLP Cm VIEERMNQSLSLLYKVPYIAEEIQDEI------? APP Ga VIEERMNQSLGLLYKVPGVADDIQDQV-ELLQREQ------592 APLP1 Ol VIEERMNQSLALLYKDPMLAEELHSDIQELVKSER------477 apl-1 Ce YIDLRINGTLAMLRDFPDLEKYVRPIAVTYWKDYR------DEVS449 appl Dm DIDRAVNQSMTMLKRYPELSAKIAQLMNDYILALRSKDDIPGSSLGMSEEAEAGILDKYR627 *: :* :: :* * : :

BLASTP results for APLP of Cm to medaka genome Smallest Sum High Probability Sequences producing Highest-scoring Segment Pairs: Score P(N) N ENSORLP00000002978 pep:novel chromosome:MEDAKA:13 APLP2 812 1.1e-73 1 ENSORLP00000011186 pep:novel chromosome:MEDAKA:21 APP 668 2.8e-59 1 ENSORLP00000008229 pep:known chromosome:MEDAKA:16 APLP1 655 4.8e-58 1

146 Results

The relationships among the four groups A-D are puzzling, because trees of the families provided by ENSEMBL contradict each other. From the APP family tree, groups B and C would be closest and result in a topology (A)(BC). The trees of the GRAMD1, TAGLN and would result in (AB)(C), backed by the (ambiguous) ARHGAP tree, while the KIRREL tree might support an (A)(BD) topology (Fig. 73 p179). Moreover, trees are dependent on the used taxa. In the case of the APP family for example, the clear (A)(BC)-topology can possibly be traced back to changes in mammalian APLP1 which renders the whole APLP1 branch to an outgroup, because mammalian genes by far outnumber other taxa. With a more balanced dataset, using APP family genes of three teleosts (medaka, zebrafish, pufferfish), clawed frog, green anole lizard, chicken and three mammals (human, mouse, platypus), the (A)(BC)- and the (AB)(C)-topology become nearly equally likely (Tab. 33A p179) and without mammalian and avian genes the (AB)(C)-topology becomes most likely more clearly (Tab. 34B) using TreePuzzle 5.2 [471] for evaluation of pre-defined trees. Using the same datasets, PhyML 3.0 [472] calculated an (A)(BC) topology when mammalian and avian APP family members (Fig. 74A p180) were included and an (AB)(C) topology when excluded (Fig. 74B), however with far less branch support by bootstrapping. Likewise, the other family trees were questioned with the same species to evaluate different pre-defined topologies. For GRAMD1 (Tab. 34A p181), the best by a small difference was (BC)(A), thus contradicting the tree provided in the ENSEMBL database, while for TAGLN (Tab. 34B) the tree (CD)(B) was confirmed with even less difference on the second best. Both for ARHGAP (Tab. 34C) and KIRREL (Tab. 34D), the ENSEMBL topologies were confirmed, however, only 25% of the ARHGAP sequences passed the test on the sequence composition whereas in the other datasets at least around 90% passed this test (not shown). For the balanced TAGLN dataset from above, likelihood mappings by TreePuzzle 5.2 favored (D:TAGLN2)(B:TAGLN,C:TAGLN3) (Fig. 75A p182) while with the mammal- dominated dataset of all TAGLN members, the result is less clear (Fig. 75B). Similarly, likelihood mapping of the balanced dataset and mammal-dominated dataset of KIRREL gave also different results. The balanced dataset displayed a slight preponderance for (A:KIRREL2,B:KIRREL3)(D:KIRREL) (Fig. 75C), and thus questions the ENSEMBL topology, while the dataset of all entries reproduced (A)(BD) (Fig. 75D).

147 Results

Actually, since for the APP, GRAMD1 and TAGLN families (AD)(BC) can be suspected, the KIRREL family should be found in the topology (AD)(B). A hint, that despite diverged sequences indeed KIRREL2:A and KIRREL:D maybe originated in the second whole genome duplication can be gained from their consensus patterns, which seem to be closer to each other, especially in teleosts, than to the pattern of KIRREL3:B (Fig. 76A p183). At least, alignment of the human KIRREL members (with the homolog kirre of fruitfly) shows no topology preference at all (Fig. 76B), again hinting on short interim time of the WGDs. Regarding (AB)(C) or (A)(BC) for the GRAMD1 family, the GRAMD1 members‟ consensus patterns of both teleosts and tetrapods rather confirm GRAMD1(A)(BC) (Fig. 76C). An interesting family, possibly involving also local duplications, includes ERF:A, ETV3:D, ETV2:A, GABPA:C, ETS1:B and ETS2:C. A tree generated by TreePuzzle 5.2 confirms the (AD)(BC) topology provided by ENSEMBL, although not with very high branch support (Fig. 77 p184).

10 Additional figures and tables from chapters 7-9

ANNE,Hs NXIVIXIPEGFCVCLLLILXFGNTAIILTXLHAYLDALXKXKLKIXXXRNFLSPSSLTLPYLWSSSPPRXVGPWARRGK CFTK VEEXVSIMSYFGHRGGTWYCSHMGRSQLVDXVGKKGKTRIFVREKRKKPQVGRIVNLVSPVSGGEAGLLVCRPRRAPPVLCLL GALHLHLLCPRVHTISLIRISHSIFYTFXKRKFXSLWLDYEKGLDVKMSLAPKNHIIKIISHASYPNKXARNNKCHLPFEMYS LAILSKILHXNSMKXNENVSIXXWFXKSVTFVHLCFLFRMKXWRLVSVXKQCSTKSWRXREFT ERCVCVGLKTKFPLLGMYKP LFCRKDSVDXYRSTSNTRGVETPFSPDSTVWLXAQXNSSRALLQLGPVLTKAGSQLGWGRXGQRVCPTVGGHWYSVCFCFVSF CFLRQGLVLSLSWSAVAXSWHCYSXSTPPTWATFSLVQRCTVGFXVGSRKHELVEHGEAGSQVTKTKPIIHXSHPPPPHHSIX STSKPGCEEKEGTLEDLSSVVLCLLPXPVPEYLSQPA RNTTKVTLKSMYIASYRRELCVSSLFCSSSLFLXFLNTFLEIWVSV SFEHTTITLDSHSIYIFINFHXRIIKAVXYLLWILRIGFLLPSATNLPVGLSVFRQXLCILLQMVVIQEEPLFFENKLLEAFA GVGGILATVLVVYRYRIACCEGTDCFVGKRNLPGERVLCCIVRDLASQLNEPWVKVSVK SYLG Fig. 61: Translation of “exon 6” and its 5‟-flanking region, divided by M309 (black highlight) with stretches similar to APP family members in yellow, the translation of c3‟Alu in red and potential former transmembrane domain in dark yellow. Reading frames 1, 2 and 3 in superscript, normal and subscript.

148 Results

Fig. 62: Alignment of ANNE to APP family member isoforms with similar lengths. Similar stretches are highlighted in yellow, c3’Alu in red and manual operations in grey. APLP1 Ol ------LLCFLPVGSLLLTGLASLGSAVVSRGISLPVSAVNGPSPQGAEPQIAMFCGRQL APLP1 Ac ------AALGPALGTPQVAMFCGKLV APLP2 Tr ------HTPHTPLLSLCIPSIALAANAGTGFAVAEPQVAMFCGKLN APLP2 Hs MAATGTAAAAATGRLLLLLLVGLTAPALALAGYIEALAANAGTGFAVAEPQIAMFCGKLN APP Tr ------ALSYNLFNSLIGYLSLYANVYPPQVPTDVSMGLLAEPQVAMFCSKLN APP Hs ------MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLN ANNE Hs NXIVIXIPEGFCVCLLLILXFGNTAIILTXLHAYLDALXKXKLKIXXXRNFLSPSSLTLP

APLP1 Ol LYMNVQTGQWEPDPQGRQGCFKEPSEILSFCQEVHIPDWVQIRGVFPKNPXXXXPVPIPG APLP1 Ac LHLNVQTGRWEPDASGTHTCFRTTDEIRSYCQEVYPELQITNAVEGTQPVT-IDNWCKKG APLP2 Tr MHVNIQTGRWEPDPSGTKSCVGTKEGVLQYCQEMYPEFQITNVVEADQQIRIENWCKKKK APLP2 Hs MHVNIQTGKWEPDPTGTKSCFETKEEVLQYCQEMYPELQITNVMEANQRVSIDNWCRRD- APP Tr MHINVQSGKWEPDPTSTKSCISTKEGILQYCQEVYPELQITNVVEANQPVSIQNWCKMGR APP Hs MHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGR ANNE Hs YLWSSSPPRXVGPWARRGKCFTKVEEXVSIMSYFGHRGGTWYCSHMGRSQLVDXVGKKGK

APLP1 Ol <-->WSKKGWGHLIVLPYRCLEGEYVSETLLVPDRCRFLHREQMDACESYVYWHNIAKEACTAD APLP1 Ac <-->RPKCKGHQHIVVPYQCLVGEFVSEALLVPDKCKFLHQEKMDSCETYLYWHSVAKEACSEE APLP2 Tr <-->--ACKGHAHIVVPYKCLVGEFVSDVLLVPEKCKFFHKERMDLCVSHQQWHGVAKEACSKS APLP2 Hs <-->--KKQCKSRFVTPFKCLVGEFVSDVLLVPEKCQFFHKERMEVCENHQHWHTVVKEACLTQ APP Tr <-->KHCRSHVHIVVPYRCLVG-EFVSDALLVPDKCKFLHQERMNQCESHLHWHTVAKESCGDR APP Hs <-->KQCKTHPHFVIPYRCLVGWEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEK ANNE Hs TRIFVREKRKKPQVGRIVNLVSPVSGGEAGLLVCRPRRAPPVLCLLGALHLHLLCPRVHTISLI

APLP1 Ol SLELHSYGMLLPCGDHFRG-VEYVCCPGRGSSGGKGEAEEKTTAGGLQALTPQTSGKLIS APLP1 Ac DLELHSYGMLLPCGADRFRGVEYVCCPSRPPPIRLEQATDAPQILPRLREEALHSNKVPT APLP2 Tr TMVLHSYGMLLPCGIDKFHGTEYVCCPSTHAGNNSPAPPPSQEDDEDEEM-EDEEIDEAD APLP2 Hs GMTLYSYGMLLPCGVDQFHGTEYVCCPQTKIIGSVSKEEEEEDEEEEEEE-DEEEDYDVY APP Tr TMNLHDYGMLLPCGIDRFRGVEFVCCPAEAER-DVDSAEKDTDDSDVWWGGAENDYSDNR APP Hs STNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDDSDVWWGGADTDYADGS ANNE Hs <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<RISHSIFYTFXKRK

APLP1 Ol VTDVEEADTLDEEDMDEEDDEEEAISVKDQDEYEYPIDSVSSHQIPNVKAKSFFKRCRPV APLP1 Ac GHMPMEEESLEIEEEGAIDEEEEVDEEDDEDGEEIPRDGVLDSDPFPPAWDDYFVEPGID APLP2 Tr LMEEETSETPAEEQPTAKEDSADDYKDDEEEEDEEEYHYVYEDEEAVKGG--TLPTTQPT APLP2 Hs KSEFPTEADLEDFTEAAVDEDDEDEEEGEEVVEDRDYYYDTFKGDDYNEENPTEPGSDGT APP Tr YQQLVEEEEEGDEDDEEEDEVLENDQDGDGEEDEEVAEEEDDEEDDEPTTNVAMTTTTTT APP Hs EDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTS---IATTTTT ANNE Hs FXSLWLDYEKGLDVKMSLAPKNHIIKIISHASYPNKXARNNKCHLPFEMYSERCVCVGLK

APLP1 Ol TTSRPTDGVDVYFEKPVDDTEHANFLRAKTDLEERRMKRINEIMKEWAEADDQSKNLPKS APLP1 Ac SGSSGLPGERELVNSVRQRWEMPIRGQVIPKSFLKPRTSCEVTLPIGAFPNGRTSGISAA APLP2 Tr SLTICVQILAVTLLTPQPTDDVDIYFETPADDKEHSRFQRAKEQLEIRHRSRMERVRKEW APLP2 Hs MSDKEITHDVKVPPTPLPTNDVDVYFETSADDNEHARFQKAKEQLEIRHRNRMDRVKKEW APP Tr TTESVEEVVRVPTASPSSSDAIDHYLETPADENEHAHFQKAKESLEAKHRERMSQVMREW APP Hs TTESVEEVVRVPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREW ANNE Hs TKFPLLGMYKPLAILSKILHXNSMKXNENVSIXXWFXKSVTFVHLCFLFRMKXWRLVSVX

APLP1 Ol E------APLP1 Ac DGWLCWHTIGGMLPVSNNRERVGRKCSAASHGHVSFSSSSQCIEQRQRQTQRQFKPSPAS APLP2 Tr EEADRQAKN------APLP2 Hs EEAELQAKN------APP Tr EDAEREAKN------APP Hs EEAERQAKN------ANNE Hs KQCSTKSWRX------

APLP1 Ol ------RQALNEHFQSVLQTLEEQVAGERQRLVETHLARVEAILNNNRR APLP1 Ac SFLRVCLIPGTGGAAASSSLFSQHFQSILQTLEEQVARERQRLVETHLARVVALLNDNRR APLP2 Tr ------LPKAERQTLIQHFQAMVESLEEETASEKQQLVETHLARVEAMLNDRRR APLP2 Hs ------LPKAERQTLIQHFQAMVKALEKEAASEKQQLVETHLARVEAMLNDRRR APP Tr ------LPRADKKIVIQRFQEKVEALEQEAASERQQLVETHMARVEALLNDRRR APP Hs ------LPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRR ANNE Hs ------REFTLFCRKDSVDXYRSTSNTRGVETPFSPDSTVWLXAQXNSSR . : : . . : : .... : : *

APLP1 Ol VALENYLTAVQSDPPQPERVLQTLKRYMAAEQKDRR======APLP1 Ac AALESYLTAVQNDNPQPDRVLAALKRYVKAEQKDQR======APLP2 Tr LALENYLAALQADPPRPHRILQALRRYVRAENKDRQ======APLP2 Hs MALENYLAALQSDPPRPHRILQALRRYVRAENKDRL======APP Tr LALESYLTALQQQPPRPRHVFSLLKKYVRAEQKDRQ======APP Hs LALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQ======ANNE Hs ALLQLGPVLTKAGSQLGWGRXGQRVCPTVGGHWYSVCFCFVSFCFLRQGLVLSLSWSAVA *: . : . : ************************

149 Results

APLP1 Ol ======HTLRHYQHIVAVDPQKAEQMKFQVYTHLHVIEERMNQSLALLYKDPMLA APLP1 Ac ======HTLRHYQHVAAADPEKAEQMKFQVYTHLHVIEERMNQSLALLYKNPQLA APLP2 Tr ======HTIRHYQHVLAVDPEKAAQMKSQVMTHLRVIEERMNQSLSLLYKVPYVA APLP2 Hs ======HTIRHYQHVLAVDPEKAAQMKSQVMTHLHVIEERRNQSLSLLYKVPYVA APP Tr ======HTLKHFEHVRMVDPKKAAQIRPQVLTHLRVIEERMNQSLGLLYKVPSVA APP Hs ======HTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVA ANNE Hs XSWHCYSXSTPPTWATFSLVQRCTVGFXVGSRKHELVEHGEAGSQVTKTKPIIHXSHPPP *********** * :. : : : .. .: .:: ::: .

APLP1 Ol EELHSDIQELVKSERGDISELMTTSFSETRTTEELLPAESEEEKTDPILPALVLFQATEM APLP1 Ac QELRGDIEELLRSERVSTSDLLTTSISETRTTVVRTTTN------SSI APLP2 Tr DEIQDEIGESVRKLKKYTNWLLAEMGTDALAVPGNEQKTDMDQFLASISE---SQPDITV APLP2 Hs QEIQEEIDELLQEQRADMDQFTASISETPVDVRVSSEESEEIPPFHPFHP---FPALPEN APP Tr DDIQDQVELLQ-REQAEMAQQLANLQTDVRVSYGNDALMPDQELGDGQTD---LLPQEDT APP Hs EEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVE---LLPVNGE ANNE Hs PHHSIXSTSKPGCEEKEGTLEDLSSVVLCLLPXPVPEYLSQPARNTTKVTLKSMYIASYR

APLP1 Ol QPNKKEYQIFTSVCMSVEDEYDYTTSERGPTDEYEEKVHLAHFCIQINNSAELKQVN--- APLP1 Ac LYFPYHQTHVFLAVKPNDGKGNILSLECGAKGAGFAIINKIKQINKKNMVVWEIIQL--- APLP2 Tr SSEESVEVSVSEGKPYRPFQVASVGSRSEPEGESHQQHPHCPPLRGEASTNDLRDKL--- APLP2 Hs EGSGVGEQDGG------LIGAEEKVINSKNKVDENMVIDETLDVK--- APP Tr AGGVGFIHPESFNQLNTDN------QVEPVDSRPTLERGVPTRPVTGKSMEAIPDM--- APP Hs FSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEV--- ANNE Hs RELCVSSLFCSSSLFLXFLNTFLEIWVSVSFEHTTITLDSHSIYIFINFHXRIIKAVXYL

APLP1 Ol ------KPNEIASDELVRKRNSFFQKKLFLGKTFNRGAMVGLLVVAVAIAM APLP1 Ac ------KQIIYKAVEIGGLNHPLPLQEPDTLVTINRGALIGLLVVAVAVAM APLP2 Tr ------KKIIIDSLYNNYRVIHESYRPLGEDFSFGSSALIGLLVIAVAIAT APLP2 Hs ------EMIFNAERVGGLEEERESVGPLREDFSLSSSALIGLLVIAVAIAT APP Tr ------RMETEDRQSTEYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT APP Hs ------KMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT ANNE Hs LWILRIGFLLPSATNLPVGLSVFRQXLCILLQMVVIQEEPLFFENKLLEAFASYLGGVGG : .: :.

APLP1 Ol VMVISLLLVRRKPYGTISHGIVEVDPMLTPEERQLNKMQNHGYENPTYKFFEQMNWWWWW 699aa orig. APLP1 Ac VIIISLLMVRRKPYGTISHGIVEVDPMVSPEERQLNKMQNHGYENPTYKFFEEMNWWWWW 723aa orig. APLP2 Tr VIVISLVLLRKRQYGTISHGIVEVDPMLSPEERHLSKMQNHGYENPTYKYLEQMQIWWWW 692aa orig. APLP2 Hs VIVISLVMLRKRQYGTISHGIVEVDPMLTPEERHLNKMQNHGYENPTYKYLEQMQIWWWW 695aa orig. APP Tr VIVITLVMLRKKQYTSIHHGIIEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQNWWWW 694aa orig. APP Hs VIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQNWWWW 695aa orig. ANNE Hs ILATVLVVYRYRIACCEGTDCFVGKXRNLPGERVLCCIVRDLASQLNEPWVKVSVKWWWW :: *:: : : . . . * ** * : .. .: . :.: ****

Tab. 11: e-values for ANNE Score = 24.6 bits (52), Query 16 LLLLLVGLTAPALALAGYIEAL 37 e-value = Expect = 0.020, LLL+L G TA L L Y++AL Method: Compositional matrix adjust. Sbjct 13 LLLILFGNTAIILTLHAYLDAL 34 Identities = 12/22 (55%), Positives = 15/22 (68%), Gaps = 0/22 Score = 13.9 bits (24), Query 10 CFTKVEEV 17 e-value = Expect = 0.13, CF EEV Method: Compositional matrix adjust. Sbjct 11 CFETKEEV 18 Identities = 5/8 (63%), Positives = 5/8 (63%), Gaps = 0/8

Tab. 12A: identities of ANNE to human APLP1, APLP2 and APP APP family identity to ANNE in alignment coding region amino acids DNA without c3’Alu translated w.o. c3’Alu APLP1 1956 bp 651 14.7% 6.6% APLP2 2292 bp 763 14.1% 5.2% APP 2313 bp 770 12.2% 4.2%

Tab. 12B: gc-contents DNA-type gc-content g + c total bp ANNE 42.8% 935 2184 53 kbp, ANNE, 24 kbp 41.4% 32947 79669 non-processed pseudogenes 51.5%* processed pseudogenes 46.1%* adjacent genomic DNA 43.4%* APLP1 coding region 63.9% 1249 1956 APLP2 coding region 51.7% 1184 2292 APP coding region 52.3% 1209 2313 *from [461]

150 Results

Tab. 13: mammalian TTMP Species upstream exons exon 1 exon 2 exon 3 exon 4 exon 5 exon 6 total bp human 211 130 128 71 182 1556 2296 chimp. 197 130 128 71 182 1593 2303 mouse 218 130 128 71 182 1456 2206 rabbit 57 + 112 107 130 128 71 213 819 horse 67 128 71 182 55 504 cattle 138 130 128 71 218 5 1472 dog ? 130 128 71 182 ? ?

Tab. 14: Alignment of DNA regions orthologous to ANNE c3’Alu of the primates highlighted in red

Progam Package: All in 1 (AiO) Program: Align 11.04.2011 15:25:21 hr. FOF: , Default smallest area: 21, Default Limit: 1 Using the following files: Name:Human #1 Start at: 1, End at: 2181 Name:Chimpanzee #2 Start at: 1, End at: 2215 Name:Mouse #3 Start at: 1, End at: 2222 Name:Rabbit #4 Start at: 1, End at: 2594 Name:Horse #5 Start at: 1, End at: 2839 Name:Dog #6 Start at: 1, End at: 2580 Name:Cattle #7 Start at: 1, End at: 2874

4 ------CCTTGTTTGTGGTCCACTGGGGTTCGTTTGTGTGCACCTTG GAGGCAGGACCTTGCATCAAATTGTGATGTGGATTCCAGAGGGGTCTGCA 7 ------TTTGATCCC...... 5 ------TTTGATCTC...... 6 ------GTTC...... 2 ---TAGATTGTAATTTAGATTCC...... 1 ---TAGATTGTAATTTAGATTCC...... 3 TCCTGTAGTGATGGACAACTT...... 4 TGTGT...... GTGTGTGTGTGTG...... TGT...... GTAT...... A...... 7 ....T...... GGGTCTGGAAGATCCCCCCTGAAGAAGGAACTGGCAACT CACTCCAGTAC...... TATTGCC...... TGGAAAATCCC 5 ....T...... TTTTCTGTGAGTT...... TGG...... GTAT...... TTTTGGA...... TTCTACAT... 6 ...... T...... TTCTAGA...... TCCCACAT... 2 ....TGAGGGGTTTTGTGTGTGTT...... TGT...... TTCT...... C.....A...... TTCTCTAA... 1 ....TGAGGGGTTTTGTGTGTGTT...... TGC...... TTCT...... C.....A...... TTCTCTAA... 3 ...... GGTATGGCAGTGTT...... GGT...... GTCTTGTATTGTGTC.....AAATCAAGTAACTCTTCACT... 4 ...... CAATTGCC 7 ATGGATGGAGGAGCCTGGTAGGCTGCAG..TCCATG.GGGTCGCAAAGAG TCGGACACAACTGAGC...... GAC...... TTCACTTCAC 5 ...... GTAAGTGAGATCATGCAG..TATTTA.TCTTCC...... TCTGT.....CTGACT...... TAC...... ATCACTTAGC 6 ...... GTGAG...GATCATGCAA..CATTTG.TCTTTC...... TCTGT.....CTAATT...... T...... CATGCAGC 2 ...... TTTGGAAATA.C..G..G..CGATTA.TTTTGA...... CCTGA.....CTGCATGCAT..ATTTGG...... ATGCCTTATA 1 ...... TTTGGAAATA.C..G..G..CGATTA.TTTTGA...... CCTGA.....CTGCATGCAT..ATTTGG...... ATGCCTTATA 3 ...... CTTGCACAAA.C..T..GATCTCTTGCTCTGGA...... GTTCA.....CATCCTGTATGCAGACGGAGCAGTGAGAAAATACTATGGA 4 ...... TT..T...TCTAAT...... A 7 TATCATACTATCCAGAAATTCCACTTCTGAGTATTGATATGAATAAAACA AAAGTGTTAATTTGAAAAGATATATG...CACCCC...... A 5 ...... ATAAT...ACCCTC...... A 6 ...... ATGATGGTA...TC...... A 2 ...... AAAAT...AAAAACTTTACTTAAAATATA 1 ...... AAAAT...AAAA.....ACTTAAAATATA 3 ...... AAAAT...AAAA.....A...... 4 AGGATATGGGAATAAAGT...... TG...... 7 AAGTTCATTGCAGCATTA...... T...... 5 AGGTTAAGGGAATTCTTT...... TAAT...... 6 AGGTTCAGGGAATTGTTTCTTTTTAAATATTTTATTTATTTAATCATGAG AAACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGGCAGAGACACAGGC 2 ATGATAAAGGAATTTTTT...... AA...... 1 ATGATAAAGGAATTTTTT...... AA...... 3 ...... A...... 4 ...... G...... CTCTC...... CACCTGTTCAGGT 7 ...... TCACAAA...... 5 ...... GAATCACCATCCCTCCC...... CACCCCCCCCGCG 6 AGAGGGAGAAGCAGGCTCCATGCTGGGAGCCCGATGCAGGACTTGATCCC AGGACCCTGGGATCATGACCTGAGCCGAAGGCAGATGCTCAACCACTGAG 2 ...... GT....CCATCATCCCT...... CACCCTTCCC... 1 ...... GT....CCATCATCCCT...... CACCCTTCCC... 3 ...... GG....CCACCGTCCCA...... AA......

151 Results

4 CCAACATCCAAGGGCTCAAGCAACTATGGTTGAAAAAACACTCAAAAAAA GTTGCATTTGTATTGAACACGTACAGAGGTTCTTTGCTTGTTGTTATTCC 7 ..A...... GC 5 CCA...... CC 6 CCA...... CC 2 ...... 1 ...... 3 ...... 4 CCGAGCAATGCAGCCTGACGACTATCTACATAGCATTTGCATTGTGGTAG GCATAAGTAATCTGGAGGTGATTTGAAGGATGCAAGAGGATGTGCATGGG 7 C...... 5 C...... 6 C...... 2 ...... 1 ...... 3 ...... 4 CTAAGTGCAGATGCTACTCCATTCCAGGTAGGGATCCGTGTTGAGATCTC TGTTGTGATCTGTGAATGTTGTCATTCATGGGGTGCTAGAACCAATCCCC 7 ...... 5 ...... 6 ...... 2 ...... 1 ...... 3 ...... 4 GGTAGATACTGAGGGACAGCTGTGCAGCGATGATTTTGATCTGGCTAAAG GAAGATTTTGATGCCTTATAAAAATAAAAAAGACCCTGAGCATGTCATTA 7 ...... 5 ...... 6 ...... 2 ...... 1 ...... 3 ...... 4 TAAAGGGATTTCTAAAACCCTCCACCTC...... TCACCCTGTG...... AC...TCTTCACCT..TATGAATG 7 ...... AAGATAGGGAA...... GCAACCTAAGTA...... T.TCCTCGATA..GATGAATG 5 ...... CCGCCACTCCC...... CCACCCTGTGTCTTTGAGCT.TCTTCACTT..TATGGATG 6 ...... AGGTGCCCCCAAGGAATTGTTTTAATAGCTCTC ACCTCCCTTTCCCCCCTGTTTTTTTGGCCTGT.TTCACCT..TATGGATG 2 ...... TATCTTTGGTCT.TCTTCACCTCCTA..GATG 1 ...... TATCTTTGGTCT.TCTTCACCTCCTA..GATG 3 ...... 4 GGTGGGA...... AATTCTCTTTCACAAAGGTCA...... GA...... CTTTTTTCACAGA.. 7 GATGAGG...... AAGA...... TGTGGTATATGTATATGCAAG GAAATATTACTCAGCCATAAAAAGAATGGAATTTTGCCATTTGCAACAAC 5 GATGGGG.CCTGGGGATAAGGAGGGGAAGTCTGGTTTACAAATATGAAAG ...... AGCAGGA...... GTTATTTTGGACATC 6 TTTGGGGGCCCAGGGATGAGGAGAGAAAGTTTGTTTTACAAATATAAAA...... TGG..... 2 AGTGGGG.CCCTGGGCTAGGAGGGGGAAGTCTGTTTTACAGAGGTCGAAG ...... AATAGG...... TC 1 AGTGGGG.CCCTGGGCTAGGAGGGGGAAGTCTGTTTTACAAAGGTCGAAG ...... AATAGG...... TC 3 ...... G ...... AACCTC...... TC 4 ...... AG..TCTGTACCATGAGTTATTTT GACACA...... GA.TGAGGTCCTTGGTACTATTTTCTCACGTGGGAA 7 ATGGATGAATCTAGAT...... 5 ATGGGTTATTTTGGACATCATGGGTTAGTTTGGACATCATGAGTTATTTT GGACAT...... GAGTTAGTTATTTTGTACTGTTTTCTCACATGCGAA 6 ...... CAGCATGAGTTATTTT AGACATCTCAACATGAGTTAGATATTTGGTAGTGTTTTCTCATATGGGAA 2 AG...... CATCATGAGTTATTTT GGACACA...... GAGG.AGGTACTTGGTACTG..TTCTCACATGGGAA 1 AG...... CATCATGAGTTATTTT GGACACA...... GAGG.AGGTACTTGGTACTG..TTCTCACATGGGAA 3 TG...... C...... 4 GG.GATCTCCTAGTAGACAATGTCGGGG.CAAAGAGCAAACACATGA..T ATGTGAGAGAGAAAAG.GAAGA...... 7 ...... GCCAGTATGC...... T GAGTGAAGTCAAAGAG.AACAAAACATCATATGATTTCATTCGTATGTAG 5 GGAG.TCAGCTAGTAGACCAAGTGAGGAAC.AAAGGCAAAAACTGGATTT ATGTGAGAGAAAAGAG.AAAAA...... 6 GGAG.TCAGCTAGTAGACTAAATGAGAAAC.AAAGGCACAGACCAGATTT GTGTGAGAGAAAAGAGGAAAAA...... 2 GGAG.TCAGCTAGTAGACTGAGTAGGGAAA.AAGGGCAAAACCAGGATTT TTGTGAAAGAGAAGAGGAAAAA...... 1 GGAG.TCAGCTAGTAGACTGAGTAGGGAAA.AAGGGCAAAACCAGGATTT TTGTGAGAGAGAAGAGGAAAAA...... 3 ...... AGAGCAGCAGAATGT...... 4 ...... ACTCCAGGCAG...... 7 ATCTAGAAATAAAAACAAATGAAGAAACACCATAAAACAGAAACAGACTC ATAGATTCAGAGAACAAACTGGTGGTTACCAGAGGGAAGCAGGTGGGGGT 5 ...... GCTCCAGGCAA...... 6 ...... ACTGTAGGCAA...... 2 ...... GCCCCAGGTAG...... 1 ...... GCCCCAGGTAG...... 3 ...... CTGCCAGG...... 4 ...... 7 ACGAGCTAGTAGGTAACAGAACTTAAAAGGTACAAACATCCAATTATAAA TAAGGCATGAGGACATACTGTGCAGTATAGGGAATACTGTGACTAGAACT 5 ...... 6 ...... 2 ...... 1 ...... 3 ...... 4 ...... TGAGGA..GTGGGTCTTTTTGGCACACTTCTG.GCTCAACTGCTGAGTCA 7 ATAGCATTAATAGTTCTGTATGGTGACAGACAGAGAGGAACTAGATCATC TGAGAATGATGAATCTTGTTTT.ACAAATCTAGGGTAAACTATGG..... 5 ...... TGAGAATAGTGAATCTTGTTTGCACATTTTTGAGGTAAACTGTTGAGCTG 6 ...... TGAGAACAGTGAATCTTGGCTGCACATTTTTGGGCTGAACCAATGAGCTG 2 ...... GGAGAATAGTGAATTTTGTCAGT...... 1 ...... GGAGAATAGTGAATCTTGTCAGT...... 3 ...... ATCTA.T.A...... 4 GCTG..GCA..A...G..CGAGCATGGGT.GTTCTGTCC...... CT TCC...... AAGGCTTTCTGTAGTATCAAG...... GA 7 GCTGCTGGCAGAGAGG..CCAGTGTGTGC.ATTCTGCCCTTGAAAAGACT ACCGGGGGTGTCAAGGGCAACGGTTGAGGTTGTTTACAGATTCCCCAGAA 5 GCTGCTGAC..AGAGG..CCAGGGTGTGC.CTTCTGTCCTTGAAAGGACT GCCTGGGGAGTAGAGGAAAACCATTTTGGTTGTTTAAAGATTCTCCAGGA 6 CCTGTTGGC..GGAGGAGCCACTATGTGAGC.TCTGTCCTTAAAAGGACT GCTTGGT.ATCAAA.GAAAATCATTTGGGTTGTTTATAGATTGTCCAGGA 2 ...... CTTGTCAGTGGT GGCGAGGCTG..GA...... 1 ...... CCTGTCAGTGGT GGCGAGGCTG..GA...... 3 ...... TG..AA......

152 Results

4 A...... AACCTCCCAGGGG 7 AGC..GC.AGAGTTTATCCAAAGAATATT.GCTTCTC.AATCAGTTCAGT TTTGTCCCCC.....A...... G...... GGG. 5 AACATGG.AGAGTTTAGTCAAAGAGCACTAG.TTTTCAACCACGGGCAAT TTTGCGCCCCCCCCCACCCCCACCGCCTTCCCCCACCAACCGCCCCGGGA 6 AACTTGGGA.AGTTTAGCCAAGGAGCACTAA.TTCTCAAGTAGGGGCAAC TTTGTGCCCCCACT...... G...... CACCCCAGGGG 2 ...... 1 ...... 3 ...... 4 TATTTGGCAACACCGGGCGACATTTATAGTTGAAA...... GCG TATGAGAACCGAGCACCTACCAG...GTGTA.AGCCAGGGATTCCACGCA 7 TATTTGGCAAGGTCCGGAGACA.TTTTAATTGTCACAATTGG.GAGTGTA TGTGGGATCCTAGCATCTA...GGATGCCAGAGGCCAGGGATTC.TTTAA 5 TGTTTGGCAATGTCTGGAGACGTTTTTAGTTATCACAACTGG.GAATGTC TGTGGGATACTGGCATCTA...G..TGCGTAGAGCCAGGGCTTCTTGTAA 6 TGTTTGGCAATGTCTGAAGACGTTTTTAGTTGTCCTGACGGGTG..TGTA TGTGAGATATTAGCGTCTG...... GGGTAGCGTGAGGGATTCTTCTGA 2 ...... CTCCTG...G..TCTGTAGACCCCGG...... 1 ...... CTCCTG...G..TCTGTAGACCCCGG...... 3 ...... CACCTC...C..ACCCAAGCCAA...... 4 ACACGCGGCCGTG.....CACATAGGGCAGCTCTGCATCGCAGTCATGCA GCCCGACCGTCAGCTGCGAATGTGGCTGCACAGCCCACAG..GTTCGCGT 7 ACCT.....CTT..ACAAAGCACAGGACAGATTCTGA...... CAGCAAAGGAAGATCCAGCCT..CAAACAT..CAACCA.. 5 ACAT.....CCT..ACAACACACATGACATCTCTCTA...... CAACAAAGAATGATCCAGAAC..CAAACAT..CAACC... 6 ACAT.....CTTGTAATGCACAGATGG...CTCTCTA...... CGACAAAGAATTACCCAG..C..CCATAATGGCAACC... 2 ...... CGG...... 1 ...... CGG...... 3 ...... 4 GTGCAGCTGCGTCAGAGCTTCCACTTGGGCCACTGCTCTGCAGCGTC..T ...... 7 ...... CC.AGAACCCCACTGTGGAG..TACGT GGGCTCCCCGTGTCTTGTCCTCA.GT..CATTGCCCAAATGCCTCAGCCT 5 ...... CTGAGAACACAGCTGTGGAGCCTA..T GGGCTCTCTGTGTAGTGTCCCCACGT..CCTTGCATGGTCACCTCCCCCT 6 ...... GCGAGGACCCTGTGGTAGAGCG....T GGACTCTCTGTGTGGTGTCCCCAGGT..CCTTACATGGGCATGTCCTCCT 2 ...... GCTCCCCCTGTGTTGTGTCTGCTTGGGGCTTTGCATCTGCACCTGCTGTG 1 ...... GCTCCCCCTGTGTTGTGTCTGCTTGGGGCTTTGCATCTGCACCTGCTGTG 3 ...... CCACC...... 4 CCCTCAGCCGAAT.TCCCCAC...... A.CCATGT 7 T....AGGCCAGC.TCTGCACCATTTCCCGCATT.AG...... AAT...... TTCTCA.CTGTAT 5 CAGTCAGGCCAGG.TCCACACCATTTCT.GTCGTCAG...... AAT...... TTCTCA.AGATAT 6 TGGTCAGGCCCAG.TCCACACCATTTCC..TCATCAG...... ACT...... TTCTCA.TAA... 2 CC...... CTAGGGTTCACACCATTTCT.CTCATCAG...... AAT...... TTCTCACAG.TAT 1 CC...... CTAGGGTTCACACCATTTCT.CTCATCAG...... AAT...... TTCTCACAG.TAT 3 ...... TCCCACAATATCC.CTCATCAGTTTCTCCTGGCGC ACTGATGGTGATTTGAGGGGCTAACACTTGACAGTTATTGTCACAG.GAG 4 ACCG..TATGCATTTG.AGAAGGCGGGAATTATAGAGA.TACTAGT.ATG ATTCA.ATTATTAAACAGA.ACTGAATTATAAAGTCTC...... ATCG.. 7 ATTGTGCTTATATTAAAAAAAAAATGAAAATTTAGAAA.TA.TTTTTATG ATT.AGACTATGGGAAAAAGATTGACTGTTAAAATGTCCTTTGCATCC.A 5 ATTG..CACACGTTTT.AAAAAACTGAAAATTTAGAGA.TA.TTTT.ATG GTT.AGATCATGAAAAAGA.ACTAAATGTTAAAATGTCCTCAGCATCC.G 6 ...... T.AGCAAACTGAAAATTTAGAGGTTA.GTA..... GTT.AAATTATGAAAAAGG.ACTGAGTATTTAAATATCTTCAGCACCC.A 2 ATTT..TACACGTTTT.AAAAA.CGGAAATTTCAGAG...... TTT.ATG GTT.AGATTATGAAAAGGG.ACTGGATGTTAAAATGTCCTTAGCACCC.A 1 ATTT..TACACGTTTT.AAAAA.CGGAAATTTTAGAG...... TTT.ATG GTT.AGATTATGAAAAGGG.ACTGGATGTTAAAATGTCCTTAGCACCC.A 3 ATTA..CAAAG...... AGGGG.CCAA.ATGTTAAAATGCCCTCATCATCTAA 4 ...... GGATGAT..CTTT....CTTGCATTGTATCC.AACAA GTAAGCAAACAATA.....TATGCTATCCGC...... 7 A..AAGCCCTCATCAAAATG..TTATTTATCATGAATTATGCCAAAGTAA ATAAGCAAACAATG.....TGTTATTTC..CTTTTTTTTTTTTTTAATGT 5 G..AAGGCCTCGTGAAAATG..TTAT....CATGAATTATACCAGAACAG ATGAGCAAACAACG.....TGTTATTTC..C...... 6 A..AAGGCCTCAC.AGAATG..TTATTTACCAAGAATTAGGCCAAAACAA ATGAGCAAT....G.....TGTTATTTG..C...... 2 AAAACCACATCATCAAAATTATTTCT....CATGCATCCTACCCAAACAA ATAAGCAAGAAACAATAAATGTCATTTG..C...... 1 AAAACCACATCATCAAAATTATTTCT....CATGCATCCTACCCAAACAA ATAAGCAAGAAACAATAAATGTCATTTG..C...... 3 GAGGCTACATC....AACATGTTTAT....CATGAATCACAGCACAATAA GTGAGCATCCAATAAACATT...ATTTG..A...... 4 ...... 7 GGTGGGGGTTGTCTTACAGTTTTGAAAGAGCTTACCTACCCTCAACTTTC CTCATTCCTGTTTAGTTTTAAACAAACAAAAAGCAGCAAAAATCAAAAAC 5 ...... 6 ...... 2 ...... 1 ...... 3 ...... 4 ...... CTTTTGAAAAGTGTTCAGA. 7 AAAGCAAAATTTTATCTATCCCCAGTGCAGGGGGGGAACCCCAACTTAAC TACCCAGGTCTAAACCCGGTCTGTTATTTCCTTTTGAAAGGAATTCAGG. 5 ...... CTTTTGAAAAGTATTCAGA. 6 ...... TTTGCGTAAAGCATTCAGA. 2 ...... CTTTTGAAATGTATTCAGA. 1 ...... CTTTTGAAATGTATTCAGA. 3 ...... CTTTTGAAGAGTATTCAGCA 4 ...... GT...TG TG..TA...... TGT.TCGTGTGTC...... TGTGTATGCAT 7 ...... AG...TT ...GTG...... AACC...TGTGTG...... TGTGTGTATGT 5 ...... GT...TG TGGGTG...... TGTCT.GTGTGTC...... TGTGTGTGTGT 6 ...... GG...TA GGTGTGTGCGCGCGTGCACTTGTAT.GCGTGTC...... CGCGTGTGT.. 2 ...... GCG.GTG TGTGTG...... TGTGT.GTGTGTGTGTGTGTGTGTGTGTGT 1 ...... GCG.GTG TGTGTG...... TGT...... T 3 TACACACACACACACACACACACACACACACACACACATATATGTGTGTG TGTGTG...... TGT...... G 4 TGATTGGC...... GA...GTAAA.CTCCCTCTGTT AGGAGTT.AC.AATA.TTCCAGTTTTAGATAAGACTTTATATTCAAACCC 7 TACCTGGA...... GA...GTAAATC.TCCTCTGTT AAGAGTTTACAAATA.TTCCAGTTTTACATTGGAGTTCATAATCAAACCC 5 TGACTGAA...... GA...ATAAA.CTTCCTCTGTT AGGAATTTACAAACA.TTCCAATTTTACTTAAGAGTTTATATTCAAACCC 6 TGATCGAA...... AA...GGAAA.CTTCCTCTGCT AGGAATTTCCAGGCA.TTTCAATTTTGCATAAGAGTTTATATTCAAACCC 2 GGACTGAA...... GAC..A.AAA.TTTCCTCTGTT AGGAATGTATAAACC.TTGCAATTTTATCTAAGATTTTATACTAAAATTC 1 GGACTGAA...... GAC..T.AAA.TTTCCTCTGTT AGGAATGTATAAACC.TTGCAATTTTATCTAAGATTTTACACTAAAATTC 3 TGTGTGTATGTATTTGTGCATGCATACAGAGAGT.GAA.ATTTTTCTATT CAGAAT.TGATGACGTTTCCAATTTGGTCTAAGATTATATACTCAGACTC 4 C...... ATAG...... CTGAAATCATCAAC...... 7 A....GTGAAATAAAATAAAAA...... C....AT...... AAATGT 5 A....GTGAAATAAAATAAAAA...... CCCAAATGTTTAAAAGGC GAAAGATTTATACGATCTGAAGAGAAACTAGAGTATAAAAACCCAAATGT 6 AACGGGGGAAA.AAAATGAAAG...... CATAAATCTTTAAAA... .AAGAATCTTTAA...... 2 A....ATGAAATGAAATGAAAA...... CGTAA...... GC 1 A....ATGAAATGAAATGAAAA...... CGTAA...... GC 3 T....ATGACACCATATGTAAATACAAATGTCCAGAG...... TA

153 Results

4 ...ATTTAATCAGGA.TTTTAAAT.ATTAGTGAC...... TTTTGTT....CG.. 7 TTAGAATAATTATGCCTTTTTTTAAACG.GTGAC...... TCTTGTT....TC.. 5 TTAAAATAATCATGA.TCTTAAAAAATTAGTGAC...... TTTTATT....TG.. 6 ..AAAATAATCACGG.TTTTAAAAAATGAGTGAC...... CTCCGTT....TG.. 2 ATTTAATAGTGGT.....TTTAAAAATCAGTAAC...... TTTTGTTCATCTG.. 1 ATTTAATAGTGGT.....TTTAAAAATCAGTAAC...... TTTTGTTCATCTG.. 3 ATTTCTTTG.GCT.....TTTGAAAATTAGTGACCAGCCTAGTTTCACTT CTGCTGCTGTGATAAAAACACAATTTCAGGCTCCATTTTGTTCCTGCGGG 4 ...... 7 ...... 5 ...... 6 ...... 2 ...... 1 ...... 3 GAATTCAGGGATGCAGGAACTTGAAGTTTCTAGTCACGTCATAACTACAG TCAAGAGCCAAAGGGAAAGAAATGCTTCCGGGCTTCATGCTCAGCCATTC 4 ...... 7 ...... 5 ...... 6 ...... 2 ...... 1 ...... 3 TCTATACTCTGACACAATCCAGAGCTCCAGGCTCGGGAATGTTCCTGCCC ACTTTCAGATTGAGTCTTCTCATATCAACCAAGGCAATCAGCAGTCCCCC 4 ...... 7 ...... 5 ...... 6 ...... 2 ...... 1 ...... 3 ACTGTCACATCCACAGGCCAATCTGACCTAGGCAATCCCTCATCGAGATT CTTTGCCCAGGTGACTCTAGTTTGGATCAAACTAAGCACACAGTGACTTT 4 ...... TCTCTGTC..TTCTTTTCAGAGTGAAGCG...... CCATT...GAGTTC.T..GGCGAGGAAGA 7 ...... TAT..TTC..CTTTT..CA.....GAATGATGAAGGTTGA TCTATGTCTGACAGTAGCACTCTGCTGAAC....T.T..TGGGATGCGGA 5 ...... TCT..GTA..CTTTTTTCAGAATGGAGTGGTGGAGGCTGG TCTTCGTCTGAAAGCAGCACTCCCCT...CAG.TT.T..GGAGATGGAGA 6 ...... TCT..GTATTCTCTTTCCAGAATGAAGTGGAGGATGTTGG TCTGTATCTGAAAGCAGCGCTCCACT...GAG.TT.TTTGGAT...GAGA 2 ...... TGT..TTT..CTTTTT..AGAATGAAGTGATGGAGGCTGG TCTCTGTCTGAAAGCAGTGCTCTACC...AAG.TCCT..GGAGATGAAGA 1 ...... TGT..TTT..CTTTTT..AGAATGAAGTGATGGAGGCTGG TCTCTGTCTGAAAGCAGTGCTCTACC...AAG.TCCT..GGAGATGAAGG 3 GATTTGCCTTTGT..TTT..CTTTTT..AGAATGAAGTGATGG.GGGTGG TCTGCGTCGGAAAGC.....TCTGCT...GAG.TCTC..AGA...... 4 GAAGTCCTTCTGTTGT...... GGATTCAT...GT AGAAGCAGCTGGC..ATTGGC..CAGAGGGG.ATGAA...... CTGTCC 7 GAGTTCATTCCACTTT..ACA..GA.AGAGATCCTGAGAATTAAC...GT GGAAGGA.CT..CAAAATGAT..GAGAGGCG.TGGAGG.CGGTCCTTTTT 5 GTATTCATCCCGTTCT..ATG..GACA.ATATCCGGTGAATTAAC...GT AGGA....CTTGC..ATTGAC..TAGAGGGG.TGGAGA.TGATCCTGTTT 6 GAATGCATTTTGCTTTCCA....GAGA.AGATCCTGTGAATTACCAGAGA AGGA....CTGGC..AATGAC..GTGAAGGGGTGGAGGCTG.TCCTTTTT 2 GAATTCACTC..TGTT..TTGCAGAAA.AGATTCTGTGGATTAATACAGA AGCA....CCAGC..AACAAC..CAGAGGGG.TGGAGAC...TCCTTTCT 1 GAATTCACTC..TGTT..TTGCAGAAA.AGATTCTGTGGATTAATACAGA AGCA....CCAGC..AACA.C..CAGAGGGG.TGGAGAC...TCCTTTCT 3 ...... TGGAAAACAATTCAGA AGGG....AATCT..AGCAGTGACCGGAAGG.AGGAGACAG....TTCTT 4 TTTCTGTCTGTGA...... T..T.....C.T.....GTACAGTTAC GAAGTCTGGCTCCACATCCAGCA...... CAACAG..CTCC.....AAA 7 CTCCAGCTTCTGC...... ACAG.....T.TACAGAGTCTGC...... TTCAGTGTTTAACAAAACAG..TACT.....CCA 5 CTCCTGTTTCTGC...... A..G.....C.T.....GTGATG...... TCCAGTG...... AAACAG..CGCC.....CAA 6 CTCCTGATTCTGC...... A...... CTT.....GGGAAG...... TCCGGGC...... TCTGCGTCCAGCAAGAGCAT 2 CTCCCGATTCTGC...... A..GTCTGGC.T.....CCAA.G...... CCCAGTA...... AAACAG..CTCC.....TGA 1 CTCCCGATTCTAC...... A..GTCTGGC.T.....CTAA.G...... CCCAGTA...... AAACAG..CTCC.....CGA 3 CCACCGATTCTACGCAGATGTGAA..GTCT.GC.G.....TCCA.T...... CACAGAA...... CAGCAGA....C.....TGA 4 CC.CTGCCCCTGGTCGAGCACTGGGTCCAGACTTGGCAAAG.CAGGAAGT CAGAAA..GCGGTTGGGAG...... 7 GCTCTGCCCA.G...... CTGGGTCCGGACA...... GGGTGGT...... 5 GCGCTGCCCCGG...... CTGGGTCCAGTCCTGACAAAGGCAGGAATC CAGATGGGGCGGGGGTGAT...... 6 GTGGGGGCAAAG...... CTGGGTGTAGG.....CAAAGGCAGGAAGT CAGACGG...... 2 GCACTGCTTCAG...... CTGGGTCCAGTCTTGACAAAGGCAGGAAGC CAGCTAGGGTGGGGGCGATAGGGTCAGCGGGTATGTCCCACTGTTGGAGG 1 GCACTGCTTCAG...... CTGGGTCCAGTCTTGACAAAGGCAGGAAGC CAGCTAGGGTGGGGGCGATAGGGTCAGCGGGTATGTCCCACTGTTGGAGG 3 GAGCTGTCCCCG...... CTGGGTGCAGG...... 4 ...... GGGATGGGGCAGGCAGATG...... G 7 ...... GTGGTGGGGC.G...GCTG...... G 5 ...... GTGATGGGGT.G...GGTG...... G 6 ...... T.G...GGTG...... G 2 TCACTGGTATTCTGTTTGTTTTTGTTTTGTTTTGTTTTGTTTTTTGAAAC AGGGTCTCGTTCTGTCGCTTAGCTGGAGTGCGGT.G...GTGT...... G 1 TCACTGGTATTCTGTTTGTTTTTGTTTTGTTTCGTTTTGTTTTTTGAGAC AGGGTCTCGTTCTGTCGCTTAGCTGGAGTGCGGT.G...GCGT...... G 3 ...... AGCTAGAGGTCTGG.G...GTGAATCTACG 4 ...ACC....CCACAGTCTGAGG....CTGCTGTTCTCCAAGCACCGGC. TGCCTCCACCCACGCGTGGTT.AT.TTTCTCTGCAGTGCTGAGGC..GGT 7 CAGA.C....CTGCAGGGGGAGG.TCCCTCC...... ACCCACAATG. GACATGC...... TCTTTTTAGGGCAAGGGG..TT. 5 ...ACC....TTGCAGTGTGAGG.TCACTGCTGTTCCTGAAGCACTAGC. TGCCTCCACCCACACGTGGGC.G.CTTTCTCTTTAGTGCAAGGGT..GG. 6 ...AAC...... TCGCTGCTATTCTTAGAGCACC.GCT TGCTTCCACCTACATGGGGAC.A.CTTACTCCATAGTGCAGAGGT..GAC 2 ...ATC....ATG...... GCTCACTGCTATTCTTGAAGCACT...... CCACCCACCTGGG..C.TACTTTTTCTTTAGTGCAGAGGT..GCA 1 ...ATC....ATG...... G..CACTGCTATTCTTGAAGCACT...... CCACCCACCTGGG..C.TACTTTTTCTTTAGTGCAGAGGT..GCA 3 ...ATCCCACATGAGAT...... CCCCACTGCCTTTGAAGCACTGGCT GCTT.CTACTAGCACATG..CATGCTTTTCCTGGGTTGCAGAGATAAGCA 4 .GC.ACTGCCTCCTTGCAGGCAAGATCTG...C.T.AGCCTGTGCTGCAG ...... GAGCAGGGAGAGGCAGGTGGCCGGGGGAGGGA.GC.GCCG..AG 7 ...... CTCTCAGGCAGGA..TCCCGC.C.AACCTGGGCTGGAG GAGAGAGAACAGGGAGAGGC...CAA....GTGAGGGA.GA.GTTC..AG 5 ...... CTTTTAGGCAGAA..TT...C.T.AACCTGTGCTGGTG GAGAGAGAAGAGGGAGAGGCAGGCAGCTTGGTGAGGGC.GA.ACTG..AG 6 ...... CTTTCAGGCGGG...TC...C...ACTCCGTGCAGGTG GGAAGAGT.GT...... GCAGGCAGGAGGTGAGGGACCG...CAC..AG 2 .CT.GTCTT...CTTTTAGGTGGGA..TCG..CGTAAGCATGAGCTGGT...... AGAGCACGGAGTGGCAGGCAGCCAGGTTAGGA...AGACTA..AG 1 .CT.GTCTT...CTTTTAGGTGGGA..TCG..CGTAAGCATGAGCTGGT...... AGAGCACGGAGAGGCAGGCAGCCAGGTTACGA...AGACTA..AG 3 TCTGGCTTT...GTTTTAGGCAGGT..TCT..GTTAACCCC.AGCTGAT...... GGGGTGAATCCAAGCT.GCATCAGGGTAGCCA...AGGGGAGGAG 4 ...... CTGATCACTG..TGAAATTCCC..CTTCCCTC...... TGCCATTGCACTTGATC...TACCTCTA.AGCCTGTGGGTGAGGAA 7 ...... CCAGTCACT...... CTCCTCC..CTCTCAACCCTACATTT ...... GACC...TACCTCCAGA.CAAGGCTGTGAGGAA 5 ...... CCAATCTCTCCCTGACCCCGCC..CCCCCCACCCCACCCTT .CCAT.CCATTGTATTGGTTG...TACCTCT.GAGCAAGGCTGTGAGGAA 6 ...... CTAATCACTCACTGAAATC.CT..TCTCCCACTCGCCCAGG .CC....CGATGTGATGTATGATCTACCTCT.AAGCAGGACTGTGAGGG. 2 ...... CCAATTATTCACTGAAGTCACCCTCCTCCCCTCCCACCATT .CGAT...... TTGATC....TACCTCT.AAGCCAGGCTGTGAAGAA 1 ...... CCAATTATTCACTGAAGTCATCCTCCTCCCC.CCCACCATT .CGAT...... TTGATC....TACCTCT.AAGCCAGGCTGTGAAGAA 3 GTAGATGGACCAGTTGTTTACTGAAGCCCTC.TC..CCCA.CTCACAAGT AC.AT...... CTGATG....GACCCCT.GAGCCAGGGCAAGAGGAA

154 Results

4 AAGA...... A.GGT.ATTTCACAAGACTTAAACGGCATAG. .CTGTGT.CTATTTCCATGATC.....TGTACTTGGG...T..GTCTTGG 7 AAGGCGTGCACTTTGTCAAAGGT.AATTAA.CAT...T...... GT TCATGT..CTACTTCCAAGACACGTTGACTTGTCACA...T..CTCG..G 5 AGGG...... AAGGC.ACGTTA.AAGACCTAAGCGGCCTAGT GCGGCG..CTACTTCCATGACC.....ACTAGCTGCA...T..CTCTCAG 6 ...... AGGC.ACCTGG.GAGACCAAAGCAGCTAAGT TCTGGGT.CTGGTTCCAGAACC.....TGCGGCTGCA...TA.C.CTTAG 2 AAGG...... AAGGC.ACTTTAGAAGACCTCAGCAGTGTGGT TCTGTGT.CTACTTCCATGACC.....TGTACCTG.A..GT..ATCTTAG 1 AAGG...... AAGGC.ACTTTAGAAGACCTCAGCAGTGTGGT TCTGTGT.CTACTTCCATGACC.....TGTACCTG.A..GT..ATCTTAG 3 AAGG...... AAGGCGACTCC..AAGACCTAAGGAGTGTAGG TCT.TCACCTAACTCTACCACC.....TATACCTG.ACAGTTGAGCTCAG 4 AAAGTCAGCCTTAA.GCCTAT.CAA.CCAATGTCCCTGTATTGTAGATAT GTGTAG.C.TCTAGAAATAAGATTGCT...... 7 GAAACCAGCCTTGGCAC..AT.CAC.CCAATGTTACTGCATTATGTA...... GCACTACAGAAATGAGATGTGTG...... 5 GCAGCCAGCCTTGGTGC..AT.CAC.CCAGTGTTACTGTGTTGTATATA...... G.A.TACAGAAATAAGATGTGTAGGGGCTGGCCCCATGGCTGGGT 6 CAGACCAGCCTGAGACC..CGCCAT.CCAACATTACTGTGTTATGTGGA...... G.C.TACAGAAATGAGATGCGT...... GT 2 CCAGCCAGCCTTAG.GC..AC.ACCACCAAGGTTACTTTGAAATCTATG...... T.A.TA...... TAGC...... T 1 CCAGCCAGCCTTAG.GA..AC.ACCACCAAGGTTACTTTGAAATCTATG...... T.A.TA...... TAGC...... T 3 TCAGGCGTATCGTG.GA...... GT...... ATTATATA...... G.A.GA...... AAGA...... T 4 ...... GATTCAC...... 7 ...... 5 GGTTAAGTTCGCGTGCTCTGCTGCAACGGCCCAGGGTTTCGCTGGTTCGA ATCCTGGGCGTGGACATGGCACTGTTCATCAAGCCATGCTGAGGTGGCGT 6 TTCTTACTTCGC...... CC...... 2 AGTTACAGACGGGAGCTATG...... 1 AGTTACAGACGGGAGCTATG...... 3 GATT...GA...... 4 ...... 7 ...... 5 CCCACGTGCCACAACTAGAAGGACCCACAACTAAAAACATGCAACTATGT ACCGGGGGGCTTTGGAGAGAAAAAGGAAAAATAAAATCTTTAAAAAAAAA 6 ...... 2 ...... 1 ...... 3 ...... 4 ...... AATTATTTTGCATCTCAT.CACTGTTC .TGATGACTCC....CGAAGACCTCTTATGGAAACA...... 7 ...... TTTCTTAATTATTTCCCTGCA..T.AA...TTA CTG.TGTCTCCTGAACAC....CTCTTTTGGAGACA.CA...... 5 AATGAAAGGAGATGTGTTTCTTTAATTATTTTGCATCTCCT.CATTGTTC CTG.TGACTCCTGGACACACACCTCTTTTGGAAACCTCCTT...... 6 ...... CTCCT.CATTGCAC CTG.TGACTC.TGGA...ACACCCCTTTTGGAAACG...... 2 ...... TGTTTC.TTCATTATTTTGCAGCTCCT.CGTTGTTC CTG.TGATTCCTGAACAC....CT.TTTTGGAAA...... 1 ...... TGTTTC.TTCATTATTTTGCAGCTCCT.CGTTGTTC CTG.TGATTCCTGAACAC....CT.TTTTGGAAA...... 3 ...... TGTGTC.TTAATTGTTTCTCATCTT.TGCATTGTTG CTG.AGATGACT....AT....CT.TTTTAAAAAACAAACAAACAAACAA 4 ...... TGAGTCCCTGTGAGTTTTGAGG...... ATACTG.TC.G..TGATTTTGGATGGTCACCCC...... AGGTGT 7 ...... GGTCCCTGTGAATTTTGAAC...... ACA.TG.GC.TCTTCATTTTGGATAGTTTGTCC...... ATTTAT 5 ...... T.TGGGTCCTTGCAAGTTTTGAGT...... ACAATG.TC.A..TCATTTTGGATAGTGTCCCC...... ACTTAT 6 ...... TGGGTCCTTGAGCGTTTTGAGTTTTGAAC ACACTGCTG....TCGTTTGGGATCATTTTCCC...... ACTCAA 2 ...... TATGGGTCTCTGTGAGTTTTGAGC...... ACACTA..CTA..TCACTTTGGATAGTTACTCC...... ATTTAT 1 ...... TATGGGTCTCTGTGAGTTTTGAGC...... ACACTA..CTA..TCACTTTGGATAGTCACTCC...... ATTTAT 3 ACAAACAAACAAACAAACATA....TCTCTTTGAGTTTTGGCC...... AGGGTG..CC...TCTTTTAAGATTGCCACTCATGTGGGTTATTATTGTT 4 .ATTTTCATAAACTTC...... CATCA.GAGA..ATCAT.TC ACACTCCTTAA.CAGCGGCT.CTTG...... 7 .ATTTTCATCAGCTTC...... CATCAGACAATCGT....TA AGACTGCTTAC.CATCTGCTAGAGGTAG..TTCATGTTGAC.TTTTTGTT 5 .ATTTTCGCAAACTTC...... CATT.GGCAA..GTCC.CTG AGACTGCTTCC.CATCTGCT.GTGG.ATTCTACCCCTTGCG.TTTTTATT 6 CAGTTTCTCCAGCTTC...... CATC.AG.AG..GATCACTG AGGCTGCTTAC.CTCGTGC...... ACGCTGGCTCATGGC.TTTTTATT 2 .ATTTTTATAAACTTC...... CATTA.GAGA..ATCA.TTA AGGCTGTTTAA.TATCTGCT.CTGG.ATATTACGCATTG.GCTTTTTGTT 1 .ATTTTTATAAACTTC...... CATTA.GAGA..ATCA.TTA AGGCTGTTTAA.TATCTGCT.CTGG.ATATTACGCATTG.GCTTTTTGTT 3 .ATTGTTATTATTATTATTATTATTGAAGTCATCA.AAGG..ATCA.TTA AGACTACTTAAATTTCTGCT.ACTG.ATACCACACACCA.GCTTTGTGTT 4 ...... ATGGATTGTGTGTC...TCATTACAGATGG.TCCCGAAACAGGAGGGCC 7 ATCTGGGGT..CA...CTA..GCCTTCCTGTAGGACTCAGTGTCTTCAAG CACTGATTATGTATC...TTATTGAAGATGGTTGT.GAAATAGGAAGAAC 5 GTCCAGACT..TG...CTA..GCCTTCCTGTTGGACTCTGAGTCTTCAGG CATTGATTGTGTATC...TGATTGAAGGTGGCTGA.GCAACAGGAAGAAC 6 GTCC...CT..TGGGACTA..GGCTTCGTCCAAAACTCAGTGTCTGCAGG CACTGGTTGTGTATC...TTACTGAGGGTGG.TTG.GAAACAGAAAGAAC 2 GCCTAG..TGCTA...CAA..ACCTTCCTGTGGGACTCAGTGTCTTCAGG CAATGATTGTGTATCCTGTTACGGATGAT...TGT.GATACAAGAAGAAC 1 GCCTAG..TGCTA...CAA..ACCTTCCTGTGGGACTCAGTGTCTTCAGG CAATGATTGTGTATCCTGTTACAGATGGT...TGT.GATACAAGAAGAAC 3 ACCTGG..TATCA...CCAGGA..TTCCTGTAGGACTCAATTTCTT...G AGATGTTTGCGTGTCTTCCTGCAGATGAT...CGC.TAAGCAG..AGAAT 4 CC...TCTCCTCCTTT...TG.AGATAAGCTGTTGGAAGATTAATGCC.. ..ATA..T...CTTTTGGGG..CTAGGAGGAAT..AT..TAACAATGT.T 7 C....ACTC.....TC...AG.AACTAAACTGCTGGAAGTTTAATGCCAG ATCCT..TTGGCAGGTGAGGGTTG.GGGGGAACAAG....AGGGACTG.T 5 C....TCTG.....TT...TG.AAATAAACTGTTGGAAGCTTAGTGCC.. ..CAC..T...CATTTGCAGGGCG.GGGGGAAT..GT..TAGTGACTG.G 6 CACTTTCTG.....TT...TG.AAGTAAATTGTGGGAGGCTGAATGCC.. ..ACT..T...TGTTTGCAGGCTG.GGGGGAAT..GT..TAGCGACTGTG 2 C....ACTC.....TTCTTTGAAAATAAACTCTTGGAAGCTTT.TG.C.. ..CAG..C...TATTTGGGGGGTA.GGAGGAAT..AT..TAGCAACTG.T 1 C....ACTC.....TTCTTTGAAAATAAACTCTTGGAAGCTTT.TG.C.. ..CAG..C...TATTTGGGGGGTA.GGAGGAAT..AT..TAGCAACTG.T 3 T....TCTG.....TCCTTTTGAAA....CTGTCCAAAACAGT.AT.G.. ..CAAGTC...CCTTGGAGGGACA.GAAGGAAG..CTACT.G..ACTG.T 4 ACTCGTTG...... TCTACAAATCAGGAG....ACAGA GTATTTGTTTTGTT.....GGG..AAAATTGCTTTGCTGGAAG.TTATCA 7 GTTGGTCGCCTACAGACATAGAATTA.CCTGTCAAGAGGGA....ACAGA GTGTC....TTGTTA.....GG..AAAATAACGTTGCTGGAAAATTTCCA 5 ATTGGTCGTCTGTGGATATAGGATT.TCCTGTCATGAGGGA....ACAGA GT..C...TTTGTT.....GGG..AAAATTGTTTCCCTGGAAATTTTTCA 6 .TTGATCATCTACGGACGTAGAATTGTCCAG.CACGAGGAA....ACAGA GT..G...TTTGTTTCCTTGGAAAAAAATTGTTTG.CAGGAAATTTTCCA 2 ATTGGTTGTCTACAGATACAGAATT.GCCTGTTGTGAGGGA....ACTGA TTGT....TTTGTT.....GGG..AAAA...... GAAATTTACCA 1 ATTGGTTGTCTACAGATACAGAATT.GCCTGTTGTGAGGGA....ACTGA TTGT....TTTGTT.....GGG..AAAA...... GAAATTTACCA 3 ATCAGTCACCTATGGATATAG.ATT.GCCTGTCATAAGACAACAGAATAT TTGT....TTTGTT.....GGG..GAAATTGCCTTCCTGGAAATTTTCTG 4 GGAGAAGGAGT....T.T.GTGGTC.ATAAAAGAAGAAGGTCCTA...TC CCT..AAT.....AA...... TTAGCGTTG.GGGAG.AAGTCAGTG 7 GGCAA....GT....T.CTGTGGTCATGTAATG.AGAGGTTCATA...GC C...... T.....CACGATCTCAACTGGCCCCGGTGGATG.ATGTCAGGG 5 GGAGAAAGAGT....T.TTGTGCCC.TGTGATG.AGAGACCCTTA...GC CCC..GAT.....ATGTCAGTTAACTTGGCCTTG.GGTGG.AAGTCAGTG 6 GGAGAGAGTGT....TGTGGTCCCA...TAAGG.AGATGTTCTTA...GC TCCAGGAT.....C..TCAGTTAAATTGGCCGTG.GGTAG.ACGTCAGTG 2 GGAGAAAGAGT....T.TTATGC...TGTATTG.TGAGAGATCTC...GC C...... T.....C..TCAGTTAAATGAGCCCTG.GGTTA.AAGTCAGTG 1 GGAGAAAGAGT....T.TTGTGC...TGTATTG.TGAGAGATCTC...GC C...... T.....C..TCAGTTAAATGAGCCCTG.GGTTA.AAGTCAGTG 3 GGAGAGTTAGTCTCAT....TGT...TGTATAA.TGAGAAATTCCCTAGT C...... TGATACC..TCAGATAGATGAGGCATG.GAT.ACAGGTCATTA 4 .TGAGTGGC 7 GAGGGC--- 5 CTGGGGGC- 6 TGGGGGGC- 2 .TGAAGGGC 1 .TGAAGGGC 3 .TGGCAGGC

155 Results

1 ERCVCVCVCVCVCVWT ... 1 RGVETPGSPDSAVWLQAQXNSSXALLQLGP ... TQMXSLRPS------QNSRLRAVPAGC ... 2 ELQARECSCPLRLSLL ... 2 RG 3 -SCVCSCVCVCIDWRV ... 3 RGDELSFLSVILYSYEVWLHIQHNSSNPAP ... 4 QSCGCVCVSVCVL--- ... 4 RGVEMILFLLFLQLXCPVKQRPSAAPAGSS ... 5 ELXTCVCVYVT--WRV ... 5 RGVEAVL FLQLLHSYRVCGSVXQNSTPALP ... 6 EVGVCARALVCVSACV ... 6 EGVEAVLFLLILHLGSPGSASSKSMWGQSW ...

GVGGILATVLVVYRYRIACCEGTD-CFVGKRNL----PGERVLCCIVRDLASQLNEPWVKVSVKG 1 ENKLLEAFASYLG 2 LSKTV----CKSLGGTEGLLTVSVTYGYRLPVIRQQNICFVGEIAGLEI-GWESXSHCCIMRNSLVXYLRXMRHGYRS 3 PFEISCWKIN--ALLGLGGILTMLLVVYKSGDRVFVL---LGKLLCWKLS-GEGVCGHKRRRSYPXXLALGRSQCEWQ 4 ENKLLEAXCPLICRAGGMLVTGLVVCGYRISCHEGTESL-LGKLFPWKFG-GERVLCPVMRDPXPRYVSXLGLGWKSS 5 ELNCWKFNARSFGGLGGTRGTVLVAYRHRITCQEGTECLVRKITLLENGQASSVVMXXEVHSLTISTGPGGXCQGRAA 6 XSKLWEAECHFVCRLGGMLATVLIIYGRRIVQHEETECCGLGKKLFAGNFPGESVVVPXGDVLSSRISVKLAVGRGXT

1:chimpanzee 2:mouse 3:rabbit 4:horse 5:cattle 6:dog

Fig. 64: Stretches with similarity to APP family members (yellow and dark yellow) in mammalian regions orthologous to ANNE in order of appearance in the sequence. The orthologuos CV-repeat is best conserved in chimpanzee.

Tab. 15: Calculation of the age of ANNE

1. ANNE has a length of 2094 bp without c3‟Alu 2184 sites - 90 sites from c3‟Alu = 2094

2. of these around 100 (estimated) are not substituted 2094 sites - 100 not substituted = 1994 total substitutions

3. of 1994 total substitutions a fraction was substituted during the past 40 my 0.8∙10-9/site/year · 2094 sites · 25∙106 years = 42 substitutions from 25 mya until now 1.2∙10-9/site/year · 2094 sites · 15∙106 years = 38 substitutions f. 40 mya u. 25 mya

4. the rest was substituted before 40 mya 1994 - 42 - 38 = 1914 substitutions before 40 mya

5. the time it takes to substitute 1914 out of 2094 bp with rodent or chicken rates 4.6∙10-9/site/year · 2094 sites · X∙106 years = 1914 substitutions

XRodentRate = 199 my 3.6∙10-9/site/year · 2094 sites · X∙106 years = 1914 substitutions

XChickenRate= 254 my

6. the calculated ages with rodent or chicken rates

Age RodentRate = 40 my + 199 my = 239 my

AgeChickenRate= 40 my + 254 my = 294 my

156 Results

Tab. 16: CLUSTAL 2.0.12 multiple of TTMPs

TTMP Ga --MAIEMKTIQTN---GAEVTGN----GESVACTMPDATEKDGFLEQTTSANPDPEPCTP TTMP Tn MDEDVEMIQISTDRSSGATYFSNERVNGEDVRISFGEAPEVDGLLRPTSGVRERAALERG TTMP Gg -MSWLRACYRDRGPRTETEAMKRQSSGGEHEVIELPEVNGEESTADHKKPLNPQVP---- TTMP Hs isof.2 ------MDLAQPSQPVDELELSVLERQPEENTPLNGADKVFPSLDEEVP .* : :

TTMP Ga PSQGTRTSTLAEICSKSRISRE------RLKLLLII--VVIIVFIAVVFVISLAV TTMP Tn SSE--QTSRPQEEHLMCRIQRELKQAVFPNIFPKVQLWMIIGLLLFIIVIIAVILISLAV TTMP Gg -----ATSKERDQWKSCRKIIFW------KCKLWMVL--TTIFVVLFLVILISLAL TTMP Hs isof.2 P----AEANKESPWSSCNKNVVG------RCKLWMII--TSIFLGVITVIIIGLCL : . .. : :* ::: ::: : *::*.*.:

TTMP Ga CSAIYEDSDGDFDSSLFKVPRSFNGSFRLPNRVFTEGLVTLSSNESKGLAAELQHKLADL TTMP Tn CAALHEDEDEKFDPSLFKVPLYHNGTFRLPNQDFTKDLINMSSTQSQVLAASLQEKIAGF TTMP Gg YSNVYTDEDD------YWYTDELLQNYHNFSGKFNLLCGLPHVFSEDIIKRITDV TTMP Hs isof.2 AAVTYVDEDE------NEILELSSNKTFFIMLKIPEECVAEEELPHLLTERLTDV : : *.* : . . . :. : .:::..

TTMP Ga YKSSPALGRYFSQAEIQAFRNG------SVIADYQLMFLMPEEQQDQLRNVTLSREMVYN TTMP Tn YTSSPALGRYFSEASISAFRDGENDQSPSVIAHYRLKFLMPEEEEDQLRNFTLSREMVFN TTMP Gg YSSSPALGRYFRSAKVDYFSNE------SSTVFYQLEFFVPPSTEGFMEN-VMNPDFIRN TTMP Hs isof.2 YSTSPSLGRYFTSVEIVDFSGE------NATVTYDLQFGVPSDDENFMKY-MMSEELVLG *.:**:***** ...: * . . . * * * :* . :. :. :. ::: .

TTMP Ga VFRQFLYDLE------SEDSGPMYIEPVSLHMFVRV TTMP Tn VFRQLLYDQE------ADAAEELYIAHDSLRTF--- TTMP Gg VLLQNIYDEEDTSNPGTSECTRLKLDPVSLTST--- TTMP Hs isof.2 ILLQDFRDQN------IPGCESLGLDPTSLLLYE-- :: * : * : . : : **

Tab. 17: Clusters upstream and downstream of tetrapodal C3orf52

Human Gene Clusters that Exhibit Conserved Synteny in Elephant Shark

cluster# Hs chr location (outdated?) genes gene entries name 372 3 113,200,332-113,282,621 2 ENSG00000144834 TAGLN3 ENSG00000176040 TMPRSS7 373 3 113,734,236 -113,842,667 2 ENSG00000144848 ATG3 ENSG00000091986 CCDC80

Gene human chr and location medaka chr and location stickleback chr and location TAGLN3 3+:111,717,511-111,732,734 20+:15,537,083-15,539,828 groupXXI+:7,478,290- 7,481,292 TMPRSS7 3+:111,753,693-111,800,116 20+:15,561,501-15,569,340 groupXXI+:7,503,400- 7,508,618 ATG3 3-:112,251,356-112,280,893 scaffold829+:20,737-25,271 groupI-:26,899,191- 26,906,427 CCDC80 3-:112,323,407-112,368,377 21-:13,604,386-13,633,220 groupXVI-:17,945,381- 12+:14,555,970-14,556,696 17,954,249 12+:14,559,308-14,562,488 groupXIV+:7,298,550- 7,301,754 groupXIV+:7,296,709- 7,298,441

157 Results

Tab. 18: BLASTP-hit against CRLF2-isoform 3 with an Alu copy as exon 5

CRLF2 isoform 3, Alu copy (exon 5) highlighted in red MGRLVLLWGAAVFLLGGWMALGQGGAAEGVQIQIIYFNLETVQVTWNASKYSRTNLTFHY RFNGDEAYDQCTNYLLQEGHTSGCLLDAEQRDDILYFSIRNGTHPVFTASRWMVYYLKPS SPKHVRFSWHQDAVTVTCSDLSYGDLLYEVQYRSPFDTEWQTQSRSVTQAGVQWCDLCLL QPSPPRFKRFSCLSLPSSWDYRHPPPRLANFCIISRDGVSPCWPGWSRTCDLR

C3orf52 downstream exon 5 3’-5’ rf1 translation: 1142 to 1208 (+) Aligned to CRLF2 Database location : ENSP00000370978 167 to 233 (+) Genomic location : chr X 1317793 to 1317993 (-) Alignment score : 461 E-value : 1.2e-38 Alignment length : 67 Percentage identity: 80.60

C3orf52 dse5:1142 VTQAGVQXCDLGSPQPLPPRFKXFSCLSLPSSWDYKHLPPCPANFCIFSRDGVSPCWPGL 1201 VTQAGVQ CDL QP PPRFK FSCLSLPSSWDY+H PP ANFCI SRDGVSPCWPG CRLF2 exon 5: 167 VTQAGVQWCDLCLLQPSPPRFKRFSCLSLPSSWDYRHPPPRLANFCIISRDGVSPCWPGW 226 Compared to whole AluJb 5’-3’ rf1 translation: FFFFFFFFFFETGSRSVAQAGVQWRDHGSLQPRPPGLKRSSCLSLPSSWDYRRAPPRPANFCIFCRDGVSPCCPGW

C3orf52 dse5:1202 SRTPDLR 1208 SRT DLR CRLF2 exon 5: 227 SRTCDLR 233 Compared to whole AluJb 5’-3’ rf1 translation: SRTPGLKXSSRLGLPKCWDYRREPPRPA

Tab. 19: EST-entries which contain parts of ANNE Accession No. Origin bp in ANNE DNA Hs#S38397521 HLUNG2 924(start of exon 6)-1113 Hs#S433777 fetal_liver_spleen 1777-2184 (end) Hs#S580576 human tonsillar cells enriched for 1368-1735 germinal center B cells Hs#S730655 ovary tumor 1246-1739 Hs#S2003398 851(before exon 6) -1429 Hs#S4224816 1831-2184 (end) Hs#S11142937 fetal_liver_spleen 1755-2184 (end) Hs#S15491979 B CELLS (RAMOS CELL LINE) 1031-1390 Hs#S19879670 COT 25-NORMALIZED 1007-1252

158 Results

Marker 500 bp 8642213 400 bp TTMP GAPDH 300 bp ANNE1 ANNE2 ANNE3 200 bp 100 bp

Fig. 65: Experimental TTMP transcript expression in 3d HESC9. 4 of 5 targeted amplicons are highlighted cyan in transcript variant 2 below. Exon starts are colored green, the c3‟Alu red. The start of exon 6 (large and cyan font on green highlight) was detected after exon 3 so that exons 4 and 5 are missing as in transcript variant 1. The stop codon for TTMP

isoform 1 is highlighted grey and the first and last bases of the antisense mRNAs in violet. TFIIB and RXR binding sites are in bold violet and red letters, respectively. TTMP transcript variant of isoform 2 gaggcggggaggtcgctcgactcgccggcgctgtggcctcccgcggagccgctcagactttccctgccggcacatggacctgg cccaaccctcacagccagtagacgagctggagctctcggtgctcgagcggcagccagaagagaacacgcctctcaatggtgcc gacaaggtcttcccttctttggacgaggaggtccccccggccgaggctaacaaggaaagcccctggagctcctgtaataagaa tgtggttggaagatgcaaactgtggatgatcatcacctccattttcctaggtgtcattacagtgatcatcataggcttatgtc ttgctgcagtaacttatgttgatgaagatgaaaatgaaatacttgaattatcatcaaacaaaacattcttcatcatgctgaag attccagaggagtgtgttgctgaagaggaattgcctcacctgctcaccgaaaggctcacagatgtgtacagtacatcgccctc tctgagtcgttattttacttcagttgaaatagtggacttcagtggtgaaaatgccacagtaacgtatgacctgcaatttgggg ttccatcagatgatgaaaattttatgaagtatatgatgagtgaggagttggtgctgggcattttgctacaggatttccgtgat cagaatatacctggttgtgagagtctggggcttgatccaacatccctcttgctctatg aatgaagtgatggaggctggtctctgtctgaaagcagtgctctaccaagtcctggagatgaagggaattcactctgttttgc agaaaagattctgtggattaatacagaagcaccagcaacaccagaggggtggagactcctttctctcccgattctacagtctg gctctaagcccagtaaaacagctcccgagcactgcttcagctgggtccagtcttgacaaaggcaggaagccagctagggtggg ggcgatagggtcagcgggtatgtcccactgttggaggtcactggtattctgtttgtttttgttttgtttcgttttgttttttg agacagggtctcgttctgtcgcttagctggagtgcggtggcgtgatcatggcactgctattcttgaagcactccacccacctg ggctactttttctttagtgcagaggtgcactgtcttcttttaggtgggatcgcgtaagcatgagctggtagagcacggagagg caggcagccaggttacgaagactaagccaattattcactgaagtcatcctcctcccccccaccattcgatttgatctacctct aagccaggctgtgaagaaaaggaaggcactttagaagacctcagcagtgtggttctgtgtctacttccatgacctgtacctga gtatcttagccagccagccttaggaacaccaccaaggttactttgaaatctatgtatatagctagttacagacgggagctatg tgtttcttcattattttgcagctcctcgttgttcctgtgattcctgaacacctttttggaaatatgggtctctgtgagttttg agcacactactatcactttggatagtcactccatttatatttttataaacttccattagagaatcattaaggctgtttaatat ctgctctggatattacgcattggctttttgttgcctagtgctacaaaccttcctgtgggactcagtgtcttcaggcaatgatt gtgtatcctgttacagatggttgtgatacaagaagaaccactcttctttgaaaataaactcttggaagcttttgccagctatt tggggggtaggaggaatattagcaactgtattggttgtctacagatacagaattgcctgttgtgagggaactgattgttttgt tgggaaaagaaatttaccaggagaaagagttttgtgctgtattgtgagagatctcgcctctcagttaaatgagccctgggtta aagtcagtgtgaagggcagctgtgtgcgggcacgagccagagtgtctgcctcagactagatttgacttgagttctttatgacc caggactctggataatgtgaatttgctttcctatttaactagaagatacatgtactatagatcattgtctcattttagtgatt gttccttaaactagtgaaactagtggatttctcttcttcctctttattttctgcatgttaaatgtgaaccttagtgtatttgt attttgtagaaaataatgaaaaattttaatggagaatgatttaaaaacatttacaatacattaaaaaaaaaaaaaaaaaaa

Tab. 20: qPCR data and sequencing products of detected amplicons Amplicon well Fluorophore amplicon ct-value average Stdev

GAPDH C04 SYBR GAPDH 20,86 20,60 0,50 TTMP C05 SYBR TTMP 31,89 32,04 0,31 ANNE1 C06 SYBR ANNE1 34,88 34,40 0,47 ANNE2 C07 SYBR ANNE2 32,89 33,14 0,38 ANNE2 C08 SYBR ANNE2 33,58 33,24 0,49 8642213 D04 SYBR 8642213 57,11 62,04 9,57 GAPDH D05 SYBR GAPDH 20,92 TTMP D06 SYBR TTMP 31,83 ANNE1 D07 SYBR ANNE1 34,37 ANNE2 D08 SYBR ANNE2 32,95 ANNE3 E04 SYBR ANNE3 33,72 8642213 E05 SYBR 8642213 73,07 GAPDH E06 SYBR GAPDH 20,03 TTMP E07 SYBR TTMP 32,4 ANNE1 E08 SYBR ANNE1 33,95 ANNE3 F04 SYBR ANNE3 33,24 ANNE3 F05 SYBR ANNE3 32,75 8642213 F06 SYBR 8642213 55,93

159 Results

Amplicon TTMP Sequencing products using either TTMP fwd or TTMP rev primers

TTMP fwd ------GAA...... 3 TTMP rev A TCCTTTCGGTGAGCAGGGGAGGCAATTCCTCTTCAGTTACACGCCTCTCA 279

TTMP fwd ...... CTCTTCTTGGACGAGGAGGT.CCCCCGGCC 32 TTMP rev ATGGTGCCGACAAGGTCTTCCCTTCTTTGGACGAGGAGGTCCCCCCGGCC 229

TTMP fwd GAGGCTAACAAGGAAAGCCCCTGGAGCTCCTGTAATAAGAATGTGGTTGG 82 TTMP rev GAGGCTAACAAGGAAAGCCCCTGGAGCTCCTGTAATAAGAATGTGGTTGG 179

TTMP fwd AAGATGCAAACTGTGGATGATCATCACCTCCATTTTCCTAGGTGTCATTA 132 TTMP rev AAGATGCAAACTGTGGATGATCATCACCTCCATTTTCCTAGGTGTCATTA 129

TTMP fwd CAGTGATCATCATAGGCTTATGTCTTGCTGCAGTAACTTATGTTGATGAA 182 TTMP rev CAGTGATCATCATAGGCTTATGTCTTGCTGCAGTAACTTATGTTGATGAA 79

TTMP fwd GATGAAAATGAAATACTTGAATTATCATCAAACAAAACATTCTTCATCAT 232 TTMP rev GATGAAAATGAAATACTTGAATTATCATCAAACAAAACATTCTTCATCAT 29

TTMP fwd GCTGAAGATTCCAGAGGAGTGTGTTGCTGAAGAGGAATTGCCTCACCTGC 282 TTMP rev GCTGAAGAT.CCA...GAGAGTGTGGGAAAAG------1

TTMP fwd TCACCGAAAGA 293 TTMP rev ------1

Amplicon ANNE1 Sequencing products using either ANNE1 fwd or ANNE1 rev primers

ANNE1 fwd ------GCCGGTCGGCAGAG...... CAGCTAG 21 ANNE1 rev TGCACTGCTTCAGCTGGGTCCAGTCTTGACAAAGGCAGGAAGCCAGCTAG 115

ANNE1 fwd GGTGGGGGCGATAGGGTCAGCGGGTATGTCCCACTGTTGGAGGTCACTGG 71 ANNE1 rev GGTGGGGGCGATAGGGTCAGCGGGTATGTCCCACTGTTGGAGGTCACTGG 65

ANNE1 fwd TATTCTGTTTGTTTTTGTTTTGTTTCGTTTTGTTTTTTGAGACAGGGTCT 121 ANNE1 rev TATTCTGTTTGTTTTTGTTTTGTTTCGTTTTGTTTTT.GAGACAGGGTCT 16

ANNE1 fwd CGTTCTGTCGCTTAGCTGGAGTGCGGTGGCGTGATCATGGCACT 165 ANNE1 rev CGTCGCGCAG...... ATTCC------1

Amplicon ANNE2 Sequencing products using either ANNE2 fwd or ANNE2 rev primers

ANNE2 fwd ------GGGG...... 4 ANNE2 rev TGTGTTTTTGTTTCATAGGGGGGAGATACAGAGCTTAGGGACATTTAAGT 227

ANNE2 fwd ...... TC...... 6 ANNE2 rev AGACGCGAACCCCCCCCCCCTGGTCTTTTAAAGGGCCCTCCCATTCTTCG 177

ANNE2 fwd ...... ATA 9 ANNE2 rev CGCCCCGCTAAGAGGCAGGCAGCCAGGTTACGAAGACTAAGCCAATTATT 127

ANNE2 fwd TTACTGAGTCATCCTCCTCT.CCCCACCATTCGATTTGATCTACCTCTAA 58 ANNE2 rev CACTGAAGTCATCCTCCTCCCCCCCACCATTCGATTTGATCTACCTCTAA 77

ANNE2 fwd GCCAGGCTGTGAAGAAAAGGAAGGCACTTTAGAAGACCTCAGCAGTGTGG 108 ANNE2 rev GCCAGGCTGTGAAGAAAAGGAAGGCACTTTAGAAGACCTCAGCAGTGTGG 27

ANNE2 fwd TTCTGTGTCTACTTCCATGACCTGTACCTGAGTATCTTAGCCAGCCAGCC 158 ANNE2 rev TTCTGTGTCTACT.CCATGAGCCCT...... TT------1

ANNE2 fwd TTAGGAACACA 169 ANNE2 rev ------1

Amplicon ANNE3 Sequencing products using either ANNE3 fwd or ANNE3 rev primers

ANNE3 fwd ------AGGCGGTGAGATATTAGC.AC 20 ANNE3 rev TCTTGGAAGCTTTTGCCAGCTATTTGGGGGGTAGGAGGAATATTAGCAAC 132

160 Results

ANNE3 fwd TGTATTGGTTGTCTACAGATACAGAATTGCCTGTTGTGAGGGAACTGATT 70 ANNE3 rev TGTATTGGTTGTCTACAGATACAGAATTGCCTGTTGTGAGGGAACTGATT 82

ANNE3 fwd GTTTTGTTGGGAAAAGAAATTTACCAGGAGAAAGAGTTTTGTGCTGTATT 120 ANNE3 rev GTTTTGTTGGGAAAAGAAATTTACCAGGAGAAAGAGTTTTGTGCTGTATT 32

ANNE3 fwd GTGAGAGATCTCGCCTCTCAGTTAAATGAGCCCTGGGTTAAAGTCAGTGT 170 ANNE3 rev GTGAGAGATCTCGCCTCTCAGT.AAATGACCG------1

ANNE3 fwd GAAGGGCAGC 180 ANNE3 rev ------1

Tab. 21 :Antisense mRNA transc. factor binding sites,>85% similarity Factor (very high similarity) Start(pos.rev.)End(pos.rev.) String Rev.Comp. TFIIB 1444 1451 ACTGAGAGctctcagt TFIIB 1371 1378 TCTGAGGCgcctcaga RAR-beta 2409 2416 TGACCCTAtagggtca RAR-beta,RXR-alpha,RAR-alpha1 2382 2382 2381 2389 2390 2390 GTGACCTCCAtggaggtcac RAR-beta,RAR-alpha1,RXR-alpha 2013 2012 2012 2020 2021 2020 TACAGGTCATatgacctgta RAR-beta:RXR-alpha 1433 1444 GGGCTCATTTAAttaaatgagccc RAR-beta 1340 1347 CTGGGTCAtgacccag Alu: derived of 7SL RNA, AluJb earliest Alu type 7SL retinoic acid response element hexamer sites: AGGCTGgaGGATCGcttgAGTCCAggAGTTCT

Tab. 22: Possible detection of 8642213

Targeted Amplicon TTMP/ANNE (1840 bp) not found Best hit for both sequencing products potentially transcript of Map Viewer Gnomon model: 8642213 Model predicted by Gnomon on Homo sapiens, GRCh37.p2-Primary Assembly (chromosome 1, NT_032977.9): mCG114749, isoform CRA_a [Mus musculus] Chromosome 1 45.2 mbp

Sequencing products using either TTMP fwd or ANNE3 rev primers

TTMP fwd ------CCAGT...... CTGGCGATGCTCTCAGA 22 ANNE3 rev TTAAAAGCCTTTTAAATGGGGGCCAGAAATCCCCGGCCGAAGCTTTCAAG 450

TTMP fwd G..CATCAACAATGCCGAAAAGAGAGG.CAAACGCCAGGTGCTTCTTAGG 69 ANNE3 rev GGCC.TTAACAAATCCGAAAAAAGAGGGCAAACCCCGGG.GCTTTTTTGG 402

TTMP fwd CCATGCTCCAAAGTCATCGTCCAGTTTCTCACTGTGATGATGAAGC.ATG 118 ANNE3 rev CCCAGCTCCAAAATTATTGTCCCGTTTTTCCCCGGGAAGAAGAAGCCA.G 353

TTMP fwd GTTACATTGGCTAATTTGAAATCACTGATGATCACAGAGCTGGGAAAATT 168 ANNE3 rev GGTTCATTGGGTAATTTGAAATTCCTGAAGGTTACAGAGCCGGGGAAAAT 303

TTMP fwd GTTGTG.AACCTCACAGGCAGGCTAAACAAGTGTGGAGCGATCAGCCCCA 217 ANNE3 rev TTTGGGAACCCTCCCGGGC.GGGTTAACAAGGGGGGGGGGATTAACCCCC 254

TTMP fwd GATTTGATGTGCAACTCAAAGATCTGGAAAAATGGCAGAATAATCTGCTT 267 ANNE3 rev GATTTTATGTGCAAATTAAAAATTTGGAAAAAAGGCCGAAAAATTTGCTT 204

TTMP fwd .CCATCCCGCCAGTTTGATTTCATTGTACTGACAACCTCAGCTGGCATCA 316 ANNE3 rev TCC.TCCCCCCCGTTTGATTTTATTGTTCCGGCAACCCCCGCCGGCCTCC 155

TTMP fwd TGGACCATGAAGCAAG.ACGAAAACACACAGGAGGGAAAATCCAGGGATT 365 ANNE3 rev TGGGCCCTGGAGCCAGGAGGAAACCCCCCGGGGGGGAAAA.CCCGGGATT 106

TTMP fwd CTTTTTCTAGGGATGTAATACATATATTTACAAATAAAATGCCTCAAGGA 415 ANNE3 rev TTTTTTTTAGGGAAGTAAAAAAAATATTTTAAAAATAAAA.CCCCAAGGG 57

161 Results

TTMP fwd CAAAAAAAAAGGGGGGCGGGGC.TTGGAGGTCCCCCCCTAGAATTCCCGC 464 ANNE3 rev AAAAAAAAAAAGTGAGCTGGGCATGGCA.GTGCACACCTAGTAATCCAGC 8

TTMP fwd CCCCCCGGGGGGTTAATA 482 ANNE3 rev ...... GTAGAGT----- 1

TTMP fwd product: 96% identity to RPS15AP11 ribosomal protein S15a pseudogene 11 in 8642213 transcript

Features flanking this part of subject sequence: 1549 bp at 5' side: 40S ribosomal protein S8 3513 bp at 3' side: bestrophin-4 Score = 765 bits (414), Expect = 0.0 Identities = 449/465 (97%), Gaps = 6/465 (1%) Strand=Plus/Minus

Query 6 CTGGC-GATGCTCTC-AGAGCATCAACAATGCCGAAAAGAGAGGCAAACGCCAGGTGCTT 63 ||||| ||||||||| |||||||||||||||||||||||||||||||||||||||||||| Sbjct 15218287 CTGGCAGATGCTCTCAAGAGCATCAACAATGCCGAAAAGAGAGGCAAACGCCAGGTGCTT 15218228

Query 64 CTTAGGCCATGCTCCAAAGTCATCGTCCAGTTTCTCACTGTGATGATGAAGCATGGTTAC 123 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 15218227 CTTAGGCCATGCTCCAAAGTCATCGTCCAGTTTCTCACTGTGATGATGAAGCATGGTTAC 15218168

Query 124 ATTGGCTAATTTGAAATCACTGATGATCACAGAGCTGGGAAAATTGTTGTGAACCTCACA 183 |||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 15218167 ATTGGCGAATTTGAAATCACTGATGATCACAGAGCTGGGAAAATTGTTGTGAACCTCACA 15218108

Query 184 GGCAGGCTAAACAAGTGTGGAGCGATCAGCCCCAGATTTGATGTGCAACTCAAAGATCTG 243 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 15218107 GGCAGGCTAAACAAGTGTGGAGCGATCAGCCCCAGATTTGATGTGCAACTCAAAGATCTG 15218048

Query 244 GAAAAATGGCAGAATAATCTGCTTCCATCCCGCCAGTTTGATTTCATTGTACTGACAACC 303 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 15218047 GAAAAATGGCAGAATAATCTGCTTCCATCCCGCCAGTTTGATTTCATTGTACTGACAACC 15217988

Query 304 TCAGCTGGCATCATGGACCATGAAGCAAGACGAAAACACACAGGAGGGAAAATCCAGGGA 363 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 15217987 TCAGCTGGCATCATGGACCATGAAGCAAGACGAAAACACACAGGAGGGAAAATCCAGGGA 15217928

Query 364 TTCTTTTTCTAGGGATGTAATACATATATTTACAAATAAAATGCCTCAAGGACaaaaaaa 423 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 15217927 TTCTTTTTCTAGGGATGTAATACATATATTTACAAATAAAATGCCTCAAGGACAAAAAAA 15217868

Query 424 aaGGGGG-GCGGGGCTTGG-AGGTccccccctag-aattcccgcc 465 || | | || |||| ||| || | | | ||||| |||||| ||| Sbjct 15217867 AAAAGTGAGCTGGGCATGGCAG-TGCACACCTAGTAATTCCAGCC 15217824

ANNE3 rev product

Features flanking this part of subject sequence: 1550 bp at 5' side: 40S ribosomal protein S8 3507 bp at 3' side: bestrophin-4

Score = 281 bits (152), Expect = 6e-73 Identities = 375/480 (78%), Gaps = 26/480 (5%) Strand=Plus/Plus

Query 8 GCTGG-ATTACTAGGTGTGCACTGCCATGCCCAGCTCACtttttttttttc-ccttgggg 65 ||||| |||||||||||||||||||||||||||||||||||||||||||| ||||| || Sbjct 15217825 GCTGGAATTACTAGGTGTGCACTGCCATGCCCAGCTCACTTTTTTTTTTTGTCCTTGAGG 15217884

Query 66 --ttttatttttaaaatattttttttACTTCCCTaaaaaaaaaaTCCCGGG-TTTTcccc 122 |||||||| |||| ||| | | |||| |||||| ||||| |||||| || ||||||| Sbjct 15217885 CATTTTATTTGTAAA-TATATGTATTACATCCCTAGAAAAAGAATCCCTGGATTTTCCCT 15217943

Query 123 cccGGGGGGTTTCCTCCTGGCTCCAGGGCCCAGGAGGCCGGCGGGGGTTGCCGGAACAAT 182 || | | | |||| || || || || || ||| || ||| || | ||||| | | ||||| Sbjct 15217944 CCTGTGTGTTTTCGTCTTG-CTTCATGGTCCATGATGCCAGCTGAGGTTGTCAGTACAAT 15218002

162 Results

Query 183 AAAATCAAACgggggggA-GGAAAGCAAAtttttcggccttttttccaaatttttaattt 241 ||||||||| || |||| |||| ||| ||| ||| ||| |||||||| || ||| | || Sbjct 15218003 GAAATCAAACTGGCGGGATGGAA-GCAGATTATTCTGCCATTTTTCCAGATCTTTGAGTT 15218061

Query 242 GCACATAAAATCGGGGGTTAATccccccccccTTGTTAACCC-GCCCGGGAGGGTTCCCA 300 |||||| ||||| |||| | ||| | || | |||||| | || ||| | |||| ||| || Sbjct 15218062 GCACATCAAATCTGGGGCTGATCGCTCCACACTTGTTTAGCCTGCCTGTGAGG-TTCACA 15218120

Query 301 A-AATTTTCCCCGGCTCTGTAACCTTCAG-GAATTTCAAATTAC-CCAATG-AACCCTGG 356 | ||||||||| | |||||| | | |||| || ||||||||| | |||||| |||| || Sbjct 15218121 ACAATTTTCCCAG-CTCTGTGATCATCAGTGA-TTTCAAATT-CGCCAATGTAACCATG- 15218176

Query 357 CTTCTTCTTCCCGGGGAAAAACGGGACAATAATTTTGGAGC-TGGGCCAAAAAAGCCCCG 415 |||| || || | | || |||| |||| || | |||||||| ||| || || |||| || Sbjct 15218177 CTTCATCATCACAGTGAGAAACTGGACGATGACTTTGGAGCATGG-CCTAAGAAGCACCT 15218235

Query 416 GG-GTTTGCCCTCTTTTTTCGG-ATTTGTTAAGGCCCTTGAAAGCTTCGGCCGGGG-ATT 472 || ||||||| ||| ||||||| ||| ||| | || ||||| ||| || ||| ||| ||| Sbjct 15218236 GGCGTTTGCC-TCTCTTTTCGGCATT-GTTGATGCTCTTGAGAGCATCTGCCAGGGCATT 15218293

Tab. 23A: C3orf52 EST Profile, modified from NCBI Hs.434247 Breakdown by Body Sites Breakdown by Health State Pool name gene EST/ total EST Pool name gene EST/ total EST transcripts per million transcripts per million Ascites 24 1 / 40015 colorectal tumor 8 1 / 114246 Bladder 33 1 / 29757 gastrointestinal tumor 8 1 / 119369 Blood 8 1 / 123478 germ cell tumor 15 4 / 263845 Embryonic tissue 18 4 / 215722 head and neck tumor 7 1 / 136302 Intestine 25 6 / 234472 kidney tumor 29 2 / 68959 Kidney 18 4 / 211777 Leukemia 31 3 / 95842 Liver 4 1 / 207743 liver tumor 0 0 / 96359 Lung 41 14 / 336974 lung tumor 48 5 / 103127 Lymph 0 0 / 44270 Lymphoma 55 4 / 71755 lymph node 65 6 / 91610 Normal 10 35 / 3360307 Mouth 14 1 / 67052 ovarian tumor 13 1 / 76682 Ovary 9 1 / 102051 pancreatic tumor 9 1 / 104616 Pancreas 4 1 / 214812 skin tumor 16 2 / 124949 Placenta 7 2 / 280825 uterine tumor 11 1 / 90257 Prostate 10 2 / 189345 Breakdown by Developmental Stage Skin 14 3 / 210574 Embryoid body 42 3 / 70761 Testis 15 5 / 330442 Blastocyst 0 0 / 62319 Thymus 73 6 / 81131 Fetus 17 10 / 564012 Tonsil 0 0 / 16999 Neonate 0 0 / 31097 Trachea 19 1 / 52413 Infant 0 0 / 23620 Uterus 4 1 / 232878 Juvenile 0 0 / 55556 Adult 6 12 / 1939121

163 Results Tab. 23B: Array data with immunological background in NM_024616 GD record Experiment GDS2819 Leukemic white blood cells and various RNA preparation protocols GDS2643 Waldenstrom's macroglobulinemia: B lymphocytes and plasma cells GDS3558 Deferasirox effect on leukemia cell line: dose response GDS2501 B-cell chronic lymphocytic leukemia cell type and prognosis GDS3089 Tretinoin effect on promyelocytic leukemia cell line GDS3057 Acute myeloid leukaemia GDS2251 Myeloid leukaemia cell lines GDS3329 Cytogenetically normal acute myeloid leukemia_test set, Expression profiling by array GDS843 Adult acute myeloid leukemia: bone marrow and peripheral blood expression profiles (SHDJ) GDS841 Adult acute myeloid leukemia: bone marrow and peripheral blood expression profiles (SHCO) GDS1604 Ionizing radiation effect on monocytic leukemia cells GDS3471 B-precursor acute lymphoblastic leukemia tumors sensitive and resistant to ionizing radiation GDS2908 T-cell prolymphocytic leukemia with inv(14)(q11q32) GDS2794 T-cell acute lymphoblastic leukemia cell line response to Notch receptor inhibition GDS1324 T-cell acute lymphoblastic leukemia with CALM-AF10 fusion GDS3487 Transcription factor CREB depletion effect on myeloid leukemic cell line GDS3690 Atherosclerotic Coronary Artery Disease: circulating mononuclear cell types GDS2154 Inflammatory dilated cardiomyopathy GDS3628 Rheumatoid arthritis and response to anti-TNF alpha therapy: blood GDS420 Severe combined immunodeficiency (HG-U133A) GDS3553 Monocytes and macrophages (RNG/MRC) GDS3555 Monocytes and macrophages (Illumina) GDS3554 Monocytes and macrophages (Affymetrix) GDS3203 Monocyte to macrophage differentiation GDS2429 Monocyte differentiation to macrophage and subsequent polarization (HG-U133A) GDS2494 Rapamycin effect on a glucorticoid-resistant T cell lymphoblastic leukemia cell line: time course GDS3558 Deferasirox effect on leukemia cell line: dose response GDS559 Inflammatory bowel disease (HG-U133A) GDS1330 Crohn disease and ulcerative colitis comparison GDS1615 Ulcerative colitis and Crohn's disease comparison: peripheral blood mononuclear cells GDS3268 Colon epithelial biopsies of ulcerative colitis patients GDS2014 Ulcerative colitis GDS2042 Celiac disease: intraepithelial cytotoxic T lymphocytes GDS3646 Celiac disease: primary leukocytes GDS502 Inflammatory cytokine effect on primary colon endothelial cells GDS3518 Chronic myelogenous leukemia response to imatinib: Philadelphia chromosome positive CD34+ cells GDS1048 Lymphoblastoid cell lines from CEPH/Utah families GDS3042 Imatinib effect on K562 leukemia cell line (I) GDS3048 Imatinib effect on K562 leukemia cell line (VII) GDS2952 Rheumatoid arthritis response to anti-tumor necrosis factor treatment: whole blood GDS289 Chronic obstructive pulmonary disease GDS2563 Cigarette smoking effect on T lymphocytes GDS2935 Allergic contact dermatitis: time course GDS1753 Colchicine anti-inflammatory effect: time course and dose response GDS785 CD4+ T cell differentiation (HG-133A) GDS751 Natural killer cell expression profiling GDS3191 Natural killer cell response to interleukin-2 treatment: time course GDS3497 Interleukin-12 effect on peripheral blood mononuclear cells GDS3559 Occupational benzene exposure: peripheral blood mononuclear cells (HG-U133A) GDS2778 1,2,4-benzenetriol effect on peripheral blood mononuclear cells in vitro GDS3691 Sustained olive oil consumption effect on peripheral blood mononuclear cells GDS2883 LEDGF/p75 transcription factor deficiency effect on T-cell line GDS3342 Curcumin effect on oxidatively stressed monocyte cell line: time course GDS2649 HIV infection effect on CD4+ and CD8+ T cells GDS3088 Leukotriene B4 effect on monocytes: time course GDS3469 Leukotriene D4 effect on monocytes GDS731 Leukotriene LTD4 effect on macrophage and endothelial cells GDS3676 Quercetin effect on CD14+ monocyte GDS3499 TREM-1 activation effect on monocytes in vitro GDS3005 Macrophage response to interleukins 1 and 6

164 Results

GDS3686 Colony stimulating factor 1 effect on monocyte derived macrophage GDS2866 Monocyte-derived macrophages of COPD patients response to fine and ultrafine particles GDS2180 M-CSF and GM-CSF differentiated macrophages response to bacillus Calmette-Guerin (Subset A) GDS3626 Macrophage migration inhibitory factor depletion effect on kidney cell line GDS3496 Alveolar macrophages of cigarette smokers GDS1269 Cigarette smoking effect on alveolar macrophage GDS3193 Monocyte-derived dendritic cell response to VAF347 GDS2847 Anisomycin effect on leukemia cell line: time course GDS3429 Azaspiracid-1 effect on T lymphocyte cell line: time course and dose response GDS3433 Azaspiracid-1 effect on T lymphocyte cell line: time course GDS3207 Chronic loneliness effect on peripheral blood leukocytes GDS2951 Hypoxia effect on peripheral blood lymphocytes GDS3383 Chronic stress effect on peripheral blood monocytes GDS2756 Measles: peripheral blood mononuclear cells GDS2754 Exhaustive exercise effect on peripheral blood mononuclear cells and T lymphocytes GDS3115 Heart failure: peripheral blood mononuclear cells GDS2168 HIV viremia effect on monocytes GDS3085 Gram-positive and gram-negative sepsis: leukocytes GDS3399 Staphylococcal superantigen toxins effect on peripheral blood mononuclear cells GDS3298 Peripheral blood monocyte response to Francisella tularensis infections GDS2856 Peripheral blood-derived monocytes response to lipopolysaccharide GDS1215 Acute myeloblastic leukemia cells response to all trans retinoic acid and valproic acid GDS2676 CD38-positive and CD38-negative chronic lymphocytic leukemia cells GDS2601 Alzheimer blood mononuclear cells GDS2887 Moderate stage Huntington's disease lymphocytes GDS810 Alzheimer's disease at various stages of severity GDS2821 Parkinson‟s disease: substantia nigra GDS2795 Alzheimer's disease: neurofibrillary tangles GDS3128 Parkinson's disease: substantia nigra GDS1331 Huntington's disease: peripheral blood expression profile GDS1332 Huntington's disease: peripheral blood expression profile (Codelink Uniset 20K) GDS1036 Microglial cell response to interferon-gamma: time course GDS1313 GFAP-negative lamina cribrosa cell response to TGF-beta1 GDS2954 Mycobacterium tuberculosis-derived lipopeptide effect on monocytes and dendritic cells: time course GDS3428 Immature dendritic cell response to butanol fraction of Echinacea purpurea: time course GDS2749 Immature dendritic cell response to Aspergillus fumigatus infection in vitro GDS2750 Immature dendritic cell response to hypoxia in vitro GDS2453 PPAR-gamma activation and RAR-alpha inhibition effect on dendritic cells GDS1249 Toll-like receptor agonists synergistic effect on dendritic cells: time course GDS2216 Lipopolysaccharide antagonist effect on lipopolysaccharide-stimulated dendritic cells: time course GDS3595 Macrophage response to H1N1 and H5N1 influenza viral infections GDS1684 Cardiac allograft rejection: time course GDS2310 Exercise effect on white blood cells GDS962 Peripheral blood mononuclear cells and the effect of exercise GDS3717 NOTCH antagonist SAHM1 effect on T-ALL cell lines GDS2832 Sphingosine-1-phosphate effect on embryonic stem cells GDS3300 SOX transcription factor overexpression in embryonic stem cells GDS3470 miR-122 overexpression effect on embryonic stem cells GDS3513 Embryonic stem cell-derived cardiomyocytes GDS3516 Nodular lymphocyte-predominant Hodgkin lymphoma: lymphocytic and histiocytic cells GDS2926 Megakaryocytic differentiation: time course GDS2513 Nicotinamide effect on -induced megakaryocyte differentiation in vitro GDS3318 Sickle cell disease: platelets GDS552 Essential thrombocythemia megakaryocytes GDS3518 Chronic myelogenous leukemia response to imatinib: Philadelphia chromosome positive CD34+ cells GDS855 Transforming growth factor beta effect on CD34+ hematopoietic stem cells GDS2378 UTP and alpha-chemokine CXCL12 effect on CD34+ hematopoietic stem cells GDS2397 Idiopathic myelofibrosis: hematopoietic CD34+ stem cells GDS1401 CD34+ hematopoietic cells expanded in artificial matrix of fibrillar collagen I GDS2118 Myelodysplastic syndromes: CD34+ cells GDS1520 Mast cell activation via the Fc(epsilon)RI GDS1257 Sickle cell plasma effect on pulmonary artery endothelial cells (HG-U133A)

165 Results

Tab. 23C: Grouping of array entry data Data cluster Total broken down on cell types (difference to total in array entries in Tab. 16B) Total Monocyte Macrophage Lymph.: B,T,NK- Dendritic cell Embry. s.c. Cells ≈100 31 14 18cell 8 4

Effectors PAMPs 13 HIV, Bact. (4) H1N1, H5N1 HIV Bact. (2), Fung. Hormones 12 Leukotr. (2), IL-12 Leu., IL-1/6,CSF1 IL-2 TLR(1) agonist sphingosine Transc. factors 6 CREB, TREM-1 LEDGF/p75 PPAR-γ, RAR-α miR-122, Sox

Effect Differentiation 14 Mono./Macro. (2) CD4+ T cell Immature (3) Hypoxia/Exerc. 5 (1) (1) (1) Leukemias 24 Myeloid (10) Lymphocytic (14) Diseases 31 Intest(1),Neuro(1) Intest(1),Neuro(1)

Tab. 23D: Array data with with prostate cancer in NM_024616

GD record Experiment GDS1973 Various prostate cell types GDS2971 Hemiasterlin analog HTI-286 effect on docetaxel-resistant prostate cancer cell line GDS1697 DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine effect on prostate cancer cell lines GDS3155 Dasatinib resistant and sensitive prostatic cancer cell lines GDS3095 Zinc effect on malignant and non-malignant prostate cell lines GDS2547 Metastatic prostate cancer (HG-U95C) GDS1390 Prostate cancer progression after androgen ablation GDS1699 Androgen sensitive and insensitive prostate cancer cell lines: expression profiles GDS2782 Androgen response element specific DNA-binding polyamide effect on dihydrotestoterone-stimulated prostate cell line GDS1699 Androgen sensitive and insensitive prostate cancer cell lines: expression profiles GDS1439 Prostate cancer progression GDS721 Prostate adenocarcinoma response to radiation (HG-U95C) GDS3634 miR-205 expression effect on prostate cancer cell line GDS1736 Arachidonic acid effect on prostate cancer cells GDS1746 Primary epithelial cell cultures from prostate tumors GDS2865 Metastatic prostate tumor model GDS3111 Prostate cancer cell line response to dihydrotestosterone: time course GDS1423 Lunasin effect on prostate epithelial cells GDS723 Prostate adenocarcinoma response to radiation (HG-U95E)

166 Results

Tab. 23E: Grouping of small molecule effectors DRUGS Azaspiracid-1 effect on T lymphocyte cell line: time course and dose response Azaspiracid-1 effect on T lymphocyte cell line: time course Deferasirox effect on leukemia cell line: dose response Chronic myelogenous leukemia response to imatinib: Philadelphia chromosome positive CD34+ cells Imatinib effect on K562 leukemia cell line entries (I);(II),(III),(IV),(V),(VI),(VII) Tretinoin effect on promyelocytic leukemia cell line Monocyte-derived dendritic cell response to VAF347 ALLERGENS Allergic contact dermatitis: time course Anisomycin effect on leukemia cell line: time course Immature dendritic cell response to butanol fraction of Echinacea purpurea: time course Colchicine anti-inflammatory effect: time course and dose response Curcumin effect on oxidatively stressed monocyte cell line: time course Sustained olive oil consumption effect on peripheral blood mononuclear cells Quercetin effect on CD14+ monocyte SMOKE COMPONENTS Monocyte-derived macrophages of COPD patients response to fine and ultrafine particles Alveolar macrophages of cigarette smokers Cigarette smoking effect on alveolar macrophage Cigarette smoking effect on T lymphocytes Occupational benzene exposure: peripheral blood mononuclear cells (HG-U133A) 1,2,4-benzenetriol effect on peripheral blood mononuclear cells in vitro RECEPTOR EFFECTORS T-cell acute lymphoblastic leukaemia cell line response to Notch receptor inhibition NOTCH antagonist SAHM1 effect on T-ALL cell lines Rheumatoid arthritis and response to anti-TNF alpha therapy: blood Rheumatoid arthritis response to anti-tumor necrosis factor treatment: whole blood PPAR-gamma activation and RAR-alpha inhibition effect on dendritic cells Lipopolysaccharide antagonist effect on lipopolysaccharide-stimulated dendritic cells: time course Toll-like receptor agonists synergistic effect on dendritic cells: time course LIPID-DERIVED HORMONES Acute myeloblastic leukemia cells response to all trans retinoic acid and valproic acid Sphingosine-1-phosphate effect on embryonic stem cells Leukotriene B4 effect on monocytes: time course Leukotriene D4 effect on monocytes Leukotriene LTD4 effect on macrophage and endothelial cells HORMONE COFACTORS Nicotinamide effect on thrombopoietin-induced megakaryocyte differentiation in vitro UTP and alpha-chemokine CXCL12 effect on CD34+ hematopoietic stem cells

Tab. 24: CLUSTAL 2.0.12 multiple sequence alignment of C3orf52 to medaka MyD88 C3orf52 Tn ---MDEDVEMIQISTDRS------SGATYFSNERVNGEDVRISFGEAPEVDGL C3orf52 Ga -----MAIEMKTIQTN------GAEVTGN----GESVACTMPDATEKDGF MyD88 Ol MAGCSSDVDLWTVPLVALNVTVRRKLGLYLNPKNTVAADWMTVAEEMGFTYLEIKNYEAS ::: : .: .*.: : : : :.

C3orf52 Tn LRPTSGVRERAALERGSSE--QTSRPQEEHLMCRIQRELKQAVFPNIFPKVQLWMIIGLL C3orf52 Ga LEQTTSANPDPEPCTPPSQGTRTSTLAEICSKSRISRE------RLKLLLII--V MyD88 Ol KNPTKTVLEDWQARSKDASVGKLLSILTEVERKDVVEDLRPQIDEDVRKYLESLQRKSEP . *. . :. : : .: ::

C3orf52 Tn LFIIVIIAVILISLAVCAALHEDEDEKFDPSLFKVPLYHNG-TFRLPNQDFTKDLINMSS C3orf52 Ga VIIVFIAVVFVISLAVCSAIYEDSDGDFDSSLFKVPRSFNG-SFRLPNRVFTEGLVTLSS MyD88 Ol PLQVAEVDSCVPRTPERFGITVEDDPDGSPEMFDAFICYCQDDFHFVCEMIRELEQTDHK : : : . .: :.* . ...:*.. . *:: . : : . .

C3orf52 Tn TQSQVLAASLQEKIAGFYTSSPALGRYFSEASISAFRDGENDQSPSVIAHYRLKFLMPEE C3orf52 Ga NESKGLAAELQHKLADLYKSSPALGRYFSQAEIQAFRNG------SVIADYQLMFLMPEE MyD88 Ol LKLCVFDRDVLPGSCVWTITSELIEKRCKRMVVVISDEYLESDACDFQTKFALSLCPGAR : : .: . :* : : .. : : .. :.: * : . C3orf52 Tn EEDQLRNFTLSREMVFNVFRQLLYDQEADA---AEELYIAHDSLRTF--- C3orf52 Ga QQDQLRNVTLSREMVYNVFRQFLYDLESED---SGPMYIEPVSLHMFVRV MyD88 Ol NKRLIPVIYKTMKKPFPTILRFLTVCDYTKPCTQAWFWIRLAKALSLP-- :: : . : : : .: ::* : ::* . :

167 Results A B Nucleotides in Permutations for number sequence (N) of identical nucleotides (I) S ↓ P Pascal‟s triangle 1 1 1 1 2 1 2 1 3 1 3 3 1 4 1 4 6 4 1 … .….………. e.g. for N=4 I: 0 1 2 3 4 Permut.: 1 4 6 4 1 Random prob. for I=1: 1 3 I 0.25 ∙0.75 ≈0.11 Permut prob. (P1): Fig. 67: Probability density (P) and cumulative distribution (S) for 0.11∙4=0.44 variable identity (I) of sequences with 100 nucleotides. Summed prob. (S): A. Graph. B. Method. C. Graph data. P0+P1=S1 S4=1 ident. random permut. Permut.p Summed P ident. random permut. Permut. Summed P bases: prob.: for I rob. up to I bases: prob.: for I prob. up to I I I I 0.25 (PI): (=SI) I 0.25 (PI): (=SI) x random x random p. 0.75100-I p. x 0.75100-I x permut. permut. 0 3.2E-13 1 3.2E-13 3.2E-13 50 4.5E-37 1E+29 4.5E-08 ≈1 1 1.1E-13 100 1.1E-11 1.1E-11 51 1.5E-37 9.9E+28 1.5E-08 ≈1 2 3.6E-14 4950 1.8E-10 1.9E-10 52 5E-38 9.3E+28 4.6E-09 ≈1 3 1.2E-14 161700 1.9E-09 2.1E-09 53 1.7E-38 8.4E+28 1.4E-09 ≈1 4 4E-15 3921225 1.6E-08 1.8E-08 54 5.5E-39 7.3E+28 4.1E-10 ≈1 5 1.3E-15 7.5E+07 9.9E-08 1.2E-07 55 1.8E-39 6.1E+28 1.1E-10 ≈1 6 4.4E-16 1.2E+09 5.2E-07 6.4E-07 56 6.1E-40 4.9E+28 3E-11 ≈1 7 1.5E-16 1.6E+10 2.3E-06 3E-06 57 2E-40 3.8E+28 7.8E-12 ≈1 8 4.9E-17 1.9E+11 9.1E-06 1.2E-05 58 6.8E-41 2.8E+28 1.9E-12 ≈1 9 1.6E-17 1.9E+12 3.1E-05 4.3E-05 59 2.3E-41 2E+28 4.6E-13 ≈1 10 5.4E-18 1.7E+13 9.4E-05 0.00014 60 7.6E-42 1.4E+28 1E-13 ≈1 11 1.8E-18 1.4E+14 0.00026 0.00039 61 2.5E-42 9E+27 2.3E-14 ≈1 12 6E-19 1.1E+15 0.00063 0.00103 62 8.4E-43 5.7E+27 4.8E-15 ≈1 13 2E-19 7.1E+15 0.00143 0.00246 63 2.8E-43 3.4E+27 9.6E-16 ≈1 14 6.7E-20 4.4E+16 0.00296 0.00542 64 9.3E-44 2E+27 1.8E-16 ≈1 15 2.2E-20 2.5E+17 0.00566 0.01108 65 3.1E-44 1.1E+27 3.4E-17 ≈1 16 7.5E-21 1.3E+18 0.01003 0.02111 66 1E-44 5.8E+26 6E-18 ≈1 17 2.5E-21 6.7E+18 0.01652 0.03763 67 3.5E-45 2.9E+26 1E-18 ≈1 18 8.3E-22 3.1E+19 0.02539 0.06301 68 1.2E-45 1.4E+26 1.6E-19 ≈1 19 2.8E-22 1.3E+20 0.03652 0.09953 69 3.8E-46 6.6E+25 2.5E-20 ≈1 20 9.2E-23 5.4E+20 0.0493 0.14883 70 1.3E-46 2.9E+25 3.8E-21 ≈1 21 3.1E-23 2E+21 0.0626 0.21144 71 4.3E-47 1.2E+25 5.3E-22 ≈1 22 1E-23 7.3E+21 0.07494 0.28637 72 1.4E-47 5E+24 7.1E-23 ≈1 23 3.4E-24 2.5E+22 0.08471 0.37108 73 4.7E-48 1.9E+24 9.1E-24 ≈1 24 1.1E-24 8E+22 0.09059 0.46167 74 1.6E-48 7E+23 1.1E-24 ≈1 25 3.8E-25 2.4E+23 0.0918 0.55347 75 5.3E-49 2.4E+23 1.3E-25 ≈1 26 1.3E-25 7E+23 0.08827 0.64174 76 1.8E-49 8E+22 1.4E-26 ≈1 27 4.2E-26 1.9E+24 0.08064 0.72238 77 5.9E-50 2.5E+22 1.5E-27 ≈1 28 1.4E-26 5E+24 0.07008 0.79246 78 2E-50 7.3E+21 1.4E-28 ≈1 29 4.7E-27 1.2E+25 0.058 0.85046 79 6.5E-51 2E+21 1.3E-29 ≈1 30 1.6E-27 2.9E+25 0.04575 0.89621 80 2.2E-51 5.4E+20 1.2E-30 ≈1 31 5.2E-28 6.6E+25 0.03444 0.93065 81 7.2E-52 1.3E+20 9.6E-32 ≈1 32 1.7E-28 1.4E+26 0.02475 0.9554 82 2.4E-52 3.1E+19 7.4E-33 ≈1 33 5.8E-29 2.9E+26 0.017 0.97241 83 8E-53 6.7E+18 5.3E-34 ≈1 34 1.9E-29 5.8E+26 0.01117 0.98357 84 2.7E-53 1.3E+18 3.6E-35 ≈1 35 6.4E-30 1.1E+27 0.00702 0.99059 85 8.9E-54 2.5E+17 2.3E-36 ≈1 36 2.1E-30 2E+27 0.00422 0.99482 86 3E-54 4.4E+16 1.3E-37 ≈1 37 7.1E-31 3.4E+27 0.00244 0.99725 87 9.9E-55 7.1E+15 7.1E-39 ≈1 38 2.4E-31 5.7E+27 0.00135 0.9986 88 3.3E-55 1.1E+15 3.5E-40 ≈1 39 7.9E-32 9E+27 0.00071 0.99931 89 1.1E-55 1.4E+14 1.6E-41 ≈1 40 2.6E-32 1.4E+28 0.00036 0.99968 90 3.7E-56 1.7E+13 6.4E-43 ≈1 41 8.8E-33 2E+28 0.00018 0.99985 91 1.2E-56 1.9E+12 2.3E-44 ≈1 42 2.9E-33 2.8E+28 8.3E-05 0.99994 92 4.1E-57 1.9E+11 7.6E-46 ≈1 43 9.8E-34 3.8E+28 3.7E-05 0.99997 93 1.4E-57 1.6E+10 2.2E-47 ≈1 44 3.3E-34 4.9E+28 1.6E-05 0.99999 94 4.5E-58 1.2E+09 5.4E-49 ≈1 45 1.1E-34 6.1E+28 6.7E-06 ≈1 95 1.5E-58 7.5E+07 1.1E-50 ≈1 46 3.6E-35 7.3E+28 2.7E-06 ≈1 96 5E-59 3921225 2E-52 ≈1 47 1.2E-35 8.4E+28 1E-06 ≈1 97 1.7E-59 161700 2.7E-54 ≈1 48 4E-36 9.3E+28 3.7E-07 ≈1 98 5.6E-60 4950 2.8E-56 ≈1 49 1.3E-36 9.9E+28 1.3E-07 ≈1 99 1.9E-60 100 1.9E-58 ≈1 50 4.5E-37 1E+29 4.5E-08 ≈1 100 6.2E-61 1 6.2E-61 1

168 Results

Tab. 25: APLP1 position on Chr19: 36.35 mbp APLP1 exon positions in alignment file by exon 1 start

start end Start end 2559410 2559696 exon 1 2 286 2560580 2560723 exon 2 1170 1313 2561809 2561941 exon 3 2399 2531 2562150 2562262 exon 4 2740 2852 2562525 2562658 exon 5 3115 3248 2562771 2562949 exon 6 3361 3539 2563396 2563526 exon 7 3986 4116 2564552 2564626 exon 8 5142 5216 2565417 2565575 exon 9 6007 6165 2565654 2565782 exon 10 6244 6372 2567430 2567529 exon 11 8020 8119 2568631 2568738 exon 12 9221 9328 2568995 2569021 exon 13 9585 9611 2569497 2569567 exon 14 10087 10157 2569803 2569865 exon 15 10393 10455 2569987 2570130 exon 16 10577 10720 2570256 2570709 exon 17 10846 11299 Tab. 26: ALUs and other repetitive elements in APLP1 genomic sequence chr19 part chr19 part start end dir. type family: bp with ident. 2559411 2559453 1 43 + GC_rich Low_complexity 1 to 43 2559600 2559643 190 233 + (CTG)n Simple_repeat 3 to 46 2561415 2561726 2005 2316 + AluSq2 SINE/Alu 1 to 312 2563761 2564072 4351 4662 C AluSq2 SINE/Alu (0) to 312 2564157 2564459 4747 5049 C AluY SINE/Alu (9) to 302 2566072 2566241 6662 6831 C AluSx SINE/Alu (8) to 304 2566242 2566533 6832 7123 C AluSx SINE/Alu (13) to 299 2566534 2566673 7124 7263 C AluSx SINE/Alu (171) to 141 2566707 2566987 7297 7577 C AluJr SINE/Alu (21) to 291 2567609 2567853 8199 8443 C MLT1H2 LTR/ERVL-MaLR (8) to 481 2567951 2568000 8541 8590 C MLT1H2 LTR/ERVL-MaLR (385) to 164 2568125 2568425 8715 9015 + AluSq2 SINE/Alu 6 to 307 2569176 2569475 9766 10065 + AluJr SINE/Alu 1 to 299 2569611 2569727 10201 10317 C MSR1 Satellite (25) to 115

Tab. 27: 31 alignments 4 1908406 2566255 62 1193208 2561416 Block Chr3 Chr19 182 1908422 2566269 84 1193298 2561517 size start start 66 1908604 2566744 5 1193393 2561615 QUERY 1 QUERY 7 49 1193401 2561620 168 706063 2561404 61 1035292 2566562 16 1194785 2564146 33 706231 2561573 29 1035354 2566623 76 1194827 2566259 60 706264 2561616 57 1036077 2568167 21 1194903 2566338 12 711397 2563207 120 1036136 2568227 QUERY 12 37 713779 2568118 33 1036256 2569404 11 1856636 2563756 230 713839 2568179 7 1036289 2569439 264 1856668 2563800 QUERY 2 6 1036296 2569451 92 1858609 2568150 86 1712483 2563882 QUERY 8 51 1858704 2568244 59 1712593 2566594 33 2133310 2564403 QUERY 13 23 1725231 2568150 7 2133358 2566069 2 2185913 2562927 67 1725254 2568175 11 2133378 2566076 61 2185915 2564001 100 1725344 2568264 100 2133391 2566252 7 2186143 2566228 8 1725445 2568364 21 2133492 2566352 7 2186152 2566241 31 1725453 2568373 7 2133517 2566507 11 2186162 2566248 QUERY 3 27 2133527 2566523 134 2186181 2566269 121 1962628 2563837 53 2133565 2566562 23 2186315 2566404 23 1964297 2566061 39 2133622 2566615 76 2186338 2566562 4 1964320 2566085 6 2133667 2566655 70 2189707 2568134 73 1964356 2566354 166 2134544 2568117 33 2190404 2568204 66 1964429 2566562 4 2134710 2568284 101 2190464 2568263 QUERY 4 59 2134715 2568288 8 2190566 2568364 4 2253589 2564148 22 2134774 2569404 40 2190574 2568373 43 2253594 2564152 5 2134797 2569428 110 2190663 2569323 199 2253637 2566274 QUERY 9 QUERY 14 23 2253857 2566492 152 647132 2563784 253 1459385 2561416 247 2256578 2568117 13 647287 2563937 16 1461012 2563751 47 2256826 2568364 2 647300 2563951 17 1461036 2563780 QUERY 5 8 647302 2566553 53 1461055 2563800 27 2104528 2564160 80 647320 2566572 45 1461137 2563884 28 2104557 2564190 132 647414 2566694 56 1461192 2563929 66 2104587 2564221 QUERY 10 75 1461248 2564374 16 2105622 2566061 28 2116251 2563909 17 1463763 2569247 29 2105640 2566081 126 2116279 2566697 10 1463798 2569281 QUERY 6 38 2116715 2568119 163 1463811 2569291 45 2105669 2566273 172 2116755 2568166 QUERY 15 51 2105733 2566335 52 2116927 2569395 24 240593 2563905 280 1900436 2561402 14 2116982 2569447 128 240617 2564317 27 1908379 2566063 QUERY 11 QUERY 16 169 Results

56 2026368 2561412 14 2958830 2566522 QUERY 24 73 2026424 2561470 20 2958845 2566536 65 1052009 2561403 7 2026505 2561543 41 2958866 2566556 67 1052074 2561470 110 2026513 2561559 13 2958907 2566599 132 1052142 2561537 20 2026623 2561670 56 2958920 2566770 20 1052274 2561670 12 2027516 2566236 68 2959729 2566875 173 1059448 2563888 13 2027537 2566254 245 2962039 2568119 103 1061077 2566259 49 2027551 2566269 4 2962285 2568364 11 1061230 2566403 7 2027615 2566336 16 2962299 2568379 34 1061248 2566420 85 2027627 2566343 7 2962320 2568401 13 1061290 2566460 99 2027713 2566722 QUERY 20 15 1061306 2566473 QUERY 17 54 1987548 2561482 QUERY 25 231 2639269 2561438 6 1987602 2561537 5 1122228 2564142 177 2643153 2563784 6 1987612 2561550 70 1122233 2564148 101 2643330 2566562 134 1987621 2561556 21 1122303 2566297 24 2646138 2568134 7 1987766 2561691 22 1122331 2566343 13 2646162 2568159 4 1987774 2561698 32 1122385 2566394 190 2646175 2568174 6 1987778 2561709 48 1122417 2566719 10 2646366 2568364 17 1990726 2566270 41 1122466 2566769 25 2646380 2568379 54 1990757 2566300 210 1131612 2568114 8 2646408 2568405 68 1990811 2566359 38 1138090 2568324 QUERY 18 10 1990879 2566562 10 1138130 2568363 254 2708470 2561415 74 1990894 2566734 QUERY 26 46 2709233 2563800 251 1993861 2568113 90 2900523 2566562 72 2709279 2566148 51 1994113 2568364 297 2901445 2568114 19 2709392 2566231 QUERY 21 12 2902188 2569247 123 2709413 2566250 263 1767423 2563800 32 2902266 2569333 16 2709542 2566379 QUERY 22 4 2902314 2569375 33 2709559 2566395 32 2590014 2563876 52 2902319 2569379 14 2709636 2566618 75 2590050 2563912 QUERY 27 78 2710220 2566722 46 2590153 2564017 85 2346856 2564332 50 2714038 2568122 3 2590199 2565092 19 2346941 2564422 68 2714088 2568174 23 2590202 2566063 17 2371234 2566084 51 2714181 2568265 9 2590265 2566156 6 2371255 2566104 5 2714232 2568317 48 2590281 2566175 90 2371261 2566273 41 2714239 2568323 8 2590343 2566231 12 2371351 2566493 8 2714281 2568364 32 2591262 2566341 6 2371363 2566508 29 2714289 2568373 43 2591397 2566924 16 2371374 2566522 QUERY 19 QUERY 23 116 2371390 2566851 134 2955714 2561403 268 2457641 2561402 123 2380314 2568119 132 2955850 2561537 10 2459299 2566062 100 2380459 2568263 22 2957929 2564145 58 2459309 2566396 37 2380560 2568363 14 2957987 2564191 19 2459369 2566454 14 2382168 2569225 24 2958670 2566063 36 2459395 2566775 30 2382200 2569258 62 2958696 2566252 55 2461621 2568117 7 2382240 2569291 10 2958772 2566327 68 2461676 2568174 63 2382293 2569343 33 2958782 2566338 136 2461768 2568265 QUERY 28 QUERY 30 10 2767860 2566672 164 2761824 2563800 109 2766823 2561414 QUERY 31 32 2761988 2566565 13 2766932 2561524 122 2439831 2561415 7 2762020 2566599 95 2766946 2561537 153 2439955 2561537 18 2762036 2566616 12 2767085 2561678 47 2442965 2566063 23 2762055 2566634 38 2767563 2566063 98 2443053 2566150 114 2762093 2566696 17 2767601 2566266 126 2443158 2566248 QUERY 29 37 2767634 2566298 32 2443313 2566395 95 2699753 2561461 113 2767671 2566338 27 2443353 2566728 111 2699853 2561558 19 2767784 2566586 65 2443380 2566757 246 2701169 2568118 34 2767807 2566609 109 2443462 2566850 42 2701422 2568370 18 2767842 2566643

APLP1 intron 11 AluSq2 (red highlight) Chr3 QUERY 1 ...... AGGCGCGGTGGCTCACGCCTATAATCCCAGCACTTTG 42 APLP1 intron 11 AGCTATCATCTTGAGGCGCGGTGGCTCACGCCTATAATCCCAGCACTTTA 8750 Chr3 QUERY 1 GGAGGCTGAGGCGGGTGGAACG.TCTGAGGTCAGGAGTTAGAGACCAGCC 91 APLP1 intron 11 GGAGGCCGAGGCGGGTGGATCACT.TGAGGTCAGGAGTTCGAGACCAGCC 8799 Chr3 QUERY 1 TGGCCAACACAGTGAAACCCCGTCTCTACTAAAAATAAAAAAATTAGCTG 141 APLP1 intron 11 TGGC...... 8803 Chr3 QUERY 1 GGTGTGGTGGCGGGCGCCTGTAATCCCACTACTCTGCAGGCTGAGGCAGG 191 APLP1 intron 11 ...... 8803 Chr3 QUERY 1 AGAATCACTTGGAGGTGGTTGCAGTGAGCCGAGATTGTGCCATTGCACTC 241 APLP1 intron 11 ...... 8803 Chr3 QUERY 1 CAACCTGGGTAACAAGAGCAATAAGCTTTGCAACGGTGGCTCACATCTGT 291 APLP1 intron 11 ...... 8803 Chr3 QUERY 1 Alu AATCCCAGCACTTTGGGAGATCAGGAGTTCAAGACCAGCCTGGTCAACAT 341 APLP1 intron 11 AluSq2 ...... CAACAT 8809 Chr3 QUERY 1 Alu GGTGAAACCCCGTCTCTACTAAAAATACGATAATTAGCCAGGCGTGGTGG 391 APLP1 intron 11 AluSq2 GGTGAAACCCCGTCTTTACTAAAAATACAAAAATCAGCCGGGCATGATG. 8858 Chr3 QUERY 1 Alu T.GCACACCTATAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATTGC 440 APLP1 intron 11 AluSq2 TCGCTTGCCTGTAATCCCAGCTACTTAGGAGGCTGAGGCAAGAGAATTGC 8908 Chr3 QUERY 1 Alu TTGAACCAGGGAGGCAGAGGTTGCAGTGAGCAGAGATCCCACCACTGCAC 490 APLP1 intron 11 AluSq2 TTGAACCCGGGAGGTGGAGGTTGCAGTGAGCTGAGATCGCATCATTGCAC 8958 Chr3 QUERY 1 Alu TACAACCTGGGCGAC.AGAATGAGACTCCATCTCAAAAAAAAAAAAAAAA 539 APLP1 intron 11 AluSq2 TCCAGCTGGG.CAACAAGAGTGAGACTCTGTCTCAAAAAAAAAAAAACAA 9007 Chr3 QUERY 1 Alu A------540 APLP1 intron 11 AluSq2 AAAAAAAACATAATCTTGAAACTTCAGCCTCCATCCTTCCTGCCAGCAGT 9057

170 Results

Ac chr. 3 Mm chr. 16

GRAMD1C

Hs chr. 21

APP

APP

Hs chr. 3 APP GRAMD1C GRAMD1C

Fig. 68: Locations of APP and GRAMD1C in human (Hs), mouse (Mm) and the anole lizard (Ac). Modified from the Ensembl Genome Browser Synteny display.

171 Results

Hs 3 Gg 1 Hs 19

Hs 11

Hs 21

Gg 24

Fig. 69: The APP family is not assigned to a protochromosome . Chromosomes of human (Hs) or chicken (Gg) colored according to ancestral protochromosomes (left, modified from [470] and to ideograms (right, modified from NCBI), providing the gene locations.

172 Results

Ol 16 Ol 13 Ol 21 Ol 2

APLP2 APP#2

APLP1

APP#1

Fig. 70: In medaka, the APP family could possibly be assigned to protochromosomes A or F. Chromosomes of medaka (Ol) colored according to ancestral protochromosomes (left, modified from [470] compared to the gene locations.

173 Results

Tab. 28:

group A: APLP1 species genes Hs GRAMD1A ETV2:↔GABPA ARHGAP33 KIRREL2 APLP1 ERF:↔ETV3 chr 19+ 19+ 19+ 19+ 19+ 19- bp 35,485,688- 36,132,647- 36,266,417- 36,347,810- 36,359,138- 42,751,717- 35,517,375 36,135,773 36,279,724 36,358,048 36,370,699 42,759,309

Mm ERF:↔ETV3 APLP1 KIRREL2 ARHGAP33 ETV2:↔GABPA GRAMD1A chr 7 - 7 - 7 - 7 - 7 - 7 - bp 26,027,580- 31,220,001- 31,232,553- 31,307,245- 31,418,635- 31,915,146- 26,035,780 31,230,580 31,242,709 31,320,079 31,421,308 31,936,069

Ac KIRREL2 APLP1 ARHGAP33 GRAMD1A ETV2:↔GABPA ENSACAG ENSACAG ENSACAG 00000016486 00000016528 00000005483 chr GL343276.1+ GL343276.1+ GL343276.1- GL343514.1- GL343514.1+ bp 1,229,145- 1,292,325- 1,378,296- 82,341-142,933 389,882-391,152 1,261,895 1,332,462 1,399,273

St ERF:↔ETV3 GRAMD1A KIRREL2 APLP1 ARHGAP33 chr GL172845.1+ GL173269.1- GL173286.1+ GL173286.1+ GL173286.1- bp 1,421,525- 135,419-152,106 215,731- 243,622- 417,071-437,664 1,452,629 238,852 282,857

Ol ARHGAP33 APLP1 KIRREL2 ERF:↔ETV3 ETV2:↔GABPA GRAMD1A ENSORLG ENSORLG ENSORLG ENSORLG 00000006555 00000006651 00000013535 00000015920 chr 16- 16- 16- 16+ 16- 16+ bp ≈11,300,000 12,117,421- 12,178,803- 18,569,200- 25,338,242- 29,252,452- (reference to Dr) 12,127,738 12,190,540 18,608,245 25,341,848 29,264,234

174 Results

Tab. 29:

group B: APLP2 species genes Hs TAGLN GRAMD1B KIRREL3 ETS1 ARHGAP32 APLP2 chr 11+ 11+ 11- 11- 11- 11+ bp 117,070,037- 123,396,344- 126,293,254- 128,328,656- 128,834,989- 129,939,732- 117,075,503 123,498,482 126,870,405 128,457,437 129,149,219 130,014,699

Mm APLP2 ARHGAP32 ETS1 KIRREL3 GRAMD1B TAGLN chr 9 - 9 + 9 + 9 + 9- 9 - bp 30,957,142- 31,923,721- 32,443,806- 34,293,711- 40,100,818- 45,737,711- 31,019,400 32,072,084 32,565,405 34,844,301 40,338,968 45,744,141

Ac APLP2 ARHGAP32 ETS1 KIRREL3 TAGLN GRAMD1B chr GL343197.1- GL343197.1+ GL343197.1+ GL343322.1- GL343949.1- LGa+ bp 4,014,972- 4,649,175- 1,328,577- 98,102-104,039 3,310,449- 56,710-79,425 4,121,353 4,679,135 1,432,456 3,389,024

Gg KIRREL3 ETS1 ARHGAP32 APLP2 GRAMD1B TAGLN chr 24- 24- 24- 24- 24- 24+ bp 466,913-705,991 897,099-963,027 1,172,172- 1,463,164- 2,946,460- 5,349,275- 1,275,817 1,486,282 2,967,575 5,350,098

St TAGLN ETS1 ARHGAP32 GRAMD1B APLP2 KIRREL3 chr GL172675.1- GL172752.1- GL172752.1- GL172934.1+ GL173184.1+ GL173288.1+ bp 1,163,231- 1,431,886- 1,853,214- 902,747-923,557 69,421-99,778 23,597-46,252 1,169,432 1,476,135 1,994,329

Ol APLP2 ARHGAP32 GRAMD1B KIRREL3 ETS1 TAGLN chr 13+ 13- 13- 13- 13- 13+ bp 4,236,927- 11,134,152- 25,229,912- ≈25,800,000 26,218,477- 32,732,092- 4,284,378 11,142,817 25,261,516 (reference to Dr) 26,236,767 32,738,658

175 Results

Tab. 30:

group C: APP species genes Hs GAPBA:↔ETV2 APP ETS2 TAGLN3 GRAMD1C ARHGAP31 chr 21+ 21- 21+ 3 + 3+ 3 + bp 27,106,881- 27,252,861- 40,177,231- 111,717,511- 113,547,029- 119,013,220- 27,144,771 27,543,446 40,196,879 111,732,734 113,666,021 119,139,561

Mm ARHGAP31 GRAMD1C TAGLN3 GABPA:↔ETV2 APP ETS2 chr 16- 16- 16- 16+ 16- 16+ bp 38,598,453- 43,980,468- 45,711,343- 84,835,170- 84,954,685- 95,923,682- 38,713,387 44,028,058 45,724,721 84,864,024 85,173,952 95,942,658

Ac ETS2 APP GABPA:↔ETV2 TAGLN3 GRAMD1C ARHGAP31 chr 3 - 3 + 3 - 3 - 3+ 3 - bp 140,003,326- 148,080,819- 148,259,826- 167,697,308- 176,451,890- 180,926,263- 140,017,857 148,242,937 148,274,981 167,708,598 176,456,875 180,955,378

Gg ARHGAP31 GRAMD1C TAGLN3 GABPA:↔ETV2 APP ETS2 chr 1 + 1- 1 + 1+ 1 - 1+ bp 84,076,993- 86,366,367- 91,857,021- 105,994,377- 106,057,664- 111,258,337- 84,096,407 86,416,249 91,867,988 106,021,763 106,305,968 111,265,467

St GABPA:↔ETV2 APP ARHGAP31 ETS2 GRAMD1C chr GL172800.1+ GL172800.1- GL172831.1- GL173038.1+ GL173885.1- bp 853,502-863,679 905,574- 1,461,324- 287,856-300,632 81,458-90,313 1,018,253 1,486,295

Ol GABPA:↔ETV2 APP TAGLN3 GRAMD1C ARHGAP31 ETS2 chr 21+ 21- 20+ 20+ 14- 14+ bp 5,225,888- 5,233,061- 15,537,083- 15,906,431- 14,334,164- ≈28,500,000 5,231,737 5,244,256 15,539,828 15,911,553 14,345,344 (reference to Dr)

176 Results

Tab. 31:

group D: 4. Region?

species genes Hs ETV3: ↔ ERF KIRREL TAGLN2 ARHGAP30 Chr 1- 1 + 1 - 1 - bp 157,090,983- 157,963,063- 159,887,897- 161,016,736- 157,108,266 158,070,052 159,895,522 161,039,760

Mm KIRREL ETV3: ↔ ERF ARHGAP30 TAGLN2 Chr 3 - 3+ 1 + 1 + bp 86,882,515- 87,329,329- 173,319,085- 174,430,178- 86,978,669 87,344,078 173,340,429 174,437,511

Ac ETV3: ↔ ERF KIRREL Chr GL343637.1+ GL344594.1+

bp 137,383-145,465 14,413-27,051

Gg “ERF” (ETV3) KIRREL ARHGAP30 ENSGALP 00000000943 Chr 25+ 25+ 2 + bp 996,468- 1,386,409- 15,506-16,585 1,002,791 1,390,450

St TAGLN2 KIRREL ETV3: ↔ ERF ARHGAP30 Chr GL172915.1+ GL173063.1+ GL173071.1+ GL174725.1+ bp 1,408,757- 613,018-648,224 978,660- 5,578-20,508 1,443,937 1,002,463

Ol ETV3: ↔ ERF KIRREL ARHGAP30 ENSORLG 00000002508 Chr 17+ 17+ 17-

bp 2,405,795- 2,812,777- 2,988,481- 2,406,209 2,849,596 2,993,312

177 Results

A APLP1

However, APLP1

APLP2

APP

APLP1 platypus APLP1 Cterm

B mamm. APLP1

Ga APLP1 Ol APLP1

Tr APLP1 Tn APLP1

Dr APLP1 St APLP1

APLP2

APP

Ac APLP1 Oa APLP1 Nterm

Oa APLP1 Cterm Dm appl Ce apl-1

Fig. 72. A: APP family tree with unannotated members, mostly from teleosts. B: Consensus patterns of unannotated members are most similar to APLP1.

178 Results

GRAMD1A: A TAGLN2: D AHRGAP31: C

GRAMD1B: B TAGLN3: C AHRGAP30: D

GRAMD1C: C TAGLN: B AHRGAP32: B

AHRGAP33: A KIRREL2, Ol, Ac :A

KIRREL :D

KIRREL3 :B Fig. 73: Trees for GRAMD1, TAGLN, ARHGAP30-33 and KIRREL family KIRREL2, mammals :A members from the ENSEMBL database.

Tab. 33A: APP family with mammalian and avian APP family members Topology Tree # log L difference S.E. p-1sKH p-SH c-ELW 2sKH ------((AB)(C)) 1 -25627.13 0.00 <---- best 1.0000 + 1.0000 + 0.5151 + best ((BC)(A)) 2 -25627.50 0.37 5.4215 0.4570 + 0.9860 + 0.4801 + + ((CA)(B)) 3 -25633.08 5.95 3.6469 0.0520 + 0.9050 + 0.0048 - + (A(B(C))) 4 -25703.66 76.53 16.2585 0.0000 - 0.0040 - 0.0000 - - (A(C(B))) 5 -25743.89 116.76 20.0913 0.0000 - 0.0000 - 0.0000 - - (B(A(C))) 6 -25786.56 159.43 21.6019 0.0000 - 0.0000 - 0.0000 - - (B(C(A))) 7 -25761.86 134.73 19.1919 0.0000 - 0.0000 - 0.0000 - - (C(A(B))) 8 -25760.21 133.08 20.2480 0.0000 - 0.0000 - 0.0000 - - (C(B(A))) 9 -25697.22 70.09 13.3436 0.0000 - 0.0130 - 0.0000 - -

Tab. 33B: APP family without mammalian and avian APP family members Topology Tree # log L difference S.E. p-1sKH p-SH c-ELW 2sKH ------((AB)(C)) 1 -19600.99 0.00 <---- best 1.0000 + 1.0000 + 0.7788 + best ((BC)(A)) 2 -19605.56 4.56 5.6684 0.2150 + 0.9580 + 0.2180 + + ((CA)(B)) 3 -19609.84 8.85 4.2505 0.0170 - 0.8610 + 0.0032 - - (A(B(C))) 4 -19680.92 79.93 15.8145 0.0000 - 0.0060 - 0.0000 - - (A(C(B))) 5 -19729.89 128.89 21.2180 0.0000 - 0.0000 - 0.0000 - - (B(A(C))) 6 -19762.98 161.99 21.4597 0.0000 - 0.0000 - 0.0000 - - (B(C(A))) 7 -19746.84 145.85 20.0060 0.0000 - 0.0000 - 0.0000 - - (C(A(B))) 8 -19734.38 133.39 20.5678 0.0000 - 0.0000 - 0.0000 - - (C(B(A))) 9 -19666.51 65.51 12.8955 0.0000 - 0.0250 - 0.0000 - - log L: logarithmized maximum likelihood The columns show the results and p-values of the following tests: 1sKH - one sided KH test based on pairwise SH tests (Shimodaira-Hasegawa 2000, Goldman et al., 2001, Kishino-Hasegawa 1989) SH - Shimodaira-Hasegawa test (2000) ELW - Expected Likelihood Weight (Strimmer-Rambaut 2002) 2sKH - two sided Kishino-Hasegawa test (1989)

Plus signs denote the confidence sets. Minus signs denote significant exclusion. All tests used 5% significance level. 1sKH, SH, and ELW performed 1000 resamplings using the RELL method. 1sKH and 2sKH are correct to the 2nd position after the the decimal point of the log-likelihoods.

179 Results

A B a ppl, Dm appl, Dm

APLP1 99

100 APP

77

100 APLP2 100

APLP2 100

APP 77

88 APLP1

Fig. 74 A/B: Trees for datasets with and without mammalian and avian APP family members calculated by maximum likelihood and tested by bootstrapping. Bootstrap values for branches of orthologs included.

180 Results

Tab. 34A: GRAMD1 family topology testing Topology Tree # log L difference S.E. p-1sKH p-SH c-ELW 2sKH ------((AB)(C)) 1 -25146.52 0.25 4.0509 0.4660 + 0.9830 + 0.4651 + + ((BC)(A)) 2 -25146.27 0.00 <---- best 1.0000 + 1.0000 + 0.4833 + best ((CA)(B)) 3 -25149.13 2.86 2.8971 0.1730 + 0.9260 + 0.0516 + + (A(B(C))) 4 -25242.85 96.58 15.4868 0.0000 - 0.0010 - 0.0000 - - (A(C(B))) 5 -25278.52 132.25 19.4902 0.0000 - 0.0000 - 0.0000 - - (B(A(C))) 6 -25239.51 93.24 15.8146 0.0000 - 0.0000 - 0.0000 - - (B(C(A))) 7 -25340.22 193.95 22.7915 0.0000 - 0.0000 - 0.0000 - - (C(A(B))) 8 -25227.04 80.77 16.9937 0.0000 - 0.0010 - 0.0000 - - (C(B(A))) 9 -25311.93 165.66 21.1816 0.0000 - 0.0000 - 0.0000 - - Tab. 34B: TAGLN family topology testing Topology Tree # log L difference S.E. p-1sKH p-SH c-ELW 2sKH ------((BC)(D)) 1 -4164.57 0.09 3.2130 0.4850 + 0.9750 + 0.4357 + + ((CD)(B)) 2 -4164.48 0.00 <---- best 1.0000 + 1.0000 + 0.4550 + best ((DB)(C)) 3 -4166.31 1.82 2.1837 0.2040 + 0.9270 + 0.0840 + + (B(C(D))) 4 -4186.37 21.89 8.6057 0.0100 - 0.1040 + 0.0010 - - (B(D(C))) 5 -4186.99 22.50 8.9056 0.0120 - 0.0970 + 0.0010 - - (C(B(D))) 6 -4186.39 21.90 8.1061 0.0050 - 0.0990 + 0.0000 - - (C(D(B))) 7 -4197.97 33.49 9.8747 0.0020 - 0.0190 - 0.0000 - - (D(B(C))) 8 -4175.91 11.43 8.0163 0.0930 + 0.3700 + 0.0223 - + (D(C(B))) 9 -4189.38 24.90 10.0308 0.0100 - 0.0690 + 0.0011 - - Tab. 34C: ARHGAP family topology testing Topology Tree # log L difference S.E. p-1sKH p-SH c-ELW 2sKH ------((AB)(CD)) 1 -83002.07 0.00 <---- best 1.0000 + 1.0000 + 1.0000 + best ((BC)(AD)) 2 -83186.95 184.88 20.8664 0.0000 - 0.0000 - 0.0000 - - ((CA)(BD)) 3 -83186.92 184.85 20.9015 0.0000 - 0.0000 - 0.0000 - - (A(B(DC))) 4 -83041.30 39.24 8.9968 0.0000 - 0.3810 + 0.0000 - - (A(D(BC))) 5 -83358.07 356.00 31.5090 0.0000 - 0.0000 - 0.0000 - - (B(A(CD))) 6 -83039.88 37.81 9.4058 0.0000 - 0.3950 + 0.0000 - - (B(C(AD))) 7 -83909.62 907.55 54.3433 0.0000 - 0.0000 - 0.0000 - - (B(D(CA))) 8 -83373.40 371.34 32.4269 0.0000 - 0.0000 - 0.0000 - - (C(A(DB))) 9 -83312.55 310.49 27.0889 0.0000 - 0.0000 - 0.0000 - - (C(B(AD))) 10 -83343.88 341.81 28.0348 0.0000 - 0.0000 - 0.0000 - - (C(D(BA))) 11 -83191.25 189.18 20.8205 0.0000 - 0.0000 - 0.0000 - - (D(A(CB))) 12 -83227.49 225.42 24.2963 0.0000 - 0.0000 - 0.0000 - - (D(B(AC))) 13 -83248.54 246.47 25.4556 0.0000 - 0.0000 - 0.0000 - - (D(C(AB))) 14 -83200.96 198.89 21.4565 0.0000 - 0.0000 - 0.0000 - - Tab. 34D: KIRREL family topology testing Topology Tree # log L difference S.E. p-1sKH p-SH c-ELW 2sKH ------((AB)(D)) 1 -22761.80 0.00 <---- best 1.0000 + 1.0000 + 0.9085 + best ((BD)(A)) 2 -22770.55 8.75 5.4700 0.0500 - 0.8780 + 0.0402 - + ((DA)(B)) 3 -22770.23 8.43 5.4139 0.0630 + 0.8790 + 0.0513 + + (A(B(D))) 4 -22960.90 199.10 24.2736 0.0000 - 0.0000 - 0.0000 - - (A(D(B))) 5 -22951.23 189.43 25.1193 0.0000 - 0.0000 - 0.0000 - - (B(A(D))) 6 -22830.91 69.11 13.8554 0.0000 - 0.0190 - 0.0000 - - (B(D(A))) 7 -22991.34 229.54 27.0490 0.0000 - 0.0000 - 0.0000 - - (D(A(B))) 8 -22809.78 47.98 10.6835 0.0000 - 0.1380 + 0.0000 - - (D(B(A))) 9 -22974.16 212.36 23.6877 0.0000 - 0.0000 - 0.0000 - -

181 Results

A (B)(CD) B (B)(CD)

(D)(BC) (C)(BD) (D)(BC) (C)(BD)

C (AB)(D) D (A)(BD)

(AB)(D) (AB)(D) (A)(BD) (A)(BD)

Fig. 75 A-D: A. Likelihood mappings for balanced TAGLN family dataset. B. Likelihood mappings for mammal-dominated TAGLN family dataset. C. Likelihood mappings for balanced KIRREL family dataset. D. Likelihood mappings for mammal-dominated KIRREL family dataset.

182 Results

A

KIRREL2, Ac, St KIRREL2, teleo.

KIRREL, tetra.

KIRREL, teleo.

KIRREL3, tetra. KIRREL3, teleo. KIRREL2, mam.

B

C GRAMD1A, teleo. GRAMD1A, tetra.

GRAMD1B, tetra.

GRAMD1B, teleo.

GRAMD1C, tetra. GRAMD1C, teleo.

Fig. 76: A. Consensus patterns display similarity (red box) in C-term. of KIRREL and KIRREL2. B. Alignment of human KIRREL members and fruitfly kirre. C. Consensus patterns display similarity (red box) in middle of GRAMD1B and GRAMD1C.

183 Results

ETV3:D (4. Region?) ERF:A (APLP1) ETV3L:D (4. Region?) ETV3L:D (4. Region?)

ERF:A (APLP1)

ERF:A (APLP1)

ETV3:D (4. Region?)

ETV3:D (4. Region?)

ETV3L:D (4. Region?)

ETV3:D (4. Region?)

ETS2:C (APP)

ETS1:B (APLP2)

ETV2:A (APLP1)

ETV2:A (APLP1)

GABPA:C (APP)

outgroup: ETV member of the tunicate Ciona savignyi

Fig. 77: Maximum likelihood tree of the ETV family. Branch support values included.

184 Discussion

11 Interpretations of

cell- and APP-construct-characterization and the

kinome screen for BACE1-affecting kinases

11.1 Interpretation of cell- and APP-construct-characterization

Characterization of the cells and APP-constructs was necessary in order to find out whether they can be used in the aimed screen for BACE1-affecing kinases. Basic characterization of CGC cultures demonstrated the features for which this cell type was chosen: Relatively pure isolation, with contamination of only 5% astrocytes, and easy transfection in addition to the typical neuronal morphology with long neurites enclosing the cytoskeletal proteins actin and tubulin. Neuronal functional properties of CGCs in the cultures were demonstrated by glutamate-evoked excitotoxicity and rescue with the NMDA-receptor antagonist MK- 801 as well as by botulinum toxin-evoked cell death. Aspartame up to 3 mM did not harm the CGCs as measured by reduction of MTT and the concomitant positive control with glutamate showed that app. 50% of reduced MTT were produced by astrocytes which has to be taken into account in MTT measurements of CGC cultures. The dominant-negative mutant of Reggie fused to GFP (DN-GFP-Reggie) from the group of Prof. Claudia Stürmer (University of Konstanz) did not abolish neurite outgrowth under the standard conditions for isolation of CGCs. Maybe the time-point of isolation 7 d after birth of the mice pups is too late; in the last transfection with DN-GFP-Reggie, where only 6 d old mice were available, no neurite outgrowth was seen (but also all transfected cells seemed dead). The GFP domain might also interfere with action of the mutant. Murine BACE1 was stainable with two antibodies against human BACE1 which is not surprising given the amino acid identity of 94%. Basic characterization of HEK293 and SHSY5Y with different compounds revealed higher sensitivity for SHSY5Y in the order of half of a magnitude.

185 Discussion

Optimization of transfection for the following APP-construct characterization with electroporation using GFP for easier readout, resulted in the same cell numbers and plasmid amounts as in the protocols by the manufacturer Amaxa. However, for CGCs the electroporation program for chicken neurons worked best and for HEK293 the original nucleofection solution could be replaced by Opti-MEM. In coelectroporation with GFP siRNA 4 µM were sufficient for a strong knockdown of GFP. Lipofection of HEK293 was possible with amounts of GFP-plasmid as little as 40 ng per well and colipofection with 50 nM GFP siRNA resulted in an efficient knockdown of GFP. The characterization of the different APP-constructs can be summarized and divided into morphological and pharmacological aspects. They could be visualized on CGCs by stainings with the APP antibody 6E10, like APP-Citrin and APPswe, and with the SEAP antibody, like SEAP-APPswe, SEAP-APPswe-AβK16V and SEAP-APPswe- EpoR. By judgment of these stainings transfection efficiencies between 10% and 40% were achieved, similarly as with GFP. The stainings displayed a quite uniform distribution on the cell surface with a region of highest intensity close to the nucleus, probably the Golgi-apparatus. Pharmacologically, cleavage of all APP-constructs by BACE1 was verified with the BACE1 inhibitors IPAD and AQD because they most strongly decreased Aβ production from APPswe, APP, SEAP-APPswe, SEAP-APP, SEAP-APPswe- AβK16V and SEAP-APP-AβK16V or SEAP activity from SEAP-APPswe-AβK16V, SEAP-APP-AβK16V and SEAP-APPswe-EpoR in a concentration-dependent manner and also to some extent SEAP activity from SEAP-APP-EpoR. A left-shift of the inhibition curve of one magnitude was observed for SEAP-APP and SEAP-APP- AβK16V in comparison with SEAP-APPswe and SEAP-APPswe-AβK16V, respectively, but this could be an artifact because these experiments were not repeated since SEAP-APP and SEAP-APP-AβK16V were not used in the kinome screen or test-runs of it and therefore of low priority. SEAP activity from SEAP-APPswe and SEAP-APP remained around 80% in HEK293 at concentrations of BACE1 inhibitors which abolished Aβ production and was reduced by 40% with the α-secretase inhibitor GM6001. The ability of these constructs, with SEAP having replaced the N-terminal 84% of APP, to be cleaved by α-secretases was thus proved. It also confirmed that cleavage of APP by α- secretases with the concomitant generation of SEAPα is prevailing in HEK293 [72].

186 Discussion

In CGCs, cleavage of SEAP-APPswe was slightly less dependent on α-secretases as judged by the SEAP activity remaining only at 65% with BACE1 inhibitors in the still non-toxic concentration of 0.5 µM. At 5 µM IPAD, SEAP activity reached 25% and reduction of MTT and Resazurin 75%, but since 50% of reduced MTT was produced by astrocytes, one could argue that the decrease of SEAP activity from 65% at 0.5 µM to 25% at 5 µM would more or less parallel the MTT reduction from CGCs from 50% (100%-50% from astrocytes) to 25% (75%-50% from astrocytes, assumed that 5 µM IPAD is not toxic to astrocytes). However, this would mean that only 15% of the total SEAP activity was produced by astrocytes which are probably not more difficult to electroporate than the much smaller CGCs. Alternatively, the astrocytes might express much lower amounts of BACE1 or they behaved like CGCs with respect to production of SEAP activity and/or sensitivity to 5 µM IPAD. At least at 5 µM AQD also all astrocytes were dead because no MTT or Resazurin was reduced and the highest non-toxic concentration of AQD was 0.1 µm. Surprisingly here, with both inhibitors a large difference of 30% in reduction potential of MTT or Resazurin was measured in the vehicle control with DMSO. AQD at 10 µM was repeatedly toxic with different APP-constructs as measured by a decrease in MTT reduction of 20%-30% even in HEK293. The prevailing α-cleavage was prevented with the APP-constructs which were engineered to be no more α-secretase substrates by the K16V-mutation of the α- cleavage site, SEAP-APPswe-AβK16V and SEAP-APP-AβK16V, or its substitution by the EpoR-part, SEAP-APPswe-EpoR and SEAP-APP-EpoR. The first evidence is a western blot from SEAP-APP-AβK16V-transfected HEK293 where binding of the APP antibody 6E10 occurred only to (uncleaved) SEAP-APP-AβK16V from cell lysate but not anywhere on the lane with supernatant containing cleavage products. 6E10 recognizes Aβ1-7 and therefore Aβ, sAPPα (data not shown) and also the derivative SEAPα (from SEAP-APPswe and SEAP-APP in Fig. 31D) but not sAPPβ and SEAPβ without the epitope. The second evidence is an only decent decrease in SEAP activity from SEAP-APPswe-EpoR of 5 and 10% with the α-secretase inhibitor GM6001 at 1 and 10 µM, which is paralleled by a decrease in MTT reduction. Taken together, in contrast to SEAP-APPswe and SEAP-APP, SEAP activity from cleavage of the AβK16V- and the EpoR-constructs was indeed nearly solely dependent on cleavage by BACE1, however with the exception of SEAP-APP-EpoR which nevertheless contained the EpoR-part as shown by sequencing.

187 Discussion

Around 10-30% of SEAP activity from SEAP-APPswe-AβK16V, SEAP-APP-AβK16V and 20-30% from SEAP-APPswe-EpoR were not inhibitable with BACE1 inhibitors and could be due to either other proteases or translational abortion. Potential sites of translational abortion are ligation sites from cDNA fusions, as with the fused SEAP and EpoR-part, because they sometimes give rise to codons with rare tRNAs. Moreover, in SEAP-APPswe-EpoR two tryptophanes (from the EpoR-sequence) are found at positions 3 and 6 after the APPswe/EpoR ligation which could maybe result in translational abortion when an unloaded tryptophane tRNA, especially in the condition of overexpression, is not hindered from occupying the ribosomal acceptor site. Inhibition of γ-secretase with LY450139 resulted also in a strongly reduced Aβ production from APPswe, APP, SEAP-APPswe and SEAP-APP and would probably also with SEAP-APPswe-AβK16V and SEAP-APP-AβK16V but this was not tested. LY450139 had no effect on SEAP activity from any SEAP-APP-construct, thus demonstrating that fusion of SEAP to APP did not alter the canonical sequence of APP-processing with either BACE1 or α-secretase cleaving firstly before γ-secretase can cleave. Hypothetical graphs from γ-secretase inhibition of SEAP-AβK16V- constructs should be identical to those of SEAP-APPswe or SEAP-APP with SEAP activity staying around 100% and Aβ production decreasing to around 20%, reflecting the canonical sequence of cleavage. However, whether γ-secretase does or does not cleave the EpoR-constructs after cleavage by BACE1 can not be stated from the observation that LY450139 had no effect on SEAP activity but this was assumed to be not relevant for further experiments. The normalization of SEAP activities to Aβ concentrations revealed that around 1.5 fold (14 pg/mAU / 9 pg/mAU) more SEAPβ per pg Aβ was produced with SEAP- APPswe than with SEAP-APP which is line with the data stating that Aβ with the Swedish mutation is a better BACE1 substrate. It also showed that approximately half of SEAP produced by CGCs is SEAPβ when comparing SEAP-APPswe to SEAP- APPswe-AβK16V (256 pg/mAU / 570 pg/mAU) and around 20% with HEK293 (14 pg/mAU / 65 pg/mAU), which both fit extremely well to the fraction of SEAP inhibitable by BACE1 inhibitors. However, a factor of 25 in comparison of SEAP- APPswe in CGCs and HEK293 (256 pg/mAU / 14 pg/mAU) seems already odd but the discrepancy with SEAP-APPswe-Aβ-K16V in the two cell types (570 vs. 65 pg/mAU) is really strange because it should actually be the same value provided that

188 Discussion only BACE1 liberates SEAP. Perhaps, HEK293 secrete only a minor fraction of produced Aβ, around 5% to 10%, or much of the produced Aβ oligomerizes, once the first seeds have formed due to the much higher absolute concentration in the medium than with CGCs, and is therefore not detectable with the Aβ-ELISA. Also, in the HEK293-experiment Aβ could be degraded intra- or extracellulary, maybe after reuptake, or here γ-secretase is the limiting factor in Aβ generation. From the normalization value from SEAP-APPswe-AβK16V in CGCs, which is higher than in HEK293, a SEAP activity of around 70 mAU/min per nmol SEAP(β) can be calculated from it. The normalization value from SEAP-APP-AβK16V was very low with 6 pg/mAU and is closer to the constructs with functional α-cleavage site than to SEAP-APPswe-AβK16V. The BACE1 inhibitor data however suggest that it indeed contains the α-mutation. The addition of a few different compounds to transfected HEK293 was part of the pharmacological characterization. SEAP-APPswe-transfected HEK293 at 0.78 µM actinomycin D produced half of Aβ and SEAP, with MTT reduction unchanged, were dead at 2 mM chloroquine and produced less Aβ and SEAP with 7 µM cycloheximide, normalized to MTT reduction and only when they were transfected by lipofection, not by electroporation. Thus, production of Aβ and SEAP from SEAP- APPswe was parallel with these compounds and was also found with the NF-κB inhibitor celastrol which reduced Aβ, SEAP and MTT values by half at 5 µM and completely at 10 µM. 5 µM, the concentration at which Aβ production was abolished in the inspiring publication, nearly completely decreased Aβ from APPswe and APP and also SEAP from SEAP-APPswe-EpoR, but also MTT reduction. 0.5 µM, the non- effective concentration in the publication, in contrast did not decrease Aβ from APPswe or APP whereas SEAP from SEAP-APPswe and SEAP-APPswe-EpoR was reduced by around 20% and 50%, respectively, with MTT reduction nearly unchanged. So, with a compound other than α-/γ-secretase inhibitors differing results were obtained for the construct lineage APPswe – SEAP-APPswe – SEAP-APPswe- EpoR, maybe reflecting differences in trafficking or other processes and underlining the necessity for validation of results with SEAP-APPswe-EpoR by APPswe. Also part of the pharmacological characterization and part of preparation for the kinome screen, siRNA technology was used in concert with different APP-constructs. Mainly, five siRNAs, directed against nothing (Con), GFP, APPswe, SEAP and BACE1 were employed. APPswe siRNA and SEAP siRNA were used as controls to

189 Discussion confirm that measured Aβ and SEAP were truly products of the APP-constructs and BACE1 siRNA as very specific control to confirm that the APP-constructs were indeed cleaved by BACE1. During characterization of the APP-constructs they were delivered nearly always by electroporation and mostly at 4 µM. Summarized, Con siRNA and GFP siRNA worked both as controls, with GFP seeming more suitable. APPswe siRNA achieved 80-90% reductions of Aβ and/or SEAP with APPswe, SEAP-APPswe and SEAP-APPswe-EpoR and 10-30% with APP, SEAP-APP and SEAP-APP-AβK16V. SEAP siRNA achieved 90% reductions of Aβ and/or SEAP with SEAP-containing constructs and none with APPswe and APP. BACE1 reduced Aβ from SEAP-APPswe and SEAP-APP by 60-70% but SEAP only by 20-30%, thus proving again the prevailing α-cleavage. It achieved mostly 60-70% reductions of Aβ or SEAP from APPswe, APP, SEAP-APP-AβK16V and SEAP-APPswe-EpoR, thus probably demonstrating the quite long half-life of the BACE1 protein with over 9 h [473] and maybe also a challenge with silencing relevant enzymatic activity because at least on qPCR product level it achieved a knockdown of 75%, this in comparison with 95% from the SEAP siRNA. PSEN1 siRNA on the other hand, delivered by lipofection, did not decrease SEAP activity from SEAP-APPswe-EpoR and confirmed the result with LY450139. All these reduction values were obtained with HEK293 and were higher than the SEAP reduction from SEAP-APPswe-EpoR in CGCs with 60% for APPswe siRNA and SEAP siRNA and 40% for BACE1 siRNA in comparison with GFP siRNA. Summarizing characterization of the APP-constructs, seven of the in total eight displayed the desired pharmacological properties and thus provided in concert a battery to elucidate β-cleavage which was used in the screen for BACE1-affecting kinases.

190 Discussion

11.2 Interpretation of the kinome screen for BACE1-affecting kinases

The kinome screen for BACE1-affecting kinases was, with the choice of SEAP- APPswe-EpoR as BACE1 substrate, actually a screen for kinases which affect the cleavage of an APPswe-β-cleavage-site tailored for BACE1. By choice of SEAP- APPswe-EpoR instead of APPswe or SEAP-APPswe, or rather SEAPβ instead of Aβ as primary readout, kinases were precluded which affect cleavage at the γ-secretase- site and probably all those which affect cleavage at the β-cleavage-site by affecting APPswe because in SEAP-APPswe-EpoR only 22 amino acids from APPswe were left. Moreover, with the constitutively highly active CMV-promoter transcriptional regulation of the BACE1 substrate by kinases should be. Effects of kinases should therefore be back-traceable to effects on BACE1, or, however, to those affecting trafficking of SEAP or the transmembrane domain of the Erythropoietin Receptor which demanded for validation of the results by simultaneous use of APPswe. One concern arising with the use of SEAP was that its phosphatase activity might interfere with phosphorylation in general - however, its pH optimum at pH 10.4 should prevent enzymatic activity. With regards to a future AD therapy, focus on β-cleavage and the abolition of BACE1 function seems to be safe because knockdown was well tolerated by adult mice or maybe other BACE1 functions than cleavage of APP were rescued by other proteins and should therefore also be by indirect means of a BACE1-affecting kinase. In the best case it would be a kinase which is exclusively expressed in the brain to avoid potential systemic side effects and most probably it would be a kinase which downregulates β-cleavage when inhibited since far more inhibitors than activators for kinases exist. Indirect downregulation of β-cleavage through upregulation of α- cleavage is another approach of several studies and actually a screen for up- or downregulation of α-cleavage was carried out during this study with the use of SEAP- APPswe as control. However, kinases up- or downregulating α-cleavage were only listed and not subjected to further experiments, like β-cleavage upregulating kinases. β-cleavage from SEAP-APPswe-EpoR served as control for α-cleavage from SEAP- APPswe such that kinases up- or downregulating α-cleavage were excluded when they affected β-cleavage in the same sense to a similar degree. Kinases affecting both cleavages similarly might exist and were thus excluded, however, for technical reasons like well position and slight variability in transfection efficiency this processing of data seems appropriate to reduce probably the number of false-

191 Discussion positives. Indirect downregulation of β-cleavage by α-cleavage upregulation was however not the aim of this study and anyway not possible with the desired restriction of produced SEAP to consist only of SEAPβ by abolition of the α-cleavage site. Finally, the need to screen for kinases which downregulate β-cleavage when inhibited is well met by the use of siRNAs.

The optimization of cotransfection with SEAP-APPswe-EpoR, APPswe and kinase siRNAs resulted in an experiment duration of 72 h with continous presence of 5 ng SEAP-APPswe-EpoR and 40 ng APPswe per well, 50 µM siRNA and restriction to the inner 60 wells of a 96-well-plate. These conditions, presence of 50 nM siRNA for 72 h, were applied previously on the same kinome siRNA library [446] and influenced the decision to use 50 nM instead of 100 nM. The library used in this screen, which are actually aliquots of the library used in [446], was a generous gift from the group leader Prof. Pierluigi Nicotera. Of the kinase siRNAs used at 50 nM, CSNK1 siRNA, which was demonstrated previously to achieve a very good knockdown on protein level, did not reduce SEAP activity whereas the hits MAP2K1IP1, MAP3K3 and IRAK3 from a previous screen using the same kinome siRNA library and SEAP- APPswe in SHSY5Y at Lundbeck A/S reduced it by 60%, 30% and 50%, respectively, and siRNAs against BCR, PFKM, PRKCA and STK10 by 35%, 50%, 50% and 65% respectively. Strangely, MAP2K1IP1 siRNA reduced SEAP activity already by 35% and 50% at extremely low concentrations of 0.1 and 0.5 nM, respectively, either due to enormous efficiency, off-target effects or general toxicity, in contrast to the other kinase siRNAs with first effects of less than 20% reduction beginning at 5 nM. In the screen itself these results could be reproduced only for some kinase siRNAs and not to the full extent. BCR and PFKM siRNAs did not reduce SEAP activity while MAP2K1IP1, MAP3K3, IRAK3 and STK10 siRNAs reduced it by 44%, 16%, 28% and 14%, respectively. Nevertheless, MAP2K1IP1 and IRAK siRNA, with the same sequence as in the screen by Lundbeck A/S, ended up as hits demonstrating some reproducibility with SHSY5Y and HEK293. Surprisingly, CSNK1D was also found as hit by the unbiased data analysis using only siRNA identifiers. The SEAP reduction due to MAP2K1IP1 siRNA from the control with SEAP-APPswe was, however, comparably high with 32% and with the strange effects seen, MAP2K1IP1 was therefore omitted from validation by measurement Aβ from APPswe. Regarding the

192 Discussion question whether the rather low SEAP activity of the control originated in general toxicity, it can be speculated that it might not be the case because with the plates which have been subjected to MTT measurement all wells had extremely uniform values, although this is not a proof that none of the used siRNAs exerted general toxicity. Since in the screen by Lundbeck A/S with SEAP-APPswe Aβ was the decision value and SEAP the control value, SEAP from the MAP2K1IP1 siRNA was probably quite high which leaves the possibility for cell-specific differences of MAP2K1IP1 function between SHSY5Y and HEK293. The remaining 38 kinase hits, those with low SEAP from SEAP-APPswe-EpoR and Aβ from APPswe and rather high SEAP from SEAP-APPswe, were classified on a first look as interesting or uninteresting by screening the literature when information was available. Six of these hits were very interesting because they were either linked to AD, remotely to BACE1 expression or phosphorylation, neuronal function or other processes. TRAD () is a multifunctional Rho GDP/GTP exchange factor with an important role in cytoskeletal organization and affects process initiation and outgrowth in neurons [474]. Kalirin-7 was the most prevalent isoform from in the adult rat hippocampus and was most consistently underexpressed in AD brain [448]. UDP-glucose pyrophosphorylase 2 (UGP2) transfers a glucose moiety from glucose- 1-phosphate to MgUTP and forms UDP-glucose and Mg-pyrophosphate and thus competes with UDP-N-acetylglucosamine pyrophosphorylase from the hexosamine biosynthetic pathway for UTP, which maybe could lead to less O-GlcNAcylation of transcription factors including YY1 and ultimately increase BACE1 transcription as hypothesized in the introduction. Endoplasmic Reticulum to Nucleus signaling 2 (ERN2), also called IRE3, and PERK, which regulates BACE1 translation [261], have a common molecular interface necessary and sufficient for dimerization and unfolded protein response signaling [475]. Casein kinase 1 D (CSNK1D) phosphorylated BACE1 at S498, which resulted in trafficking to late endosomal compartments [99] and enhanced interaction with GGA1 [298], and was upregulated in AD hippocampus, 30fold on protein level [299] and 24fold on mRNA level [300]. Interleukin-1 receptor-associated kinase 3 (IRAK3, IRAK-M) was interesting because it selectively attenuated p38 activation [454] which was activated in mild and severe

193 Discussion

AD cases [452], associated with amyloid deposition in an AD model [453] and because the interleukin-1 receptor 2 (IL-1R2) is a BACE1 substrate [392]. Serine-/threonine-kinase 29 (STK29, BRSK2, SAD kinase) was required in forebrain neurons to acquire polarity and endow axon and dendrites with distinct properties which is promoted by phosphorylation of Tau and could therefore maybe link Aβ generation and Tau hyperphosphorylation. In order to increase the likelihood to work on true hits in future experiments, and that as unbiased as possible, it was necessary to compile hit profiles of likely true and false hits first and then rank all hits according to the criteria found to be important. Repetition experiments with the siRNAs against CSNK1D, TRAD and STK29 were thought to suffice that the kinases revealed their true and false hit characters. These three were chosen because they had different profiles, in terms of SEAP from SEAP- APPswe-EpoR and SEAP and Aβ from APPswe, and also because they were most interesting. Data could be reproduced with CSNK1D, though not in each experiment, but not with TRAD or STK29 and demonstrated the importance of the control because CSNK1D as only one of them had in the screen high SEAP from SEAP- APPswe with around 90% and a large difference to SEAP from SEAP-APPswe- EpoR. Controlling the CSNK1D siRNA-mediated knockdown resulted in 45% decrease on qPCR product level which was not too far from the 60% for the effective siRNA against CSNK1 (D and E) [438] obtained during transfection optimization. No knockdown at all could be confirmed for TRAD siRNA which would not be too astonishing but repeated detection of the housekeeping gene Actin B at a similarly high cycle number of around 28 was. It could be reduced to a cycle number of 23 by use of only the reverse primer specific for Actin B in direct comparison with usually employed random hexamer primers. Maybe the usage of poly-T-primers, the longer, the better to bind exclusively the poly-A-tail, would result in a similar cycle number even with distances from poly-A-tail to site of qPCR amplification longer than 1000 bp, but this was not tested. Coming back to validation of hit kinases, ranking of the hits was influenced by giving the control SEAP and the Aβ values more weight in the ranking formula such that the likely false hit STK29, which was ranked higher than CSNK1D by the unweighted formula, then got a much lower rank than CSNK1D while most other high ranking hits changed only some positions. The upper half of the hits was validated with validated siRNAs and also some lower ranking to substitute those where no validated siRNA

194 Discussion was available. All of them were falsified. This fate was also shared by 16 hits identified out of the 885 remaining kinase siRNAs which were screened in parallel by Dr. Yana Chernyshova and then also validated in parallel; these hits were PRKACB, CAMK1, PRKCE, RPS6KA3, PRKCG, RPS6KB2, PRKCI, CASK, PRKCZ, TSSK3, PDK1, STK17A, PKN3, PRKAA1, PKN2, PRKAA2. CSNK1D siRNA seemed the most interesting candidate. However, the negative result of the CSNK1D/E specific inhibitor for downregulation of β-cleavage is in line with the negative result for the D-isoform of a study on regulation of Aβ generation by CSNK1 in the laboratories of the Noble Prize laureate Paul Greengard [302], CSNK1D might indeed not be a regulator of β-cleavage. Inhibition of CSNK1E resulted in increased CTFβ, hinting on a probable regulation of γ-secretase [302]. This could however not be tested with the use of SEAP-APPswe-EpoR and was also not tested with the other constructs. Similarly, the PI3K inhibitor LY294002 confirmed the negative result of the validated siRNAs against the catalytic and regulatory subunit of PI3K for regulation of β- cleavage. The p38-MAPK inhibitor showed that the effect of IRAK3 on β-cleavage, if it is true, is probably not mediated via p38-MAPK. The validated siRNA against RIPK4 seemed to upregulate α-cleavage. RIPK4 interacts with (PKC) isoform δ and activation of PKC decreased the production of Aβ [476]. However, the effect was not reproducible. The outcome of validated siRNAs vs. screen siRNAs suggests that all hits were due to off-target effects. In comparison, the experimental conditions were similar for validation of the siRNAs [451] and for this kinase hit validation with continuous presence of 25 and 50 nM for 48 h and 72 h on 4∙104 and 7.5∙104 HEK293 per 96- well, respectively. Nevertheless, the marginal possibility exists that some of the negative results for the hit kinases with the validated siRNAs could also be wrong due to single nucleotide mutations acquired during cell culture of the herein used cells [477]. Regarding off-target effects, the reduction of SEAP of SEAP-APPswe by the validated TSSK3 siRNA is intriguing because TSSK3, testis-specific serine kinase 3, is exclusively expressed in testis and not in other tissues as demonstrated [478] . Two other kinase hits of the screen were also found to be expressed in testis: TEX14, testis expressed sequence 14, and the TSSK3 paralog TSSK1B – the latter one not expressed in brain, ovary, kidney, liver or early embryonic cells [479] and in the validation with a similar effect like TSSK3, although not as strong. If the validated

195 Discussion

TSSK3 siRNA did not trigger an off-target effect, the kidney-derived HEK293 would have enlarged their repertoire of expressed kinases beyond their initial kinome set. Further, TSSK3 would be a candidate for a kinase affecting trafficking of the SEAP- part in the APP-constructs, or unmodified SEAP also, because SEAP is expressed in germ cells [480]. If this is not the reason for reduction of SEAP activity with both APP- constructs, it would have to downregulate both α- and β-cleavage and thus add one more potential side effect of systemic inhibition of BACE1: The downregulation of TSSK-triggered cleavage of proteins by BACE1 which could affect germ cells. Targeted deletion of TSSK1 and -2 resulted in infertility probably due to haploinsuffiency and in the failure to form elongated spermatids [481] and at least two BACE1 substrates are present in testis and on sperm [401]. APLP2, or rather the N-terminally truncated variant YWK-II, was found to be highly expressed in human testis and sperm [482, 483] and immunization of female rats with peptide segments of YWK-II reduced their fertility probably by interference with sperm-oocyte interaction [484], which by the way would be a potential side effect of systemic α- secretase activation. YWK-II was also shown to interact with the Müllerian-inhibiting substance [485]. This was suggested to promote sperm longevity and viability, possibly, as also suggested, via a G0-mediated pathway because the C-terminus of YWK-II interacted with G0 [486]. APP containing the KPI-domain was also expressed on sperm [401]. On the other hand, sporadic AD patients are typically not in the reproductive age and possible side effects of BACE1-inhibition in the reproductive tract would be a concern to far less patients - those at risk for Familial AD in young age. And even then: Sperm or oocytes could be frozen before begin of a therapy. Also, reduced fertility because of mutations in APP does not seem to pose a problem, probably because the mutations do not affect α-cleavage, are rescued by APLP2 or APP plays only a minor role here. The outcome of screen siRNAs vs. kinome also has to be discussed. Firstly, some kinases are probably not expressed in HEK293 and these could never become hits. Secondly, catalytic activity might be more difficult to knockdown than the protein because not only kinase and substrate concentration but also the duration of colocalization of kinase and substrate could be very important given that theoretically one single kinase molecule would suffice to rescue largely the kinase function if the colocalization lasts long enough and if it is very refractory to degradation. With

196 Discussion degradation the time-factor becomes again important. A long-lived kinase with a half- life of 30 h would, at the end of 72 h experiment duration with 100% knockdown of mRNA, have around 20% of its protein left while the area under the curve until 2.4- times the half-life would be around 50%. Thirdly, redundancy due to related genes and differential splicing could also rescue function especially with kinases because most, according to current knowledge, recognize accessible serines, threonines or tyrosines in very short motifs. However, the situation is different when other functions than the catalytic activity are more important for example when the kinase is mainly serving as scaffold protein as has already been demonstrated for at least one phosphatase containing several protein interaction domains [487]. In these cases, like in the case of the MAP2K1 Interacting Protein 1 (MAP2K1IP1), the siRNA could have a stronger effect and open maybe new pathways with relevance for AD but a hit like this would actually not be specifically against the kinome, just the proteome, and not what the screen was to a large part conducted for because most kinase inhibitors are ATP-analog-derivatives. At the same time it would and would not be off-target. Summarizing the screen results, the outcome of kinome vs. AD still has to be awaited.

197 Discussion

12 Interpretation of potential APLP-like pseudogenic fragments and gene expression data analysis of C3orf52

12.1 Interpretation of potential APLP-like pseudogenic fragments The potential APLP-like processed pseudogenic fragments (ANNE) presented, as well as the result of their analysis, have to be compared to characteristics of verified processed pseudogenes in order to be classified. These characteristics encompass properties of the sequence of processed pseudogenes on one hand and those of its genomic region on the other hand. Further, estimation of their existence in phylogenetic relevant taxa is mandatory. So, beginning with the comparison of ANNE to verified processed pseudogenes, the most important attribute of a pseudogene is similarity to its parent gene. Here, verified pseudogenes possess in average 75% and 86% sequence similarity for amino acids and for nucleotides in addition to 94% completeness in coding regions, while ANNE has only 5.3% and 13.7% identity on amino acid and nucleotide level to (human) APP family members, assuming that it is 93% complete (≈150 bp missing in alignment with APP family members). The expect-value (e-value) accounts for both similarity and completeness and in the whole genome screen only potential pseudogenes with e-values lower than the cut-off 10-10 were regarded as true [413]. An example for an achieved e-value is 3.5∙10-212 for the pseudogene 5316 with 85% similarity of its ≈450 amino acids to its parent gene ERR1, listed in the pseudogene database [413, www.pseudogene.org], from which ANNE with an e-value of 0.02 is hundreds of magnitudes away. However, as outlined in the introduction, similarity to parent genes is lost with increasing age due to accumulation of nucleotide substitutions [413]. While most verified processed pseudogenes were created after the primate-rodent divergence 85-75 mya [413], the estimated ages of ANNE with 239 or 294 my would account for far less similarity. According to D. Graur, pseudogenes older than 200 my have by now become unrecognizable [424]. In line with this assumption, the highest age for a processed pseudogene found in literature, by use of a substitution rate of 1.25∙10- 9/site/year, was calculated to over 150 my for Nanog P6 with 83% identity to its parent gene and was maybe an overestimation according to the authors [412]. So, if ANNE is truly a processed pseudogene, despite its poor level of conservation it would have been recognized with an age possibly older than 200 my. However, the

198 Discussion location of ANNE in a region with a rather low gc-content of 41.4% might have preserved it slightly better than most young processed pseudogenes typically found in isochores of 46%, anticipated that chromosomes with lower gc-content have a slower DNA-turnover rate which reduces the substitution rate [413]. The calculated ages would probably also be sufficient to erase other features of many verified relatively young processed pseudogenes like short direct repeats and a poly-a-tail of which none could be found for ANNE. Even if ANNE is a processed pseudogene, especially missing the poly-a-tail makes it difficult to judge whether ANNE was derived from an APP family member mRNA. The coding region could also have stopped further downstream, however, was supposed to end shortly downstream of a stretch reminiscent of the conserved APP family motif “PEER”. Likewise, all other stretches were also recognized, once a potential former transmembrane domain was noticed, because the author was familiar with APP-like protein sequences. This strong bias in addition to the very low e-value and the fact that so far no APLP-like pseudogene has yet been reported (as for 90% of the proteome) [413] more or less ablates a sufficient probability for an APLP-like pseudogenic origin of the sequence. Nevertheless, a hypothetical APLP-like pseudogenic origin might be worth a short discussion. For ANNE to be at least possible as APLP-like processed pseudogene, three main points have to be regarded. The first one relates to the technical feasibility for creation of an APLP-like processed pseudogene in general and the second and third specifically to the calculated ages and the presented location in context with vertebrate phylogeny and 3‟-untranslated regions (UTRs). Although processed pseudogenes have an average length of just 740 bp and are thus much shorter than ANNE and an average APP family member with over 2000 bp, the LINE1 machinery is capable of reverse transcription of its own 6 kbp full length sequence which was also confirmed for the more recent past by identification of young full length copies [411]. Processed pseudogenes with similar lengths as ANNE with 2094 bp were easily found in the human pseudogene dataset like the processed pseudogenes 251236, 238485 and 238566 with 2168, 2328 and 2083 bp, respectively [www.pseudogene.org]. Whether an APLP-like processed pseudogene would have been 5‟-truncated cannot be deduced from the found sequence, because as potential APLP-like pseudogene its mRNA would probably have had a 5‟-

199 Discussion untranslated region similar to the ones of the APP family members between lengths of 138-200 bp. Further as outlined in the introduction, germ-line expression level is regarded as crucial for the creation of processed pseudogenes. Beginning with oocytes, where retrotransposition is suspected to take primarily place [424], human APP members were not reported to be expressed in them, however, full length APP and the N- terminally truncated APLP2 variant YWK-II were found to be highly expressed in human testis and sperm [401, 482, 483]. YWK-II was shown to interact with the Müllerian-inhibiting substance [485] which appears like a mammal-specific activity. But reduced fertility of female rats immunized with peptide segments of YWK-II, probably due to interference with sperm-oocyte interaction [484] and effects on sperm longevity and viability via a G0-mediated signal transduction pathway [486], seem to hint on more basic activity which might thus have also been present in early tetrapods with the chance to become fossilized as processed pseudogene. The expression and assumed function for APP family members on sperm immediately raises the question, why no processed pseudogene of the family is known (according to the databases). Firstly, mature sperms have meiosis already completed, so that genes which are expressed after this stage in spermatogenesis should have fewer chances on pseudogene-offspring as genes expressed during the critical phases. However, no data were found regarding when exactly the APP members are expressed on sperm. Also, LINE1 is restricted to mammals which makes creation of processed pseudogenes in general and also of APP members in other vertebrate classes less likely [424]. However, even if not LINE1 or its ancestral sequence was present in the early tetrapods, reverse transcriptase‟s of any retrovirus should principally fulfill the same function and indeed (rarely) processed pseudogenes have been found in other tetrapodal lineages and fruitfly [424]. The calculated ages of either 239 or 294 my, using either rodent or chicken substitution rates, would place the hypothetical retrotransposition event in the era of the first animals regarded as mammals like Thrinaxodon, which lived around 245 mya [425], or close to the mammal-reptilian divergence 300 mya. While the possible similar creation of an intron between mammalian and chicken exon 5 of C3orf52 and its 3‟UTR in contrast to teleosts (although confirmation by amphibians and chondrichthyans lacks) rather refutes the lower age, the higher age might work out for both when the substitution rate was slightly lower in reality. Then again, the lower

200 Discussion substitution rate in chicken should have conserved a processed pseudogene of this age better than in human, which is actually not the case. In other mammals, as identified for dog in this study, potential remnants of an APLP- like pseudogenic sequence are equally poorly or even worse conserved. However, the potential former transmembrane domain was recognized and thus shared at least by all placental mammals. Examples for a general worse conservation in other mammalian taxa are obviously larger insertions, for example in rabbit, horse and cattle, and deviations from a shared gt-repeat with best conservation in chimpanzee. At this point to be discussed best, alignments of mammalian ANNE on DNA-level to calculate trees for mammalian phylogeny by AiO and CLUSTAL (not shown in results chapter) resulted in similar trees (Fig. 78). Primates were placed between ungulates and rodents, comparable as in the upper (C) of two trees from evolutionary studies on orthologous sequences of mammalian LINE1 copies [488, 421]. A C Rabbit (Oc) Cattle (Bt) Horse (Ec) Dog (Cf) Chimp. (Pt) Human (Hs) Mouse (Mm) B D Cattle Horse Dog

Fig. 78 A-D: Trees of mammalian ANNE- DNA -sequences (AB) compared to trees calculated from orthologous mammalian LINE1 sequences (CD). C and D modified from [488] and [421].

201 Discussion

The presented location of ANNE as potential APLP-like processed pseudogene has to be evaluated regarding two aspects. Firstly, it was suggested that processed pseudogenes tend not to be found in the vicinity to their parent genes [413], because it is unlikely given the lengths of genomic DNA. However, even if ANNE is relatively close to APP at least in rodents, APLP1 and APLP2 are found at completely different locations. Secondly, ANNE covers a region (“exon 6” and its 5‟-flanking region) in C3orf52 which could be called an alternatively spliced exon from the perspective of three C3orf52 transcripts, of which one is protein coding. Further the large size of “exon 6” with nearly 1500 bp in human and mouse was reminiscent of a pseudogene. However, TTMP from teleosts corresponds to human TTMP isoform 2, where “exon 6” would be nothing but the 3‟-UTR. Further, 1500 bp for a mammalian 3‟UTR is just an average length when seen in the light of more recent results [489, 490]. Also, no other pseudogenes different from Alu sequences were identified downstream of human exon 5 until the end of “exon 6”. The Alu-copy identified in ANNE (c3‟Alu) is 5‟-truncated, missing 210 of usually 300 bp, and has a probable age of 80 my as member of the AluJb subfamily [422]. Disruption by Alu or LINE copies has been reported for over 1100 processed pseudogenes [413]. To summarize, for all the strong negative reasons mentioned, an APLP-like processed pseudogene was most probably not identified, however the discussion might serve as example how ancient processed pseudogenes can be validated.

12.2 Gene expression data analysis of C3orf52

The expression of C3orf52 was analyzed both in vitro and in silico. By the in vitro data, expression in embryonic stem cells as previously reported in the expressed sequence tag (EST) data was confirmed. Interestingly, in addition to the mRNA entries from B-cells containing “exon 6” in same reading direction as in C3orf52, two mRNAs also from B-cells were reported in antisense. Further analysis of EST data revealed many entries linked to myeloid leukemia and monocyte differentiation so that TTMP might potentially contribute to disease etiology. Therefore it has to fullfill an essential function to be lost or gain a deleterious function. So far, a function for TTMP has not yet been identified. By comparative relative positions of the paralogous regions on chromosomes 3 and 11 and similar tissue- specific expression the chemokine receptor CXCR5 was suspected as potentially

202 Discussion related gene. However, TTMP and CXCR5 have 250 and 374 amino acids with only 15% identity and 27% similarity so that the two genes are most probably not strongly related. Despite this phylogenetic distance, the two genes might serve in similar pathways due to some common characteristics and were compared in order to get an idea for the function of TTMP. To start with, the expression of TTMP is highest in thymus with 73 per 1 million transcripts, followed by lymph nodes with 65 (Tab. 23A). Likewise, CXCR5 was identified as gene specifically expressed in lymphatic tissues [491]. Further, it was suggested that the primary function of CXCR5 is linked to B-cell migration and localization within lymph nodes and spleen by interaction with its specific ligand B-lymphocyte chemoattractant (BLC) [492, 493], which was confirmed in mouse. For TTMP, experimental array data with immunological background do not suggest a function in migration of B-cells (or other cells of the immune system), since only one entry is linked to migration which was an observed depletion of a kidney cell line as response to the macrophage migration inhibitory factor. So a different pathway and function have to be suspected which appear perhaps in the end as suggestion by following the array data from step to step on the most solid data clusters. The first step, choice of cell type, is easy because the monocyte- macrophage lineage data cluster doubles the lymphocyte lineage cluster (Tab. 23C). In the next step, the upstream effector cannot be decided because the data clusters PAMPs and hormones are approximately equally large. For the effect, the option is basically just differentiation, so for both monocytes and dendritic cells a main function for TTMP in differentiation seems to vaguely appear. Its function maybe however also necessary earlier in the myeloid lineage leading to leukocytes, megakaryocytes dividing into platelets, and also erythrocytes, because megakaryocytic differentiation, hematopoietic stem cells and sickle cell anemia are also mentioned 3, 6 and 2 times in the array data, respectively. One entry is from platelets in sickle cell disease, so that both megakaryocytes and erythrocytes are involved (Tab. 23B). In another entry nicotinamide had an effect on thrombopoietin-induced megakaryocyte differentiation. Further, two entries of hematopoietic stem cells are due to responses to CXCL12 (in combination to UTP) and Transforming Growth Factor-β (TGF-β). Regarding effects of hormones, TGF-beta1 elicited also a response of GFAP-negative lamina cribrosa cell and interferon-γ of microglia. In two more entries, anti-TNFα therapy for rheumatoid arthritis had an effect on blood. Further, at least for one Toll-like receptor

203 Discussion on dendritic cells, agonists showed synergistic effects in an array entry and in total 26 small molecules, 5 drugs, 5 allergens, 2 smoke components, 7 receptor effectors, 5 lipid-derived hormones and 2 hormone cofactors elicited effects (Tab. 23E). Interestingly, with regards to the lipid-derived hormones, CXCR5 showed up in a study on gene-centric association signals for lipids and apolipoproteins via the HumanCVD BeadChip [494] and with regards to an effect of CXCL12 on TTMP, CXCL13 causes CXCR5-dependent activation of PI3Kp85α in prostate cancer cells [495]. Summarized, although TTMP and CXCR5 do not seem to share very similar functions, the array data in comparison to CXCR5 might propose a function as receptor, maybe for small ligand cofactors since TTMP itself is rather small. It could thereby modify responses to hormones, in the monocyte lineage maybe responses to pathogens, leukotrienes, IL-1, IL-6, IL-12, CSF-1 or TLRs. The above array data relating to mechanistic studies were taken as hint to continue the comparison to CXCR5 with data from epidemiological studies in order to refute or suspect TTMP to be at least able to contribute to a disease. For CXCR5, contribution to disease etiology was already demonstrated by the association of overexpression to prostate cancer [496] and to a lesser degree to Burkitt‟s lymphoma [497] and to systemic lupus erythematosus and –nephritis [498]. For TTMP, 19 array data entries were related to prostate cancer, more specifically: 2 to prostate cancer progression and metastasis each, 4 to androgens, and one to dihydotestosterone, arachidonic acid and herniasterlin each [Tab. 23D]. Hence, the array data linked to aggressive stages and small ligands might suggest that a function of TTMP contributes to prostate cancer. In the transcript data from diseased tissues, lymphomas lead with 55 per million, followed by lung tumors, leukemias, and kidney tumors with 48, 31 and 29 [Tab. 23B]. In this light, the observed depletion of a kidney cell line as response to the macrophage migration inhibitory factor might bear some significance. By the array data with immunological background, TTMP was linked to leukemias, with 10 and 14 entries of myeloid and lymphatic origin, inflammatory bowel disease, arthritis and neurodegenerative diseases [Tab. 23B/C]. Further, Burkitt‟s lymphoma is characterized by large cells with traits of B-cells and a basophile-like cytoplasm whereas myeloid leukemias are characterized by reaching different stages of monocyte differentiation, ranging from no signs of differentiation to around 80% fully differentiated monocytes. So, with a putative function of TTMP in monocyte differentiation, contribution of TTMP to myeloid leukemia might be hypothesized.

204 Discussion

Mechanistically, the antisense mRNAs from germinal center B-cells could counteract expression of TTMP. Also they could actually be somehow independent exons of the immediate downstream antisense gene Germinal Center Expressed Transcript 2 (GCET2). With regards to another possible relationship of TTMP to a gene, an essential function in myeloid differentiation as cytosolic adaptor protein was established for the myeloid differentiation primary response gene 88 (MyD88)[468]. It has a similar length and in medaka it is found only 0.5 mbp downstream of GRAMD1C which led to thoughts about a possible duplication leading to TTMP, however TTMP possesses a transmembrane domain and sequence identity was also not high. Lastly, in order not to omit the connection of TTMP to lung cancer, tetramethylpyrazine reduced gene expression levels of the type 2 cytokines Il-4 (in Mm alias B-cell IgG differentiation factor) and Il-6 (in Hs alias B-cell differentiation factor) in lung cancer patients [499].

205 Discussion

13 The search for 4. APP family member and the examination of the APP family regions Following the perception, that C3orf52 is not the 4. APP family member, a second attempt to identify it on chromosome 3 was conducted, however also without success but with two insights. Gapless alignments of APP family C-termini to the region between GRAMD1C and LSAMP led to the result that theoretically, after a certain age, and due to substitution, discrimination between a true alignment to a lost sequence and any false alignment to a sequence with randomly high identity would not be possible anymore. When searching for example a 100 bp sequence, in order to be able to discriminate between true and false alignments to 100 kbp of genomic DNA, the true sequence must not be lost earlier than around 175 mya. The age, where discrimination is still possible, is probably positively correlated to the length of the searched sequence and negatively to the length of the scanned DNA, which implicates that processed pseudogenes should be found easier than lost exons of other pseudogenes of the same age. Actually, the complications associated to random similarity is just a reason for an age-dependent cut-off with regards to “recognizability” of processed pseudogenes from the above statement (there 200 mya as cut-off). The other insight can be summarized as taking special care of repeats like Alu copies is advisable before alignments of genomic regions. Unfortunately, their strong impact on the alignments was underestimated and the probable linkage between the regions around APP and GRAMD1C not known by the author at that timepoint. During the phylogenetic calculations for gene families in the three regions containing APP family members and a potential fourth region from the two WGDs, the topology testings and tree reconstructions gave ambiguous results. However, the topolgoy (AD)(BC), which is tantamount to (APLP1,?)(APLP2,APP) for the APP family, achieved slightly better results than the alternatives when the trees of all test families are considered. Regarding the question about the relationship of the APP family members, the result is thus similar as suggested by Coulson [408] with the first WGD leading to the APLP1/?-ancestor and the APLP2/APP-ancestor. The result is backed by the higher bootstrap values for the APP family topology (A)(BC) and also by the presence of other gene families like the BACE, PHLDB and IgLON families. The uncertainties in topology rather reflect the initial small phylogenetic distance due to an short interim time of the WGDs as anticipated.

206 Discussion

For the APLP identified in the elephant shark genome in thus study, it is currently unknown to the author whether it is one of the sequences reported as APP or APLP2. This APLP might be actually APLP1, but was found via BLASTN with APLP2. If it is the reported APLP2 (because maybe also identified by the Elephant Shark Genome Project via APLP2), it should be validated for example by neighboring genes. In case that it is not the reported APLP2 or APP, it should also be validated because it might be APLP1 (or APLP3 or a local duplication). One week after contacting Prof. Venkatesh from the elephant shark genome project because of this APLP and providing also the annotated medaka APLP1 in comparison, the teleost and green anole APLP1 genes were annotated (Fig. 79) which they were not at least the past four months in the ENSEMBL database. So, maybe during this study some preparatory work for database maintenance was carried out indirectly. However, the update might also be just coincidental because of a novel entry from hedgehog (so far merely a 47aa KPI-domain). Then again, the second platypus APLP1 entry (Cterm) is still included and placed along with rabbit and green anole APLP1 as branching off from a duplication together with tetrapodal APLP1.

APLP2

APP rabbit APLP1

platypus APLP1 Cterm green anole APLP1 APLP1

teleost APLP1

hedgehog KPI-domain

Fig. 79: Updated APP family tree in the ENSEMBL database, modified. Duplication, speciation and ambiguous nodes in red, blue and cyan.

207 References

14 References

[1] A. Alzheimer, Über eine eigenartige Erkrankung der Hirnrinde, Allgemeine Zeitschrift für Psychiatrie und Psychisch-gerichtliche Medizin (1907) 146-148. [2] E. Redlich, Über miliare Sklerose der Hirnrinde bei seniler Atrophie, Jahrb Psychiat Neurol (1898) 208-216. [3] M. Goedert, Oskar Fischer and the study of dementia, Brain 132 (2009) 1102-1111. [4] M. Franke (1975) in Bezirkskrankenhaus Brandenburg/Havel, Vol. M.D. [5] A. Alzheimer, Histologische Studien zur Differentialdiagnose der progressiven Paralyse., Nissls Arb 1 (1904) [6] T. Arendt, G. Taubert, V. Bigl, A. Arendt, Amyloid deposition in the nucleus basalis of Meynert complex: a topographic marker for degenerating cell clusters in Alzheimer's disease, Acta Neuropathologica 75 (1988) 226- 232. [7] O. Fischer, Miliare Nekrosen mit drusigen Wucherungen der Neurofibrillen, eine regelmässige Veränderung der Hirnrinde bei seniler Demenz, Monatsschr Psychiat Neurol (1907) 361-372. [8] M. Neumann, R. Cohn, Incidence of Alzheimer's disease in large mental hospital; relation to senile psychosis and psychosis with cerebral arteriosclerosis., AMA Arch Neurol Psychiatry 69 (1953) 615-36. [9] H.K. Hans, F. Wolfgang, W. Herbert, F. Wolfgang, I. Bernd, E.K. Helmfried, Alzheimer's second patient: Johann F. and his family, Annals of Neurology 52 (2002) 520-523. [10] A. Alzheimer, Über eigenartige Krankheitsfälle des späteren Alters, Z ges Neurol Psychiat (1911) 356-85. [11] M.B. Graeber, S. Kösel, R. Egensperger, R.B. Banati, U. Müller, K. Bise, P. Hoff, H.J. Möller, K. Fujisawa, P. Mehraein, Rediscovery of the case described by Alois Alzheimer in 1911: historical, histological and molecular genetic analysis, neurogenetics 1 (1997) 73-80. [12] G.G. Glenner, C.W. Wong, Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein, BBRC 120 (1984) 885-890. [13] F. Struwe, Histopathologische Untersuchungen über Entstehung und Wesen der senilen Plaques, Zeitschrift für die gesamte Neurologie und Psychiatrie 122 (1929) 291-307. [14] M.I. Olson, C.-M. Shaw, PRESENILE DEMENTIA AND ALZHEIMER'S DISEASE IN MONGOLISM, Brain 92 (1969) 147-156. [15] G.G. Glenner, C.W. Wong, Alzheimer's disease and Down's syndrome: Sharing of a unique cerebrovascular amyloid fibril protein, BBRC 122 (1984) 1131-1135. [16] C.L. Masters, G. Simms, N.A. Weinman, G. Multhaup, B.L. McDonald, K. Beyreuther, Amyloid Plaque Core Protein in Alzheimer Disease and Down Syndrome, PNAS 82 (1985) 4245-4249. [17] D. Goldgaber, M.I. Lerman, O.W. McBride, U. Saffiotti, D.C. Gajdusek, Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease, Science 235 (1987) 877-880. [18] R.E. Tanzi, J.F. Gusella, P.C. Watkins, G.A. Bruns, P. St George-Hyslop, M.L. Van Keuren, D. Patterson, S. Pagan, D.M. Kurnit, R.L. Neve, Amyloid beta protein gene: cDNA, mRNA distribution, and genetic linkage near the Alzheimer , Science 235 (1987) 880-884. [19] J. Kang, H.-G. Lemaire, A. Unterbeck, J.M. Salbaum, C.L. Masters, K.-H. Grzeschik, G. Multhaup, K. Beyreuther, B. Muller-Hill, The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor, Nature 325 (1987) 733-736. [20] M. Citron, T. Oltersdorf, C. Haass, L. McConlogue, A.Y. Hung, P. Seubert, C. Vigo-Pelfrey, I. Lieberburg, D.J. Selkoe, Mutation of the β-amyloid precursor protein in familial Alzheimer's disease increases β-protein production, Nature 360 (1992) 672-674. [21] M. Mullan, H. Houlden, M. Windelspecht, L. Fidani, C. Lombardi, P. Diaz, M. Rossor, R. Crook, J. Hardy, K. Duff, F. Crawford, A locus for familial early-onset Alzhelmer's disease on the long arm of chromosome 14, proximal to the a1-antichymotrypsin gene, Nat Genet 2 (1992) 340-342. [22] X.D. Cai, T.E. Golde, S.G. Younkin, Release of excess amyloid beta protein from a mutant amyloid beta protein precursor, Science 259 (1993) 514-516. [23] I. Hussain, D. Powell, D.R. Howlett, D.G. Tew, T.D. Meek, C. Chapman, I.S. Gloger, K.E. Murphy, C.D. Southan, D.M. Ryan, T.S. Smith, D.L. Simmons, F.S. Walsh, C. Dingwall, G. Christie, Identification of a Novel Aspartic Protease (Asp 2) as β-Secretase, Mol Cell Neurosc 14 (1999) 419-427. [24] X. Lin, G. Koelsch, S. Wu, D. Downs, A. Dashti, J. Tang, Human aspartic protease memapsin 2 cleaves the β- secretase site of β-amyloid precursor protein, PNAS 97 (2000) 1456-1460. [25] S. Sinha, J.P. Anderson, R. Barbour, G.S. Basi, R. Caccavello, D. Davis, M. Doan, H.F. Dovey, N. Frigon, J. Hong, K. Jacobson-Croak, N. Jewett, P. Keim, J. Knops, I. Lieberburg, M. Power, H. Tan, G. Tatsuno, J. Tung, D. Schenk, P. Seubert, S.M. Suomensaari, S. Wang, D. Walker, J. Zhao, L. McConlogue, V. John, Purification and cloning of amyloid precursor protein β-secretase from human brain, Nature 402 (1999) 537-540. [26] R. Vassar, B.D. Bennett, S. Babu-Khan, S. Kahn, E.A. Mendiaz, P. Denis, D.B. Teplow, S. Ross, P. Amarante, R. Loeloff, Y. Luo, S. Fisher, J. Fuller, S. Edenson, J. Lile, M.A. Jarosinski, A.L. Biere, E. Curran, T. Burgess, J.-C. Louis, F. Collins, J. Treanor, G. Rogers, M. Citron, β-Secretase Cleavage of Alzheimer's Amyloid Precursor Protein by the Transmembrane Aspartic Protease BACE, Science 286 (1999) 735-741. [27] R. Yan, M.J. Bienkowski, M.E. Shuck, H. Miao, M.C. Tory, A.M. Pauley, J.R. Brashler, N.C. Stratman, W.R. Mathews, A.E. Buhl, D.B. Carter, A.G. Tomasselli, L.A. Parodi, R.L. Heinrikson, M.E. Gurney, Membrane- anchored aspartyl protease with Alzheimer's disease β-secretase activity, Nature 402 (1999) 533-537. [28] D. Edbauer, E. Winkler, C. Haass, H. Steiner, Presenilin and nicastrin regulate each other and determine amyloid β-peptide production via complex formation, PNAS 99 (2002) 8666-8671. [29] H. Steiner, E. Winkler, D. Edbauer, S. Prokop, G. Basset, A. Yamasaki, M. Kostka, C. Haass, PEN-2 Is an Integral Component of the g-Secretase Complex Required for Coordinated Expression of Presenilin and Nicastrin, J. Biol. Chem. 277 (2002) 39062-39065.

208 References

[30] D.R. Borchelt, G. Thinakaran, C.B. Eckman, M.K. Lee, F. Davenport, T. Ratovitsky, C.-M. Prada, G. Kim, S. Seekins, D. Yager, H.H. Slunt, R. Wang, M. Seeger, A.I. Levey, S.E. Gandy, N.G. Copeland, N.A. Jenkins, D.L. Price, S.G. Younkin, S.S. Sisodia, Familial Alzheimer's Disease-Linked Presenilin 1 Variants Elevate Aβ1-42/1-40 Ratio In Vitro and In Vivo, Neuron 17 (1996) 1005-1013. [31] J. Hardy, D. Allsop, Amyloid deposition as the central event in the aetiology of Alzheimer's disease., Trends Pharmacol Sci 12 (1991) 383-8. [32] D.J. Selkoe, The molecular pathology of Alzheimer's disease, Neuron 6 (1991) 487-498. [33] E.H. Corder, A.M. Saunders, W.J. Strittmatter, D.E. Schmechel, P.C. Gaskell, G.W. Small, A.D. Roses, J.L. Haines, M.A. Pericak-Vance, Gene Dose of Apolipoprotein E Type 4 Allele and the Risk of Alzheimer's Disease in Late Onset Families, Science 261 (1993) 828-9. [34] K.A. Jellinger, Alzheimer 100 – highlights in the history of Alzheimer research, J Neural Transm 113 (2006) 1603- 23. [35] D. Mann, The pathological association between Down syndrome and Alzheimer disease., Mech Age Dev 43 (1988) 99-136. [36] L.R. Stanton, R.H. Coetzee, Down's syndrome and dementia, Adv Psychiatr Treat 10 (2004) 50-58. [37] J. Tyrrell, C. Mary, M. Mary, M. Janet, C. Johnston, K. Alan, M. Martin, G. Michael, A.L. Brian, Dementia in people with Down's syndrome, International J of Geriatric Psychiatry 16 (2001) 1168-1174. [38] V.P. Prasher, V.H.R. Krishnan, Age of onset and duration of dementia in people with Down's syndrome, Am J Geriatr Psychiatry 8 (1993) 915-22. [39] R.C. Barnhart, B. Connolly, Aging and Down Syndrome: Implications for Physical Therapy, PHYS THER 87 (2007) 1399-1406. [40] D.S. Smith, Health care management of adults with Down syndrome., Am Fam Physician 64 (2001) 1031-1038. [41] C. Fonseca, D. Amaral, M. Ribeiro, I. Beserra, M. Guimaraes, Insulin resistance in adolescents with Down syndrome: a cross-sectional study, BMC Endocrine Disorders 5 (2005) 6. [42] S. Vora, U. Francke, Assignment of the human gene for liver-type 6-phosphofructokinase isozyme (PFKL) to chromosome 21 by using somatic cell hybrids and monoclonal anti-L antibody, PNAS 78 (1981) 3738-3742. [43] K. Fishler, R. Koch, Mental Development in Down Syndrome Mosaicism., Am J Ment Retard 96 (1991) 345-51. [44] V.P. Prasher, J.F. Matthew, M.K. Anna, M.C.F. Elizabeth, R.J. West, P.C. Barber, A.C. Butler, Molecular mapping of alzheimer-type dementia in Down's syndrome, Annals of Neurology 43 (1998) 380-383. [45] N. Schupf, D. Kapell, B. Nightingale, J.H. Lee, J. Mohlenhoff, S. Bewley, R. Ottman, R. Mayeux, Specificity of the fivefold increase in AD in mothers of adults with Down syndrome, Neurology 57 (2001) 979-984. [46] I.F. Rowe, M.A.C. Ridler, F.B. Gibberd, PRESENILE DEMENTIA ASSOCIATED WITH MOSAIC TRISOMY 21 IN A PATIENT WITH A DOWN SYNDROME CHILD, The Lancet 334 (1989) 229-229. [47] J.M. Ringman, P.N. Rao, P.H. Lu, S. Cederbaum, Mosaicism for Trisomy 21 in a Patient With Young-Onset Dementia: A Case Report and Brief Literature Review, Arch Neurol 65 (2008) 412-415. [48] H. Yamaguchi, S. Hirai, M. Morimatsu, M. Shoji, Y. Harigaya, Diffuse type of senile plaques in the brains of Alzheimer-type dementia, Acta Neuropathologica 77 (1988) 113-119. [49] J.M.M.D. Rozemuller, P.M.D. Eikelenboom, F.C.M.D. STAM, K.P.D. Beyreuther, C.L.M.D. Masters, A4 Protein in Alzheimer's Disease: Primary and Secondary Cellular Events in Extracellular Amyloid Deposition, J Neuropath Exp Neurol 48 (1989) 674-91. [50] K. Ogomori, T. Kitamoto, J. Tateishi, Y. Sato, M. Suetsugu, M. Abe, Beta-protein amyloid is widely distributed in the central nervous system of patients with Alzheimer's disease, Am J Pathol 134 (1989) 243-251. [51] A. Probst, D. Langui, J. Ulrich, Alzheimer‟s disease: A description of the structural lesions, Brain Pathol 1 (1991) 229-239. [52] J.G. Sheng, R.E. Mrak, W.S.T. Griffin, Neuritic plaque evolution in Alzheimer's disease is accompanied by transition of activated microglia from primed to enlarged to phagocytic forms, Acta Neuropathologica 94 (1997) 1- 5. [53] G.G. Glenner, Current knowledge of amyloid deposits as applied to senile plaques and congophilic angiopathy., in: R. Katzman,R. Terry (Eds.) Alzheimer's disease: senile dementia and related disorders., Raven Press, New York, 1978, pp. 493. [54] D. Thal, W. Griffin, R. de Vos, E. Ghebremedhin, Cerebral amyloid angiopathy and its relationship to Alzheimer‟s disease, Acta Neuropathologica 115 (2008) 599-609. [55] P. Westermark, J.B. Natvig, B. Johansson, CHARACTERIZATION OF AN AMYLOID FIBRIL PROTEIN FROM SENILE CARDIAC AMYLOID, J Exp Med 146 (1977) 631-36. [56] S.-H. Lee, S.-M. Kim, N. Kim, B.-W. Yoon, J.-K. Roh, Cortico-subcortical distribution of microbleeds is different between hypertension and cerebral amyloid angiopathy, J of the Neurological Sciences 258 (2007) 111-114. [57] T.M. Revesz, J.P. Ghiso, T.B. Lashley, G.M. Plant, A.P. Rostagno, B.M.P. Frangione, J.L.M.P. Holton, Cerebral Amyloid Angiopathies: A Pathologic, Biochemical, and Genetic View, J Neuropath Exp Neurol September 2003;62(9):885-898 [58] C. Van Broeckhoven, J. Haan, t.J.A.H. BAKKE1R, A.W. W. VAN HUL, M. VEGTER-VAN DER VLIS,t, R.A.C. Roos, Amyloid , Protein Precursor Gene and Hereditary Cerebral Hemorrhage with Amyloidosis (Dutch), Science 248 (1990) 1120. [59] K. Kamino, H.T. Orr, H. Payami, E.M. Wijsman, M.E. Alonso, S.M. Pulst, L. Anderson, S. O'dahl, E. Nemens, J.A. White, e. al., Linkage and mutational analysis of familial Alzheimer disease kindreds for the APP gene region., Am J Hum Gen 51 (1992) 998-1014. [60] S. Rodziewicz-Motowidlo, P. Czaplewska, E. Sikorska, M. Spodzieja, A.S. Kolodziejczyk, The Arctic mutation alters helix length and type in the 11-28 [beta]-amyloid peptide monomer--CD, NMR and MD studies in an SDS micelle, J Struct Biol 164 (2008) 199-209.

209 References

[61] C. Nilsberth, A. Westlind-Danielsson, C.B. Eckman, M.M. Condron, K. Axelman, C. Forsell, C. Stenh, J. Luthman, D.B. Teplow, S.G. Younkin, J. Näslund, L. Lannfelt, The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation., Nat Neurosci 4 (2001) 887-93. [62] S.W. Snyder, U.S. Ladror, W.S. Wade, G.T. Wang, L.W. Barrett, E.D. Matayoshi, H.J. Huffaker, G.A. Krafft, T.F. Holzman, Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths, 67 (1994) 1216-1228. [63] R.F. Craven, C.J. Hirnle, Fluid, Electrolyte, and Acid-Base Balance, in: R.F. Craven,C.J. Hirnle (Eds.) Fundamentals of nursing: human health and function, Lippincott Williams & Wilkins; Fifth Edition edition (February 1, 2006), Philadelphia, pp. [64] M. Meyer-Luehmann, T.L. Spires-Jones, C. Prada, M. Garcia-Alloza, A. de Calignon, A. Rozkalne, J. Koenigsknecht-Talboo, D.M. Holtzman, B.J. Bacskai, B.T. Hyman, Rapid appearance and local toxicity of amyloid- β plaques in a mouse model of Alzheimer's disease, Nature 451 (2008) 720-724. [65] P. Yan, A.W. Bero, J.R. Cirrito, Q. Xiao, X. Hu, Y. Wang, E. Gonzales, D.M. Holtzman, J.-M. Lee, Characterizing the Appearance and Growth of Amyloid Plaques in APP/PS1 Mice, J. Neurosci. 29 (2009) 10706-10714. [66] M. Citron, D.J. Selkoe, Proteolytic Processing of the β-Amyloid Precursor Protein, in: C. Haass (Ed.) Molecular Biology of Alzheimer‟s Disease – Genes and Mechanisms Involved in Amyloid Generation, Harwood Academic Publishers, Amsterdam, 1998, pp. [67] E.H. Koo, S.S. Sisodia, D.R. Archer, L.J. Martin, A. Weidemann, K. Beyreuther, P. Fischer, C.L. Masters, D.L. Price, Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport, PNAS 87 (1990) 1561-1565. [68] A.W. Lyckman, A.M. Confaloni, G. Thinakaran, S.S. Sisodia, K.L. Moya, Post-translational Processing and Turnover Kinetics of Presynaptically Targeted Amyloid Precursor Superfamily Proteins in the Central Nervous System, J. Biol. Chem. 273 (1998) 11100-11106. [69] S. Tomita, Y. Kirino, T. Suzuki, A Basic Amino Acid in the Cytoplasmic Domain of Alzheimer's β-Amyloid Precursor Protein (APP) Is Essential for Cleavage of APP at the a-Site, J. Biol. Chem. 273 (1998) 19304-19310. [70] T. Yamazaki, D.J. Selkoe, E.H. Koo, Trafficking of cell surface β-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons, J. Cell Biol. 129 (1995) 431-442. [71] F.L. Graham, J. Smiley, W.C. Russell, R. Nairn, Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5, J Gen Virol 36 (1977) 59-72. [72] F.S. Esch, P.S. Keim, E.C. Beattie, R.W. Blacher, A.R. Culwell, T. Oltersdorf, D. McClure, P.J. Ward, Cleavage of amyloid beta peptide during constitutive processing of its precursor, Science 248 (1990) 1122-1124. [73] S. Lammich, E. Kojro, R. Postina, S. Gilbert, R. Pfeiffer, M. Jasionowski, C. Haass, F. Fahrenholz, Constitutive and regulated a-secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease, PNAS 96 (1999) 3922-3927. [74] F. Fahrenholz, R. Postina, a-Secretase Activation -- An Approach to Alzheimer's Disease Therapy, Neurodeg Dis 3 (2006) 255-261. [75] K. Maruyama, F. Kametani, M. Usami, W. Yamao-Harigaya, K. Tanaka, "Secretase," Alzheimer amyloid protein precursor secreting enzyme is not sequence-specific, BBRC 179 (1991) 1670-1676. [76] S.B. Roberts, J.A. Ripellino, K.M. Ingalls, N.K. Robakis, K.M. Felsenstein, Non-amyloidogenic cleavage of the beta-amyloid precursor protein by an integral membrane metalloendopeptidase, J. Biol. Chem. 269 (1994) 3111- 3116. [77] C. Haass, M.G. Schlossmacher, A.Y. Hung, C. Vigo-Pelfrey, A. Mellon, B.L. Ostaszewski, I. Lieberburg, E.H. Koo, D. Schenk, D.B. Teplow, D.J. Selkoe, Amyloid β-peptide is produced by cultured cells during normal metabolism, Nature 359 (1992) 322-325. [78] J.R. McDermott, J.A. Biggins, A.M. Gibson, Human brain peptidase activity with the specificity to generate the N- terminus of the Alzheimer β-amyloid protein from its precursor, BBRC 185 (1992) 746-752. [79] A. Thompson, F. Grueninger-Leitch, G. Huber, P. Malherbe, Expression and characterization of human β- secretase candidates metalloendopeptidase MP78 and cathepsin D in βAPP-overexpressing cells, Mol Brain Res 48 (1997) 206-214. [80] A.B. Huber, C. Brösamle, H. Mechler, G. Huber, Metalloprotease MP100: a synaptic protease in rat brain, Brain Res 837 (1999) 193-202. [81] R.B. Nelson, R. Siman, M.A. Iqbal, H. Potter, Identification of a Chymotrypsin-Like Mast Cell Protease in Rat Brain Capable of Generating the N-Terminus of the Alzheimer Amyloid β-Protein, J Neurochem 61 (1993) 567-577. [82] S.R. Sahasrabudhe, A.M. Brown, J.D. Hulmes, J.S. Jacobsen, M.P. Vitek, A.J. Blume, J.L. Sonnenberg, Enzymatic generation of the amino terminus of the β-amyloid peptide, J. Biol. Chem. 268 (1993) 16699-16705. [83] U.S. Ladror, S.W. Snyder, G.T. Wang, T.F. Holzman, G.A. Krafft, Cleavage at the amino and carboxyl termini of Alzheimer's amyloid-β by cathepsin D, J. Biol. Chem. 269 (1994) 18422-18428. [84] P. Saftig, C. Peters, K. von Figura, K. Craessaerts, F. Van Leuven, B. De Strooper, Amyloidogenic Processing of Human Amyloid Precursor Protein in Hippocampal Neurons Devoid of Cathepsin D, J. Biol. Chem. 271 (1996) 27241-27244. [85] K. Mori, Y. Ogawa, N. Tamura, K. Ebihara, T. Aoki, S. Muro, S. Ozaki, I. Tanaka, K. Tashiro, K. Nakao, Molecular cloning of a novel mouse aspartic protease-like protein that is expressed abundantly in the kidney, FEBS Letters 401 (1997) 218-222. [86] D.J. Powell, T.S. Smith, C.G. Chapman, K.E. Murphy (1998) in Bulletin 2006/38 (S.B. Pharm., Ed.), Vol. EP 0 855 444 B1, European Union. [87] H. Cai, Y. Wang, D. McCarthy, H. Wen, D.R. Borchelt, D.L. Price, P.C. Wong, BACE1 is the major β-secretase for generation of Aβ peptides by neurons, Nat Neurosci 4 (2001) 233-234.

210 References

[88] U. Bodendorf, S. Danner, F. Fischer, M. Stefani, C. Sturchler-Pierrat, K.-H. Wiederhold, M. Staufenbiel, P. Paganetti, Expression of human b-secretase in the mouse brain increases the steady-state level of b-amyloid, J Neurochem 80 (2002) 799-806. [89] U. Bodendorf, F. Fischer, D. Bodian, G. Multhaup, P. Paganetti, A Splice Variant of b-Secretase Deficient in the Amyloidogenic Processing of the Amyloid Precursor Protein, J Biol Chem 276 (2001) 12019-12023. [90] S. Benjannet, A. Elagoz, L. Wickham, M. Mamarbachi, J.S. Munzer, A. Basak, C. Lazure, J.A. Cromlish, S. Sisodia, F. Checler, M. Chretien, N.G. Seidah, Post-translational Processing of β-Secretase (β-Amyloid-converting Enzyme) and Its Ectodomain Shedding. The pro- and transmembrane/cytosolic domains affect its cellular activity and amyloid-β production, J. Biol. Chem. 276 (2001) 10879-10887. [91] J.T. Huse, D.S. Pijak, G.J. Leslie, V.M.Y. Lee, R.W. Doms, Maturation and Endosomal Targeting of β-Site Amyloid Precursor Protein-cleaving Enzyme. The Alzheimer's disease β-secretase, J. Biol. Chem. 275 (2000) 33729-33737. [92] J. Walter, R. Fluhrer, B. Hartung, M. Willem, C. Kaether, A. Capell, S. Lammich, G. Multhaup, C. Haass, Phosphorylation Regulates Intracellular Trafficking of β-Secretase, J. Biol. Chem. 276 (2001) 14634-14641. [93] G. Evin, R. Cappai, Q.-X. Li, J.G. Culvenor, D.H. Small, K. Beyreuther, C.L. Masters, Candidate g-Secretases in the Generation of the Carboxyl Terminus of the Alzheimer's Disease bA4 Amyloid: Possible Involvement of Cathepsin D, Biochemistry 34 (2002) 14185-14192. [94] J. Higaki, D. Quon, Z. Zhong, B. Cordell, Inhibition of β-amyloid formation identifies proteolytic precursors and subcellular site of catabolism, Neuron 14 (1995) 651-659. [95] R. Sherrington, E.I. Rogaev, Y. Liang, E.A. Rogaeva, G. Levesque, M. Ikeda, H. Chi, C. Lin, G. Li, K. Holman, Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease., Nature 375 (1995) 754-60. [96] G.D. Schellenberg, T.D. Bird, E.M. Wijsman, H.T. Orr, L. Anderson, E. Nemens, J.A. White, L. Bonnycastle, J.L. Weber, M.E. Alonso, Genetic linkage evidence for a familial Alzheimer's disease locus on chromosome 14., Science 258 (1992) 668-71. [97] G. Thinakaran, D.R. Borchelt, M.K. Lee, H.H. Slunt, L. Spitzer, G. Kim, T. Ratovitsky, F. Davenport, C. Nordstedt, M. Seeger, J. Hardy, A.I. Levey, S.E. Gandy, N.A. Jenkins, N.G. Copeland, D.L. Price, S.S. Sisodia, Endoproteolysis of Presenilin 1 and Accumulation of Processed Derivatives In Vivo, Neuron 17 (1996) 181-190. [98] M. Citron, Thekla S. Diehl, G. Gordon, Anja L. Biere, P. Seubert, Dennis J. Selkoe, Evidence that the 42- and 40- amino acid forms of amyloid β protein are generated from the β-amyloid precursor protein by different protease activities, PNAS 93 (1996) 13170-13175. [99] M.S. Wolfe, M. Citron, T.S. Diehl, W. Xia, I.O. Donkor, D.J. Selkoe, A Substrate-Based Difluoro Ketone Selectively Inhibits Alzheimer's g-Secretase Activity, J of Medicinal Chemistry 41 (1998) 6-9. [100] M.S. Wolfe, W. Xia, C.L. Moore, D.D. Leatherwood, B. Ostaszewski, T. Rahmati, I.O. Donkor, D.J. Selkoe, Peptidomimetic Probes and Molecular Modeling Suggest That Alzheimer's g-Secretase Is an Intramembrane- Cleaving Aspartyl Protease, Biochemistry 38 (1999) 4720-4727. [101] B. De Strooper, P. Saftig, K. Craessaerts, H. Vanderstichele, G. Guhde, W. Annaert, K. Von Figura, F. Van Leuven, Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein, Nature 391 (1998) 387-390. [102] W. Xia, J. Zhang, B.L. Ostaszewski, W.T. Kimberly, P. Seubert, E.H. Koo, J. Shen, D.J. Selkoe, Presenilin 1 Regulates the Processing of β-Amyloid Precursor Protein C-Terminal Fragments and the Generation of Amyloid β-Protein in Endoplasmic Reticulum and Golgi, Biochemistry 37 (1998) 16465-16471. [103] M.S. Wolfe, W. Xia, B.L. Ostaszewski, T.S. Diehl, W.T. Kimberly, D.J. Selkoe, Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and g-secretase activity, Nature 398 (1999) 513-517. [104] G. Thinakaran, C.L. Harris, T. Ratovitski, F. Davenport, H.H. Slunt, D.L. Price, D.R. Borchelt, S.S. Sisodia, Evidence That Levels of Presenilins (PS1 and PS2) Are Coordinately Regulated by Competition for Limiting Cellular Factors, J. Biol. Chem. 272 (1997) 28415-28422. [105] C. Kaether, C. Haass, H. Steiner, Assembly, Trafficking and Function of g-Secretase, Neurodeg Dis 3 (2006) 275- 283. [106] G. Yu, M. Nishimura, S. Arawaka, D. Levitan, L. Zhang, A. Tandon, Y.-Q. Song, E. Rogaeva, F. Chen, T. Kawarai, A. Supala, L. Levesque, H. Yu, D.-S. Yang, E. Holmes, P. Milman, Y. Liang, D.M. Zhang, D.H. Xu, C. Sato, E. Rogaev, M. Smith, C. Janus, Y. Zhang, R. Aebersold, L. Farrer, S. Sorbi, A. Bruni, P. Fraser, P. St George- Hyslop, Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and βAPP processing, Nature 407 (2000) 48-54. [107] G. Thinakaran, E.H. Koo, Amyloid Precursor Protein Trafficking, Processing, and Function, J. Biol. Chem. 283 (2008) 29615-29619. [108] W. Wasco, K. Bupp, M. Magendantz, J.F. Gusella, R.E. Tanzi, F. Solomon, Identification of a mouse brain cDNA that encodes a protein related to the Alzheimer disease-associated amyloid beta protein precursor, PNAS 89 (1992) 10758-10762. [109] W. Wasco, S. Gurubhagavatula, M.d. Paradis, D.M. Romano, S.S. Sisodia, B.T. Hyman, R.L. Neve, R.E. Tanzi, Isolation and characterization of APLP2 encoding a homologue of the Alzheimer's associated amyloid [beta] protein precursor, Nat Genet 5 (1993) 95-100. [110] C.A. Sprecher, F.J. Grant, G. Grimm, P.J. O'Hara, F. Norris, K. Norris, D.C. Foster, Molecular cloning of the cDNA for a human amyloid precursor protein homolog: Evidence for a multigene family, Biochemistry 32 (1993) 4481- 4486. [111] K. Paliga, G. Peraus, S. Kreger, U. Dürrwang, L. Hesse, G. Multhaup, C.L. Masters, K. Beyreuther, A. Weidemann, Human Amyloid Precursor-Like Protein 1: cDNA cloning, ectopic expression in COS-7 cells and identification of soluble forms in the cerebrospinal fluid, Eur J Biochemistry 250 (1997) 354-363.

211 References

[112] J. Herms, B. Anliker, S. Heber, S. Ring, M. Fuhrmann, H. Kretzschmar, S. Sisodia, U. Müller, Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members., EMBO J 23 (2004) 4106- 15. [113] B. Anliker, U. Müller, The Functions of Mammalian Amyloid Precursor Protein and Related Amyloid Precursor-Like Proteins, Neurodeg Dis 3 (2006) 239-246. [114] B.E. Needham, M.E. Wlodek, G.D. Ciccotosto, B.C. Fam, C.L. Masters, J. Proietto, S. Andrikopoulos, R. Cappai, Identification of the Alzheimer's disease amyloid precursor protein (APP) and its homologue APLP2 as essential modulators of glucose and insulin homeostasis and growth, J Pathol 215 (2008) 155-163. [115] C.S. von Koch, H. Zheng, H. Chen, M. Trumbauer, G. Thinakaran, L.H.T. van der Ploeg, D.L. Price, S.S. Sisodia, Generation of APLP2 KO Mice and Early Postnatal Lethality in APLP2/APP Double KO Mice, Neurobiology of Aging 18 (1997) 661-669. [116] S. Heber, J. Herms, V. Gajic, J. Hainfellner, A. Aguzzi, T. Rulicke, H. Kretzschmar, C. von Koch, S. Sisodia, P. Tremml, H.-P. Lipp, D.P. Wolfer, U. Muller, Mice with Combined Gene Knock-Outs Reveal Essential and Partially Redundant Functions of Amyloid Precursor Members, J. Neurosci. 20 (2000) 7951-7963. [117] U. Müller, N. Cristina, Z.-W. Li, D.P. Wolfer, H.-P. Lipp, T. Rülicke, S. Brandner, A. Aguzzi, C. Weissmann, Behavioral and anatomical deficits in mice homozygous for a modified β-amyloid precursor protein gene, Cell 79 (1994) 755-765. [118] H. Zheng, M. Jiang, M.E. Trumbauer, D.J.S. Sirinathsinghji, R. Hopkins, D.W. Smith, R.P. Heavens, G.R. Dawson, S. Boyce, M.W. Conner, K.A. Stevens, H.H. Slunt, S.S. Sisodia, H.Y. Chen, L.H.T. Van der Ploeg, β- amyloid precursor protein-deficient mice show reactive gliosis and decreased locomotor activity, Cell 81 (1995) 525-531. [119] K. Lorent, L. Overbergh, D. Moechars, B. de Strooper, F. van Leuven, H. van den Berghe, Expression in mouse embryos and in adult mouse brain of three members of the amyloid precursor protein family, of the alpha-2- macroglobulin receptor/low density lipoprotein receptor-related protein and of its ligands apolipoprotein E, lipoprotein lipase, alpha-2-macroglobulin and the 40,000 molecular weight receptor-associated protein, Neuroscience 65 (1995) 1009-1025. [120] G. Thinakaran, H.H. Slunt, L. Spitzer, M.K. Lee, S.S. Sisodia, Tissue distribution and developmental expression of the amyloid precursor protein homolog, APLP1., Soc Neurosci Abstr 21 (1995) 205. [121] A.J. Saunders, T.-W. Kim, Tanzi, R. E., W. Fan, B.D. Bennett, S. Babu-Kahn, Y. Luo, J.-C. Louis, M. McCaleb, M. Citron, R. Vassar, Richards, W. G., BACE Maps to Chromosome 11 and a BACE Homolog, BACE2, Reside in the Obligate Down Syndrome Region of Chromosome 21, Science 286 (1999) 1255a-. [122] D. Dominguez, J. Tournoy, D. Hartmann, T. Huth, K. Cryns, S. Deforce, L. Serneels, I.E. Camacho, E. Marjaux, K. Craessaerts, A.J.M. Roebroek, M. Schwake, R. D'Hooge, P. Bach, U. Kalinke, D. Moechars, C. Alzheimer, K. Reiss, P. Saftig, B. De Strooper, Phenotypic and Biochemical Analyses of BACE1- and BACE2-deficient Mice, J. Biol. Chem. 280 (2005) 30797-30806. [123] S. Eggert, K. Paliga, P. Soba, G. Evin, C.L. Masters, A. Weidemann, K. Beyreuther, The Proteolytic Processing of the Amyloid Precursor Protein Gene Family Members APLP-1 and APLP-2 Involves a-, b-, g-, and e-Like Cleavages: MODULATION OF APLP-1 PROCESSING BY N-GLYCOSYLATION, J. Biol. Chem. 279 (2004) 18146-18156. [124] D. Hartmann, B. De Strooper, P. Saftig, Presenilin-1 deficiency leads to loss of Cajal-Retzius neurons and cortical dysplasia similar to human type 2 lissencephaly, Current Biology 9 (1999) 719-727. [125] S. Guenette, Y. Chang, T. Hiesberger, J.A. Richardson, C.B. Eckman, E.A. Eckman, R.E. Hammer, J. Herz, Essential roles for the FE65 amyloid precursor protein-interacting proteins in brain development, EMBO J 25 (2006) 420-431. [126] R. Feng, C. Rampon, Y.-P. Tang, D. Shrom, J. Jin, M. Kyin, B. Sopher, G.M. Martin, S.-H. Kim, R.B. Langdon, S.S. Sisodia, J.Z. Tsien, Deficient Neurogenesis in Forebrain-Specific Presenilin-1 Knockout Mice Is Associated with Reduced Clearance of Hippocampal Memory Traces, Neuron 32 (2001) 911-926. [127] P. Wang, G. Yang, D.R. Mosier, P. Chang, T. Zaidi, Y.-D. Gong, N.-M. Zhao, B. Dominguez, K.-F. Lee, W.-B. Gan, H. Zheng, Defective Neuromuscular Synapses in Mice Lacking Amyloid Precursor Protein (APP) and APP- Like Protein 2, J. Neurosci. 25 (2005) 1219-1225. [128] D. Beltrán-Valero de Bernabé, S. Currier, A. Steinbrecher, J. Celli, E. van Beusekom, B. van der Zwaag, H. Kayserili, L. Merlini, D. Chitayat, W.B. Dobyns, B. Cormand, A.-E. Lehesjoki, J. Cruces, T. Voit, C.A. Walsh, H. van Bokhoven, H.G. Brunner, Mutations in the O-Mannosyltransferase Gene POMT1 Give Rise to the Severe Neuronal Migration Disorder Walker-Warburg Syndrome, The American J of Human Genetics 71 (2002) 1033- 1043. [129] A. Murakami-Sekimata, K. Sato, K. Sato, A. Takashima, A. Nakano, O-Mannosylation is required for the solubilization of heterologously expressed human β-amyloid precursor protein in Saccharomyces cerevisiae, Genes to Cells 14 (2009) 205-215. [130] R.J. Konrad, J.E. Kudlow, The role of O-linked protein glycosylation in beta-cell dysfunction., Int J Mol Med 10 (2002) 535-539. [131] T. Itagaki, Y. Itoh, Y. Sugai, N. Suematsu, E. Ohtomo, M. Yamada, [Glucose metabolism and Alzheimer's dementia], Nippon Ronen Igakkai Zasshi 33 (1996) 569-572. [132] P. Tremml, H.-P. Lipp, U. Müller, L. Ricceri, D. P. Wolfer, Neurobehavioral development, adult openfield exploration and swimming navigation learning in mice with a modified [beta]-amyloid precursor protein gene, Behavioural Brain Res 95 (1998) 65-76. [133] R.I. Scahill, J.M. Schott, J.M. Stevens, M.N. Rossor, N.C. Fox, Mapping the evolution of regional atrophy in Alzheimer's disease: Unbiased analysis of fluid-registered serial MRI, PNAS 99 (2002) 4703-4707.

212 References

[134] D.M. Yilmazer-Hanke, J. Hanke, Progression of Alzheimer-Related Neuritic Plaque Pathology in the Entorhinal Region, Perirhinal Cortex and Hippocampal Formation, Dementia and Geriatric Cognitive Disorders 10 (1999) 70- 76. [135] C.E. Myers, R.O. Hopkins, J. DeLuca, L.J. Wolansky, M.A. Gluck, J.M. Sumner, N.B. Moore, Learning and generalization deficits in patients with memory impairments due to anterior communicating artery aneurysm rupture or hypoxic brain injury, Neuropsychology 22 (2008) 681-686. [136] J. DeLuca, D. Pendick, The Truth About Confabulation - The rupture of a tiny blood vessel in the brain can produce distorted or erroneous memories., Memory Loss & the Brain Summer 2000 (2000) [137] D.A. Drachman, A.K. Ommaya, Memory and the Hippocampal Complex., Arch Neurol 10 (1964) 411-25. [138] A. Abi-Dargham, O. Guillin, Integrating the Neurobiology of Schizophrenia, Elsevier, Amsterdam, Boston, 2007. [139] J. Puoliväli (2000) in Department of Neuroscience and Neurology, University of Kuopio, Kuopio. [140] S. Ikonen (2001) in Department of Neuroscience and Neurology, Vol. Ph.D., University of Kuopio, Kuopio. [141] P. Davies, A.J.F. Maloney, SELECTIVE LOSS OF CENTRAL CHOLINERGIC NEURONS IN ALZHEIMER'S DISEASE, The Lancet 308 (1976) 1403-1403. [142] E.K. Perry, G. Blessed, B.E. Tomlinson, R.H. Perry, T.J. Crow, A.J. Cross, G.J. Dockray, R. Dimaline, A. Arregui, Neurochemical activities in human temporal lobe related to aging and Alzheimer-type changes, Neurobiology of Aging 2 (1981) 251-256. [143] D.M. Kaufman (2001) in Clinical neurology for psychiatrists (D.M. Kaufman, Ed.), Saunders Elseviers, Philadelphia. [144] R.C. MOHS, V. HAROUTUNIAN, Chapter 82: Alzheimer Disease: From Earliest Symptoms to End Stage, in: K.L. Davis, D. Charney, J.T. Coyle,C. Nemeroff (Ed.^Eds.) Neuropsychopharmacology: The Fifth Generation of Progress., American College of Neuropsychopharmacology, 2002, pp. [145] H. Tohgi, T. Abe, M. Kimura, M. Saheki, S. Takahashi, Cerebrospinal fluid acetylcholine and choline in vascular dementia of Binswanger and multiple small infarct types as compared with Alzheimer-type dementia, J of Neural Transmission 103 (1996) 1211-1220. [146] L. Frölich, A. Dirr, M.E. Götz, W. Gsell, H. Reichmann, P. Riederer, K. Maurer, Acetylcholine in human CSF: methodological considerations and levels in dementia of Alzheimer type, J of Neural Transmission 105 (1998) 961-973. [147] J.-p. JIA, J.-m. JIA, W.-d. ZHOU, M. XU, C.-b. CHU, X. YAN, Y.-x. SUN, Differential acetylcholine and choline concentrations in the cerebrospinal fluid of patients with Alzheimer‟s disease and vascular dementia, Chinese Medical J 117 (2004) 1161-1164. [148] E.K. Perry, The Cholinergic Hypothesis - Ten Years On, Brit Med Bull 42 (1986) 63-69. [149] J.J. Freeman, D.J. Jenden, The source of choline for acetylcholine synthesis in the brain., Life Sci 19 (1976) 949- 962. [150] E. Cohen, R.J. Wurtman, Brian acetylcholine synthesis: control by dietary choline., Science 191 (1976) 561-562. [151] S. Tucek, Problems in the organization and control of acetylcholine synthesis in brain neurons, Progress in Biophysics and Molecular Biology 44 (1984) 1-46. [152] B.K. Siesjö, Brain energy metabolism, Wiley, New York, 1978. [153] G.E. Gibson, T.E. Duffy, Impaired Synthesis of Acetylcholine by Mild Hypoxic Hypoxia or Nitrous Oxide, J Neurochem 36 (1981) 28-33. [154] O.U. Scremin, D.J. Jenden, Focal ischemia enhances choline output and decreases acetylcholine output from rat cerebral cortex, Stroke 20 (1989) 92-95. [155] O.U. Scremin, D.J. Jenden, Effects of middle cerebral artery occlusion on cerebral cortex choline and acetylcholine in rats, Stroke 20 (1989) 1524-1530. [156] D.F. Swaab, The human hypothalamus: basic and clinical aspects, Elsevier B.V., Amsterdam, 2004. [157] R. Donzelli, S. Marinkovic, L. Brigante, O. de Divitiis, I. Nikodijevic, C. Schonauer, F. Maiuri, Territories of the perforating (lenticulostriate) branches of the middle cerebral artery, Surgical and Radiologic Anatomy 20 (1999) 393-398. [158] J. Ahn, I. Han, Y. Chung, P. Yoon, S. Kim, Posttraumatic infarction in the territory supplied by the lateral lenticulostriate artery after minor head injury, Child's Nervous System 22 (2006) 1493-1496. [159] B.T. Hyman, H. Damasio, A.R. Damasio, G.W. Van Hoesen, Alzheimer's Disease, Annual Review of Public Health 10 (1989) 115-140. [160] X. Zhang, K. Zhou, R. Wang, J. Cui, S.A. Lipton, F.-F. Liao, H. Xu, Y.-w. Zhang, Hypoxia inducible factor 1a (HIF- 1a)-mediated hypoxia increases BACE1 expression and β-amyloid generation, J. Biol. Chem. (2007) M608856200. [161] M. Guglielmotto, M. Aragno, R. Autelli, L. Giliberto, E. Novo, S. Colombatto, O. Danni, M. Parola, M.A. Smith, G. Perry, E. Tamagno, M. Tabaton, The up-regulation of BACE1 mediated by hypoxia and ischemic injury: role of oxidative stress and HIF1α, J Neurochem 108 (2009) 1045-1056. [162] C. Choeiri, W. Staines, C. Messier, Immunohistochemical localization and quantification of glucose transporters in the mouse brain, Neuroscience 111 (2002) 19-34. [163] T. Arendt, Synaptic plasticity and cell cycle activation in neurons are alternative effector pathways: the 'Dr. Jekyll and Mr. Hyde concept' of Alzheimer's disease or the yin and yang of neuroplasticity, Progress in Neurobiology 71 (2003) 83-248. [164] M.T. Scharf, N. Naidoo, J.E. Zimmerman, A.I. Pack, The energy hypothesis of sleep revisited, Progress in Neurobiology 86 (2008) 264-280. [165] R. Szymusiak, Magnocellular nuclei of the basal forebrain: substrates of sleep and arousal regulation., Sleep 18 (1995) 478-500.

213 References

[166] M.L. Evans, R.J. McCrimmon, D.E. Flanagan, T. Keshavarz, X. Fan, E.C. McNay, R.J. Jacob, R.S. Sherwin, Hypothalamic ATP-sensitive K + Channels Play a Key Role in Sensing Hypoglycemia and Triggering Counterregulatory Epinephrine and Glucagon Responses, Diabetes 53 (2004) 2542-2551. [167] W.P. Borg, M.J. During, R.S. Sherwin, M.A. Borg, M.L. Brines, G.I. Shulman, Ventromedial Hypothalamic Lesions in Rats Suppress Counterregulatory Responses to Hypoglycemia, J Clin Invest 93 (1994) 1677-1682. [168] W.P. Borg, R.S. Sherwin, M.J. During, M.A. Borg, G.I. Shulman, Local ventromedial hypothalamus glucopenia triggers counterregulatory hormone release, Diabetes 44 (1995) 180-184. [169] M.A. Borg, R.S. Sherwin, W.P. Borg, W.V. Tamborlane, G.I. Shulman, Local ventromedial hypothalamus glucose perfusion blocks counterregulation during systemic hypoglycemia in awake rats., J Clin Invest 99 (1997) 361-365. [170] P. Pietrini, N.P. Azari, C.L. Grady, J.A. Salerno, A. Gonzales-Aviles, L.L. Heston, K.D. Pettigrew, B. Horwitz, J.V. Haxby, M.B. Schapiro, Pattern of cerebral metabolic interactions in a subject with isolated amnesia at risk for Alzheimer's disease: a longitudinal evaluation., Dementia 4 (1993) 94-101. [171] R. Mielke, K. Herholz, M. Grond, J. Kessler, W.D. Heiss, Clinical deterioration in probable Alzheimer's disease correlates with progressive metabolic impairment of association areas., Dementia 5 (1994) 36-41. [172] M.J. de Leon, A. Convit, O.T. Wolf, C.Y. Tarshish, S. DeSanti, H. Rusinek, W. Tsui, E. Kandil, A.J. Scherer, A. Roche, A. Imossi, E. Thorn, M. Bobinski, C. Caraos, P. Lesbre, D. Schlyer, J. Poirier, B. Reisberg, J. Fowler, Prediction of cognitive decline in normal elderly subjects with 2-[18F]fluoro-2-deoxy-d-glucose/positron-emission tomography (FDG/PET), PNAS 98 (2001) 10966-10971. [173] A. Drzezga, T. Grimmer, M. Riemenschneider, N. Lautenschlager, H. Siebner, P. Alexopoulus, S. Minoshima, M. Schwaiger, A. Kurz, Prediction of Individual Clinical Outcome in MCI by Means of Genetic Assessment and 18F- FDG PET, J Nucl Med 46 (2005) 1625-1632. [174] A. Drzezga, N. Lautenschlager, H. Siebner, M. Riemenschneider, F. Willoch, S. Minoshima, M. Schwaiger, A. Kurz, Cerebral metabolic changes accompanying conversion of mild cognitive impairment into Alzheimer's disease: a PET follow-up study, Eur J Nuclear Medicine and Molecular Imaging 30 (2003) 1104-1113. [175] S. Hoyer, Oxidative energy metabolism in Alzheimer brain, Molecular and Chemical Neuropathology 16 (1992) 207-224. [176] M.B. Schapiro, M.J. Ball, C.L. Grady, J.V. Haxby, J.A. Kaye, S.I. Rapoport, Dementia in Down's syndrome: Cerebral glucose utilization, neuropsychological assessment, and neuropathology, Neurology 38 (1988) 938-. [177] S. Hoyer, The brain insulin signal transduction system and sporadic (type II) Alzheimer disease: an update, J of Neural Transmission 109 (2002) 341-360. [178] J.-K. Jin, N.-H. Kim, Y.-J. Lee, Y.-S. Kim, E.-K. Choi, P.B. Kozlowski, M.H. Park, H.-S. Kim, D.S. Min, Phospholipase D1 is up-regulated in the mitochondrial fraction from the brains of Alzheimer's disease patients, Neuroscience Letters 407 (2006) 263-267. [179] T. Narwo, Brain Imaging, in: H. D'aenen,P. Willner (Eds.) Biological Psychiatry, 2, John Wiley & Sons Ltd., Chichester, 2002, pp. 1181-1189. [180] A.A. Capizzano, L. Acion, T. Bekinschtein, M. Furman, H. Gomila, A. Martinez, R. Mizrahi, S.E. Starkstein, White matter hyperintensities are significantly associated with cortical atrophy in Alzheimer's disease, J Neurol Neurosurg Psychiatry 75 (2004) 822-827. [181] P.-N. Wang, C.-L. Yang, K.-N. Lin, W.-T. Chen, L.-C. Chwang, H.-C. Liu, Weight loss, nutritional status and physical activity in patients with Alzheimer‟s Disease, J Neurol 251 (2004) 314-320. [182] O. Guerin, S. Andrieu, S.M. Schneider, M. Milano, R. Boulahssass, P. Brocker, B. Vellas, Different modes of weight loss in Alzheimer disease: a prospective study of 395 patients, Am J Clin Nutr 82 (2005) 435-441. [183] P. Seeger, Auf der Suche nach dem Gedächtnis - Der Hirnforscher Eric Kandel, cast: Eric Kandel, runtime: 95 min, first release: 26. may 2008 in Vienna, FILMFORM GmBH, Köln (2008) [184] A.M. Mamoon, J. Smith, R.C. Baker, J.M. Farley, Activation of Protein Kinase A Increases Phospholipase D Activity and Inhibits Phospholipase D Activation by Acetylcholine in Tracheal Smooth Muscle, J Pharmacol Exp Ther 291 (1999) 1188-1195. [185] F. Liu, Z. Liang, J. Shi, D. Yin, E. El-Akkad, I. Grundke-Iqbal, K. Iqbal, C.-X. Gong, PKA modulates GSK-3b- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners, FEBS Letters 580 (2006) 6269-6274. [186] T. Arendt, J. Stieler, A.M. Strijkstra, R.A. Hut, J. Rudiger, E.A. Van der Zee, T. Harkany, M. Holzer, W. Hartig (2003) in J. Neurosci., Vol. 23, pp. 6972-6981. [187] J. Stieler, T. Bullmann, F. Kohl, B. Barnes, T. Arendt, PHF-like tau phosphorylation in mammalian hibernation is not associated with p25-formation, J of Neural Transmission 116 (2009) 345-350. [188] L.H. Tsai, T. Takahashi, V.S. Caviness, E. Harlow, Activity and expression pattern of cyclin-dependent kinase 5 in the embryonic mouse nervous system, Development 119 (1993) 1029-1040. [189] G.N. Patrick, L. Zukerberg, M. Nikolic, S. de la Monte, P. Dikkes, L.-H. Tsai, Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration, Nature 402 (1999) 615-622. [190] L. Connell-Crowley, M. Le Gall, D.J. Vo, E. Giniger, The cyclin-dependent kinase Cdk5 controls multiple aspects of axon patterning in vivo, Current Biology 10 (2000) 599-603. [191] M. Nikolic, H. Dudek, Y.T. Kwon, Y.F. Ramos, L.H. Tsai, The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation, Genes & Development 10 (1996) 816-825. [192] M. Nikolic, M.M. Chou, W. Lu, B.J. Mayer, L.-H. Tsai, The p35/Cdk5 kinase is a neuron-specific Rac effector that inhibits Pak1 activity, Nature 395 (1998) 194-198. [193] H.K. Paudel, J. Lew, Z. Ali, J.H. Wang, Brain proline-directed protein kinase phosphorylates tau on sites that are abnormally phosphorylated in tau associated with Alzheimer's paired helical filaments, J Biol Chem 268 (1993) 23512-23518. [194] S. Hisanaga, K. Ishiguro, T. Uchida, E. Okumura, T. Okano, T. Kishimoto, Tau protein kinase II has a similar characteristic to cdc2 kinase for phosphorylating neurofilament proteins, J Biol Chem 268 (1993) 15056-15060.

214 References

[195] J. Lew, R.J. Winkfein, H.K. Paudel, J.H. Wang, Brain proline-directed protein kinase is a neurofilament kinase which displays high to p34cdc2, J Biol Chem 267 (1992) 25922-25926. [196] K.T. Shetty, W.T. Link, H.C. Pant, cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization, PNAS 90 (1993) 6844-6848. [197] D. Sun, C.L. Leung, R.K.H. Liem, Phosphorylation of the High Molecular Weight Neurofilament Protein (NF-H) by Cdk5 and p35, J Biol Chem 271 (1996) 14245-14251. [198] C. Goldsbury, M.-M. Mocanu, E. Thies, C. Kaether, C. Haass, P. Keller, J. Biernat, E. Mandelkow, E.-M. Mandelkow, Inhibition of APP Trafficking by Tau Protein Does Not Increase the Generation of Amyloid-β Peptides, Traffic 7 (2006) 873-888. [199] G.W. Hart, Y. Akimoto, The O-GlcNAc Modification, in: A. Varki, R.D. Cummings, J.D. Esko, H.H. Freeze, P. Stanley, C.R. Bertozzi, G.W. Hart,M.E. Etzler (Eds.) Essentials of Glycobiology, CSH Press, Cold Spring Harbor, 2009, pp. [200] C.S. Arnold, G.V.W. Johnson, R.N. Cole, D.L.Y. Dong, M. Lee, G.W. Hart, The Microtubule-associated Protein Tau Is Extensively Modified with O-linked N-acetylglucosamine, J Biol Chem 271 (1996) 28741-28744. [201] F. Liu, J. Shi, H. Tanimukai, J. Gu, J. Gu, I. Grundke-Iqbal, K. Iqbal, C.-X. Gong, Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer's disease, Brain 132 (2009) 1820-1832. [202] L.K. Kreppel, G.W. Hart, Regulation of a Cytosolic and Nuclear O-GlcNAc Transferase, J Biol Chem 274 (1999) 32015-32022. [203] D.C. Love, J.A. Hanover, The Hexosamine Signaling Pathway: Deciphering the "O-GlcNAc Code", Sci. STKE 2005 (2005) re13-. [204] R.M. Gryder, B.M. Pogell, Further Studies on Glucosamine 6-Phosphate Synthesis by Rat Liver Enzymes, J Biol Chem 235 (1960) 558-562. [205] L.F. LELOIR, C.E. CARDINI, The biosynthesis of glucosamine., Biochimica et Biophysica Acta (BBA) 12 (1953) 15-22. [206] W.B. Dias, G.W. Hart, O-GlcNAc modification in diabetes and Alzheimer's disease., Mol Biosyst 3 (2007) 766-772. [207] B.M. Pogell, R.M. Gryder, ENZYMATIC SYNTHESIS OF GLUCOSAMINE 6-PHOSPHATE IN RAT LIVER, J Biol Chem 228 (1957) 701-712. [208] T. Oki, K. Yamazaki, J. Kuromitsu, M. Okada, I. Tanaka, cDNA Cloning and Mapping of a Novel Subtype of Glutamine:fructose-6-phosphate Amidotransferase (GFAT2) in Human and Mouse, Genomics 57 (1999) 227-234. [209] S. Vora, Isozymes of human phosphofructokinase: biochemical and genetic aspects., Isozymes Curr Top Biol Med Res 11 (1983) 2-23. [210] R.B. Layzer, Phosphofructokinase from human erythrocytes., Methods Enzymol. 42 (1975) 110-115. [211] S. Kornfeld, R. Kornfeld, E.F. Neufeld, P.J. O'Brien, THE FEEDBACK CONTROL OF SUGAR NUCLEOTIDE BIOSYNTHESIS IN LIVER, PNAS 52 (1964) 371-379. [212] Q.K. Huynh, E.A. Gulve, T. Dian, Purification and Characterization of Glutamine:Fructose 6-Phosphate Amidotransferase from Rat Liver, Archives of Biochemistry and Biophysics 379 (2000) 307-313. [213] Y. Li, C. Roux, S. Lazereg, J.-P. LeCaer, O. Laprevote, B. Badet, M.-A. Badet-Denisot, Identification of a Novel Serine Phosphorylation Site in Human Glutamine:Fructose-6-phosphate Amidotransferase Isoform 1 Biochemistry 46 (2007) 13163-13169. [214] S. Milewski, D. Kuszczak, R. Jedrzejczak, R.J. Smith, A.J.P. Brown, G.W. Gooday, Oligomeric Structure and Regulation of Candida albicans Glucosamine-6-phosphate Synthase, J Biol Chem 274 (1999) 4000-4008. [215] K.O. Broschat, C. Gorka, J.D. Page, C.L. Martin-Berger, M.S. Davies, H.-c. Huang, E.A. Gulve, W.J. Salsgiver, T.P. Kasten, Kinetic Characterization of Human Glutamine-fructose-6-phosphate Amidotransferase I, J Biol Chem 277 (2002) 14764-14770. [216] J. Zhou, Q.K. Huynh, R.T. Hoffman, E.D. Crook, M.C. Daniels, E.A. Gulve, D.A. McClain, Regulation of glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase, Diabetes 47 (1998) 1836- 1840. [217] H.R. Graack, U. Cinque, H. Kress, Functional regulation of glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1) of Drosophila melanogaster in a UDP-N-acetylglucosamine and cAMP-dependent manner, Biochem. J. 360 (2001) 401-412. [218] Q. Chang, K. Su, J.R. Baker, X. Yang, A.J. Paterson, J.E. Kudlow, Phosphorylation of Human Glutamine:Fructose-6-phosphate Amidotransferase by cAMP-dependent Protein Kinase at Serine 205 Blocks the Enzyme Activity, J Biol Chem 275 (2000) 21981-21987. [219] Y. Hu, L. Riesland, A.J. Paterson, J.E. Kudlow, Phosphorylation of Mouse Glutamine-Fructose-6-phosphate Amidotransferase 2 (GFAT2) by cAMP-dependent Protein Kinase Increases the Enzyme Activity, J Biol Chem 279 (2004) 29988-29993. [220] A.G. Nerlich, U. Sauer, V. Kolm-Litty, E. Wagner, M. Koch, E.D. Schleicher, Expression of glutamine:fructose-6- phosphate amidotransferase in human tissues: evidence for high variability and distinct regulation in diabetes, Diabetes 47 (1998) 170-178. [221] T.C. Richards, O. Greengard, Distribution of glutamine hexosephosphate aminotransferase in rat tissues; changes with state of differentiation, Biochimica et Biophysica Acta (BBA) - General Subjects 304 (1973) 842-850. [222] S. Tarui, N. Kono, K. Uyeda, Purification and Properties of Rabbit Erythrocyte Phosphofructokinase, J Biol Chem 247 (1972) 1138-1145. [223] H. Narabayashi, J.W. Lawson, K. Uyeda, Regulation of phosphofructokinase in perfused rat heart. Requirement for fructose 2,6-bisphosphate and a covalent modification, J Biol Chem 260 (1985) 9750-9758. [224] A. Hannemann, B. Jandrig, F. Gaunitz, K. Eschrich, M. Bigl, Characterization of the human P-type 6- phosphofructo-1-kinase gene promoter in neural cell lines, Gene 345 (2005) 237-247. [225] D.L. Gee (2009) in NUTR 543 Advanced Nutritional Biochemistry, Central Washington University.

215 References

[226] G. Löffler, P.E. Petrides, P.C. Heinrich, Biochemie und Pathobiochemie, Springer Medizin Verlag, Heidelberg, 2007. [227] D.A. McClain, E.D. Crook, Hexosamines and insulin resistance, Diabetes 45 (1996) 1003-1009. [228] L.F. Hebert, M.C. Daniels, J. Zhou, E.D. Crook, R.L. Turner, S.T. Simmons, J.L. Neidigh, J.S. Zhu, A.D. Baron, D.A. McClain, Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance, The J of Clinical Investigation 98 (1996) 930-936. [229] L. Wells, K. Vosseller, G.W. Hart, Glycosylation of Nucleocytoplasmic Proteins: Signal Transduction and O- GlcNAc, Science 291 (2001) 2376-2378. [230] G. Boehmelt, A. Wakeham, A. Elia, T. Sasaki, S. Plyte, J. Potter, Y. Yang, E. Tsang, J. Ruland, N.N. Iscove, J.W. Dennis, T.W. Mak, Decreased UDP-GlcNAc levels abrogate proliferation control in EMeg32-deficient cells, EMBO J 19 (2000) 5092-5104. [231] M.D. Roos, K. Su, J.R. Baker, J.E. Kudlow, O glycosylation of an Sp1-derived peptide blocks known Sp1 protein interactions, Mol. Cell. Biol. 17 (1997) 6472-6480. [232] M. Hiromura, C.H. Choi, N.A. Sabourin, H. Jones, D. Bachvarov, A. Usheva, YY1 Is Regulated by O-Linked N- Acetylglucosaminylation (O-GlcNAcylation), J Biol Chem 278 (2003) 14046-14052. [233] A.J. Reason, H.R. Morris, M. Panico, R. Marais, R.H. Treisman, R.S. Haltiwanger, G.W. Hart, W.G. Kelly, A. Dell, Localization of O-GlcNAc modification on the serum response transcription factor, J Biol Chem 267 (1992) 16911- 16921. [234] B. Camoretti-Mercado, N.O. Dulin, J. Solway, Serum response factor function and dysfunction in smooth muscle, Respiratory Physiology & Neurobiology 137 (2003) 223-235. [235] M.A. Christensen, W. Zhou, H. Qing, A. Lehman, S. Philipsen, W. Song, Transcriptional Regulation of BACE1, the β-Amyloid Precursor Protein β-Secretase, by Sp1, Mol. Cell. Biol. 24 (2004) 865-874. [236] K. Nowak, C. Lange-Dohna, U. Zeitschel, A. Günther, B. Lüscher, A. Robitzki, J.R. Perez-Polo, S. Roßner, The transcription factor Yin Yang 1 is an activator of BACE1 expression, J Neurochem 96 (2006) 1696-1707. [237] J.S. Lee, K.M. Galvin, Y. Shi, Evidence for physical interaction between the zinc-finger transcription factors YY1 and Sp1, PNAS 90 (1993) 6145-6149. [238] J.R. Flanagan, A.M. Krieg, E.E. Max, A.S. Khan, Negative control region at the 5' end of murine leukemia virus long terminal repeats, Mol. Cell. Biol. 9 (1989) 739-746. [239] J.R. Flanagan, K.G. Becker, D.L. Ennist, S.L. Gleason, P.H. Driggers, B.Z. Levi, E. Appella, K. Ozato, Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus, Mol. Cell. Biol. 12 (1992) 38-44. [240] K. Park, M.L. Atchison, Isolation of a candidate repressor/activator, NF-E1 (YY-1, delta), that binds to the immunoglobulin kappa 3' enhancer and the immunoglobulin heavy-chain mu E1 site, PNAS 88 (1991) 9804-9808. [241] A. Gualberto, D. LePage, G. Pons, S.L. Mader, K. Park, M.L. Atchison, K. Walsh, Functional antagonism between YY1 and the serum response factor, Mol. Cell. Biol. 12 (1992) 4209-4214. [242] Q. Zhou, R.W. Gedrich, D.A. Engel, Transcriptional repression of the c-fos gene by YY1 is mediated by a direct interaction with ATF/CREB, J. Virol. 69 (1995) 4323-4330. [243] T. Bauknecht, P. Angel, H.D. Royer, H. zur Hausen, Identification of a negative regulatory domain in the human papillomavirus type 18 promoter: interaction with the transcriptional repressor YY1., EMBO J 11 (1992) 4607- 4617. [244] R. Paciucci, A. Pellicer, Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element, Mol. Cell. Biol. 11 (1991) 1334-1343. [245] X.-P. Dong, H. Pfister, Overlapping YY1- and aberrant SP1-binding sites proximal to the early promoter of human papillomavirus type 16, J Gen Virol 80 (1999) 2097-2101. [246] W. Ai, Y. Liu, T.C. Wang, Yin yang 1 (YY1) represses histidine decarboxylase gene expression with SREBP-1a in part through an upstream Sp1 site, Am J Physiol Gastrointest Liver Physiol 290 (2006) G1096-1104. [247] J. Ye, X. Zhang, Z. Dong, Characterization of the human granulocyte-macrophage colony-stimulating factor gene promoter: an AP1 complex and an Sp1-related complex transactivate the promoter activity that is suppressed by a YY1 complex, Mol. Cell. Biol. 16 (1996) 157-167. [248] F. Galvagni, E. Cartocci, S. Oliviero, The Dystrophin Promoter Is Negatively Regulated by YY1 in Undifferentiated Muscle Cells, J Biol Chem 273 (1998) 33708-33713. [249] T.C. Lee, Y. Shi, R.J. Schwartz, Displacement of BrdUrd-induced YY1 by serum response factor activates skeletal alpha-actin transcription in embryonic myoblasts, PNAS 89 (1992) 9814-9818. [250] S. Kamada, T. Miwa, A protein binding to CArG box motifs and to single-stranded DNA functions as a transcriptional repressor, Gene 119 (1992) 229-236. [251] K.M. Galvin, Y. Shi, Multiple mechanisms of transcriptional repression by YY1, Mol. Cell. Biol. 17 (1997) 3723- 3732. [252] D.L. Gumucio, H. Heilstedt-Williamson, T.A. Gray, S.A. Tarle, D.A. Shelton, D.A. Tagle, J.L. Slightom, M. Goodman, F.S. Collins, Phylogenetic footprinting reveals a nuclear protein which binds to silencer sequences in the human gamma and epsilon globin genes, Mol. Cell. Biol. 12 (1992) 4919-4929. [253] Y. Shi, E. Seto, L.-S. Chang, T. Shenk, Transcriptional repression by YY1, a human GLI-Krüppel-related protein, and relief of repression by adenovirus E1A protein, Cell 67 (1981) 377-388. [254] E. Seto, B. Lewis, T. Shenk, Interaction between transcription factors SP1 and YY1, Nature 365 (1993) [255] P.D. Ellis, K.M. Martin, C. Rickman, J.C. Metcalfe, P.R. Kemp, Increased actin polymerization reduces the inhibition of serum response factor activity by Yin Yang 1, Biochem. J. 364 (2002) 547-554. [256] C.K. Vincent, A. Gualberto, C.V. Patel, K. Walsh, Different regulatory sequences control creatine kinase-M gene expression in directly injected skeletal and cardiac muscle, Mol. Cell. Biol. 13 (1993) 1264-1272. [257] S.J. Cooper, N.D. Trinklein, L. Nguyen, R.M. Myers, Serum response factor binding sites differ in three human cell types, Genome Res 17 (2007) 136-144. 216 References

[258] D. Vlieghe, A. Sandelin, P.J. De Bleser, K. Vleminckx, W.W. Wasserman, F. van Roy, B. Lenhard, A new generation of JASPAR, the open-access repository for transcription factor binding site profiles, Nucl. Acids Res. 34 (2006) D95-97. [259] Q. Sun, G. Chen, J.W. Streb, X. Long, Y. Yang, C.J. Stoeckert, J.M. Miano, Defining the mammalian CArGome, Genome Res 16 (2006) 197-207. [260] K. Sambamurti, R. Kinsey, B. Maloney, Y.-W. Ge, D.K. Lahiri, Gene structure and organization of the human β- secretase (BACE) promoter, FASEB J. 18 (2004) 1034-1036. [261] T. O'Connor, K.R. Sadleir, E. Maus, R.A. Velliquette, J. Zhao, S.L. Cole, W.A. Eimer, B. Hitt, L.A. Bembinster, S. Lammich, S.F. Lichtenthaler, S.S. Hébert, B. De Strooper, C. Haass, D.A. Bennett, R. Vassar, Phosphorylation of the Translation Initiation Factor eIF2a Increases BACE1 Levels and Promotes Amyloidogenesis, Neuron 60 (2008) 988-1009. [262] K.A. Webster, Evolution of the coordinate regulation of glycolytic enzyme genes by hypoxia, J Exp Biol 206 (2003) 2911-2922. [263] P.W. Hochachka, P.L. Lutz, Mechanism, origin, and evolution of anoxia tolerance in animals, Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 130 (2001) 435-459. [264] S.R. Riddle, A. Ahmad, S. Ahmad, S.S. Deeb, M. Malkki, B.K. Schneider, C.B. Allen, C.W. White, Hypoxia induces hexokinase II gene expression in human lung cell line A549, Am J Physiol Lung Cell Mol Physiol 278 (2000) L407-416. [265] D.J. Discher, N.H. Bishopric, X. Wu, C.A. Peterson, K.A. Webster, Hypoxia Regulates b-Enolase and Pyruvate Kinase-M Promoters by Modulating Sp1/Sp3 Binding to a Conserved GC Element, J Biol Chem 273 (1998) 26087-26093. [266] J.L. Johnson, A. McLachlan, Novel clustering of Sp1 transcription factor binding sites at the transcription initiation site of the human muscle phosphofructokinase P1 promoter, Nucl. Acids Res. 22 (1994) 5085-5092. [267] P.W. Hochachka, The metabolic implications of intracellular circulation, PNAS 96 (1999) 12233-12239. [268] N.V. Iyer, L.E. Kotch, F. Agani, S.W. Leung, E. Laughner, R.H. Wenger, M. Gassmann, J.D. Gearhart, A.M. Lawler, A.Y. Yu, G.L. Semenza, Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1a, Genes & Development 12 (1998) 149-162. [269] H. Tanahashi, T. Tabira, Three novel alternatively spliced isoforms of the human β-site amyloid precursor protein cleaving enzyme (BACE) and their effect on amyloid β-peptide production, Neuroscience Letters 307 (2001) 9-12. [270] L. Marselli, J. Thorne, Y.-B. Ahn, A. Omer, D.C. Sgroi, T. Libermann, H.H. Otu, A. Sharma, S. Bonner-Weir, G.C. Weir, Gene Expression of Purified b-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection, J Clin Endocrinol Metab 93 (2008) 1046-1053. [271] M. Coletta, G. Amiconi, A. Bellelli, A. Bertollini, J. Carsky, M. Castagnola, S. Condò, M. Brunori, Alteration of T- state binding properties of naturally glycated hemoglobin, HbA1c, J of Molecular Biology 203 (1988) 233-239. [272] Q. Wang, C. Jin, C. Lin, H. Pang, K. Sun, [Rapid prenatal detection of Down syndrome by homologous gene quantitative PCR], Zhonghua Yi Xue Yi Chuan Xue Za Zhi 22 (2005) 209-211. [273] M. Magnani, V. Stocchi, G. Novelli, M. Dachà, G. Fornaini, Red blood cell glucose metabolism in Down's syndrome., Clin Physiol Biochem 5 (1987) 9-14. [274] A. Lapidot, A. Gopher, S.H. Korman, H. Mandel (1990) in Ninth Annual Scientific Meeting of the Society of Magnetic Resonance in Medicine, New York. [275] N.R. Cutler, Cerebral metabolism as measured with positron emission tomography (PET) and [18F] 2-deoxy-D- glucose: healthy aging, Alzheimer's disease and Down syndrome., Prog Neuropsychopharmacol Biol Psychiatry 10 (1986) 309-21. [276] M. Peled-Kamar, H. Degani, P. Bendel, R.a. Margalit, Y. Groner, Altered brain glucose metabolism in transgenic- PFKL mice with elevated phosphofructokinase: in vivo NMR studies, Brain Res 810 (1998) 138-145. [277] X. Sun, G. He, W. Song, BACE2, as a novel APP {theta}-secretase, is not responsible for the pathogenesis of Alzheimer's disease in Down syndrome, FASEB J. 20 (2006) 1369-1376. [278] E. Diaz-Rodriguez, J.C. Montero, A. Esparis-Ogando, L. Yuste, A. Pandiella, Extracellular Signal-regulated Kinase Phosphorylates Tumor Necrosis Factor alpha -converting Enzyme at Threonine 735: A Potential Role in Regulated Shedding, Mol. Biol. Cell 13 (2002) 2031-2044. [279] K.R. Bales, T. Verina, D.J. Cummins, Y. Du, R.C. Dodel, J. Saura, C.E. Fishman, C.A. DeLong, P. Piccardo, V. Petegnief, B. Ghetti, S.M. Paul, Apolipoprotein E is essential for amyloid deposition in the APPV717F transgenic mouse model of Alzheimer's disease, PNAS 96 (1999) 15233-15238. [280] M. Raftery, R. Campbell, E.N. Glaros, K.A. Rye, G.M. Halliday, W. Jessup, B. Garner, Phosphorylation of Apolipoprotein-E at an Atypical Protein Kinase CK2 PSD/E Site in Vitro, Biochemistry 44 (2005) 7346-7353. [281] D. Games, D. Adams, R. Alessandrini, R. Barbour, P. Borthelette, C. Blackwell, T. Carr, J. Clemens, T. Donaldson, F. Gillespie, T. Guido, S. Hagopian, K. Johnson-Wood, K. Khan, M. Lee, P. Leibowitz, I. Lieberburg, S. Little, E. Masliah, L. McConlogue, M. Montoya-Zavala, L. Mucke, L. Paganini, E. Penniman, M. Power, D. Schenk, P. Seubert, B. Snyder, F. Soriano, H. Tan, J. Vitale, S. Wadsworth, B. Wolozin, J. Zhao, Alzheimer-type neuropathology in transgenic mice overexpressing V717F β-amyloid precursor protein, Nature 373 (1995) 523- 527. [282] M.A. García, M. Campillos, A. Marina, F. Valdivieso, J. Vázquez, Transcription factor AP-2 activity is modulated by protein kinase A-mediated phosphorylation, FEBS Letters 444 (1999) 27-31. [283] J. Lee, K. Lau, M.S. Perkinton, C.L. Standen, S.J.A. Shemilt, L. Mercken, J.D. Cooper, D.M. McLoughlin, C.C.J. Miller, The neuronal adaptor protein X11a reduces Aβ levels in the brains of Alzheimer's APPswe Tg2576 transgenic mice, J Biol Chem 278 (2003) 47025-9. [284] J. Lee, K. Lau, M.S. Perkinton, C.L. Standen, B. Rogelj, A. Falinska, D.M. McLoughlin, C.C.J. Miller, The neuronal adaptor protein X11β reduces amyloid β-protein levels and amyloid plaque formation in the brains of transgenic mice, J Biol Chem 279 (2004) 49099-104. 217 References

[285] Z. Muresan, V. Muresan, c-Jun NH2-Terminal Kinase-Interacting Protein-3 Facilitates Phosphorylation and Controls Localization of Amyloid-β Precursor Protein, J. Neurosci. 25 (2005) 3741-3751. [286] M. Oishi, A.C. Nairn, A.J. Czernik, G.S. Lim, T. Isohara, S.E. Gandy, P. Greengard, T. Suzuki, The cytoplasmic domain of Alzheimer's amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells., Mol Med 3 (1997) 111-123. [287] A. Lai, A. Gibson, C.R. Hopkins, I.S. Trowbridge, Signal-dependent Trafficking of b-Amyloid Precursor Protein- Transferrin Receptor Chimeras in Madin-Darby Canine Kidney Cells, J Biol Chem 273 (1998) 3732-3739. [288] P. Zheng, J. Eastman, S. Vande Pol, S.W. Pimplikar, PAT1, a microtubule-interacting protein, recognizes the basolateral sorting signal of amyloid precursor protein, PNAS 95 (1998) 14745-14750. [289] Y. Sano, T. Nakaya, Physiological Mouse Brain Aβ Levels Are Not Related to the Phosphorylation State of Threonine-668 of Alzheimer's APP, PLoS ONE 1 (2006) e51. [290] M.-S. Lee, S.-C. Kao, C.A. Lemere, W. Xia, H.-C. Tseng, Y. Zhou, R. Neve, M.K. Ahlijanian, L.-H. Tsai, APP processing is regulated by cytoplasmic phosphorylation, J. Cell Biol. 163 (2003) 83-95. [291] N. Suzuki, T.T. Cheung, X.D. Cai, A. Odaka, L. Otvos, Jr., C. Eckman, T.E. Golde, S.G. Younkin, An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants, Science 264 (1994) 1336-1340. [292] K.-i. Iijima, K. Ando, S. Takeda, Y. Satoh, T. Seki, S. Itohara, P. Greengard, Y. Kirino, A.C. Nairn, T. Suzuki, Neuron-Specific Phosphorylation of Alzheimer's β-Amyloid Precursor Protein by Cyclin-Dependent Kinase 5, J Neurochem 75 (2000) 1085-1091. [293] A.E. Aplin, G.M. Gibb, J.S. Jacobsen, J.-M. Gallo, B.H. Anderton, In Vitro Phosphorylation of the Cytoplasmic Domain of the Amyloid Precursor Protein by Glycogen Synthase Kinase-3β, J Neurochem 67 (1996) 699-707. [294] Z. Muresan, V. Muresan, The Amyloid-β Precursor Protein Is Phosphorylated via Distinct Pathways during Differentiation, Mitosis, Stress, and Degeneration, Mol. Biol. Cell (2007) E06-07-0625. [295] C.L. Standen, J. Brownlees, A.J. Grierson, S. Kesavapany, K.-F. Lau, D.M. McLoughlin, C.C.J. Miller, Phosphorylation of thr668 in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3), J Neurochem 76 (2001) 316-320. [296] N. Zambrano, P. Bruni, G. Minopoli, R. Mosca, D. Molino, C. Russo, G. Schettini, M. Sudol, T. Russo, The β- amyloid precursor protein APP is tyrosine phosphorylated in cells expressing a constitutively active form of the Abl protoncogene, J. Biol. Chem. (2001) M100792200. [297] P.E. Tarr, R. Roncarati, G. Pelicci, P.G. Pelicci, L. D'Adamio, Tyrosine Phosphorylation of the β-Amyloid Precursor Protein Cytoplasmic Tail Promotes Interaction with Shc, J. Biol. Chem. 277 (2002) 16798-16804. [298] C.A.F. von Arnim, M.M. Tangredi, I.D. Peltan, B.M. Lee, M.C. Irizarry, A. Kinoshita, B.T. Hyman, Demonstration of BACE (β-secretase) phosphorylation and its interaction with GGA1 in cells by fluorescence-lifetime imaging microscopy, J Cell Sci 117 (2004) 5437-5445. [299] N. Ghoshal, J.F. Smiley, A.J. DeMaggio, M.F. Hoekstra, E.J. Cochran, L.I. Binder, J. Kuret, A New Molecular Link between the Fibrillar and Granulovacuolar Lesions of Alzheimer's Disease, Am J Pathol 155 (1999) 1163-1172. [300] K. Yasojima, J. Kuret, A.J. DeMaggio, E. McGeer, P.L. McGeer, Casein kinase 1 delta mRNA is upregulated in Alzheimer disease brain, Brain Res 865 (2000) 116-120. [301] C. Schwab, A.J. DeMaggio, N. Ghoshal, L.I. Binder, J. Kuret, P.L. McGeer, Casein kinase 1 delta is associated with pathological accumulation of tau in several Neurodeg Dis, Neurobiology of Aging 21 (2000) 503-510. [302] M. Flajolet, G. He, M. Heiman, A. Lin, A.C. Nairn, P. Greengard, Regulation of Alzheimer's disease amyloid-β formation by casein kinase I, PNAS 104 (2007) 4159-4164. [303] K.F. Gietzen, D.M. Virshup, Identification of Inhibitory Autophosphorylation Sites in Casein Kinase I epsilon, J. Biol. Chem. 274 (1999) 32063-32070. [304] Y. Li, A. Grupe, C. Rowland, P. Nowotny, J.S.K. Kauwe, S. Smemo, A. Hinrichs, K. Tacey, T.A. Toombs, S. Kwok, J. Catanese, T.J. White, T.J. Maxwell, P. Hollingworth, R. Abraham, D.C. Rubinsztein, C. Brayne, F. Wavrant-De Vrieze, J. Hardy, M. O'Donovan, S. Lovestone, J.C. Morris, L.J. Thal, M. Owen, J. Williams, A. Goate, DAPK1 variants are associated with Alzheimer's disease and allele-specific expression, Hum. Mol. Genet. 15 (2006) 2560-2568. [305] M.F. Favata, K.Y. Horiuchi, E.J. Manos, A.J. Daulerio, D.A. Stradley, W.S. Feeser, D.E. Van Dyk, W.J. Pitts, R.A. Earl, F. Hobbs, R.A. Copeland, R.L. Magolda, P.A. Scherle, J.M. Trzaskos, Identification of a Novel Inhibitor of Mitogen-activated Protein Kinase Kinase, J Biol Chem 273 (1998) 18623-18632. [306] D.R. DeSilva, E.A. Jones, M.F. Favata, B.D. Jaffee, R.L. Magolda, J.M. Trzaskos, P.A. Scherle, Inhibition of Mitogen-Activated Protein Kinase Kinase Blocks T Cell Proliferation But Does Not Induce or Prevent Anergy, J Immunol 160 (1998) 4175-4181. [307] R. Anjum, P.P. Roux, B.A. Ballif, S.P. Gygi, J. Blenis, The Tumor Suppressor DAP Kinase Is a Target of RSK- Mediated Survival Signaling, Current Biology 15 (2005) 1762-1767. [308] G. Shohat, T. Spivak-Kroizman, O. Cohen, S. Bialik, G. Shani, H. Berrisi, M. Eisenstein, A. Kimchi, The Pro- apoptotic Function of Death-associated Protein Kinase Is Controlled by a Unique Inhibitory Autophosphorylation- based Mechanism, J. Biol. Chem. 276 (2001) 47460-47467. [309] C.-H. Chen, W.-J. Wang, J.-C. Kuo, H.-C. Tsai, J.-R. Lin, Z.-F. Chang, R.-H. Chen, Bidirectional signals transduced by DAPK-ERK interaction promote the apoptotic effect of DAPK, EMBO J 24 (2005) 294-304. [310] M. Boyce, K.F. Bryant, C. Jousse, K. Long, H.P. Harding, D. Scheuner, R.J. Kaufman, D. Ma, D.M. Coen, D. Ron, J. Yuan, A Selective Inhibitor of eIF2a Dephosphorylation Protects Cells from ER Stress, Science 307 (2005) 935- 939. [311] Y. Shi, J. An, J. Liang, S.E. Hayes, G.E. Sandusky, L.E. Stramm, N.N. Yang, Characterization of a Mutant Pancreatic eIF-2a Kinase, PEK, and Co-localization with Somatostatin in Islet Delta Cells, J Biol Chem 274 (1999) 5723-5730.

218 References

[312] M. Clemens, Initiation factor eIF2 alpha phosphorylation in stress responses and apoptosis., Prog Mol Subcell Biol 27 (2001) 57-89. [313] W. Ladiges, J. Morton, C. Blakely, M. Gale, Tissue specific expression of PKR protein kinase in aging B6D2F1 mice, Mech Age Dev 114 (2000) 123-132. [314] W. Yan, C.L. Frank, M.J. Korth, B.L. Sopher, I. Novoa, D. Ron, M.G. Katze, Control of PERK eIF2a kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK, PNAS 99 (2002) 15920-15925. [315] U. Ozcan, E. Yilmaz, L. Ozcan, M. Furuhashi, E. Vaillancourt, R.O. Smith, C.Z. Gorgun, G.S. Hotamisligil, Chemical Chaperones Reduce ER Stress and Restore Glucose Homeostasis in a Mouse Model of , Science 313 (2006) 1137-1140. [316] S.L. Sabo, A.F. Ikin, J.D. Buxbaum, P. Greengard, The Amyloid Precursor Protein and Its Regulatory Protein, FE65, in Growth Cones and Synapses In Vitro and In Vivo, J. Neurosci. 23 (2003) 5407-5415. [317] K. Ando, K.-i. Iijima, J.I. Elliott, Y. Kirino, T. Suzuki, Phosphorylation-dependent Regulation of the Interaction of Amyloid Precursor Protein with Fe65 Affects the Production of β-Amyloid, J. Biol. Chem. 276 (2001) 40353-40361. [318] Z. Muresan, V. Muresan, Coordinated transport of phosphorylated amyloid-β precursor protein and c-Jun NH2- terminal kinase-interacting protein-1, J. Cell Biol. 171 (2005) 615-625. [319] M.S. Perkinton, C.L. Standen, K. Lau, S. Kesavapany, H.L. Byers, M. Ward, D.M. McLoughlin, C.C.J. Miller, The c-Abl Tyrosine Kinase Phosphorylates the Fe65 Adaptor Protein to Stimulate Fe65/Amyloid Precursor Protein Nuclear Signaling, J Biol Chem 279 (2004) 22084-91. [320] C.L. Standen, M.S. Perkinton, H.L. Byers, S. Kesavapany, K.-F. Lau, M. Ward, D. McLoughlin, C.C.J. Miller, The neuronal adaptor protein Fe65 is phosphorylated by mitogen-activated protein kinase (ERK1/2), Mol Cell Neurosc 24 (2003) 851-857. [321] T. Wahle, D.R. Thal, M. Sastre, A. Rentmeister, N. Bogdanovic, M. Famulok, M.T. Heneka, J. Walter, GGA1 Is Expressed in the Human Brain and Affects the Generation of Amyloid β-Peptide, J. Neurosci. 26 (2006) 12838- 12846. [322] B. Doray, K. Bruns, P. Ghosh, S.A. Kornfeld, Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif, PNAS 99 (2002) 8072-8077. [323] G. Tesco, Y.H. Koh, E.L. Kang, A.N. Cameron, S. Das, M. Sena-Esteves, M. Hiltunen, S.-H. Yang, Z. Zhong, Y. Shen, J.W. Simpkins, R.E. Tanzi, Depletion of GGA3 Stabilizes BACE and Enhances β-Secretase Activity, Neuron 54 (2007) 721-737. [324] M.H. Scheinfeld, S. Matsuda, L. D'Adamio, JNK-interacting protein-1 promotes transcription of Aβ protein precursor but not Aβ precursor-like proteins, mechanistically different than Fe65, PNAS 100 (2003) 1729-1734. [325] D. Nihalani, H.N. Wong, L.B. Holzman, Recruitment of JNK to JIP1 and JNK-dependent JIP1 Phosphorylation Regulates JNK Module Dynamics and Activation, J. Biol. Chem. 278 (2003) 28694-28702. [326] J.J. Song, Y.J. Lee, Cross-talk between JIP3 and JIP1 during Glucose Deprivation: SEK1-JNK2 AND Akt1 ACT AS MEDIATORS, J. Biol. Chem. 280 (2005) 26845-26855. [327] S.A. Beausoleil, M. Jedrychowski, D. Schwartz, J.E. Elias, J. Villen, J. Li, M.A. Cohn, L.C. Cantley, S.P. Gygi, Large-scale characterization of HeLa cell nuclear phosphoproteins, PNAS 101 (2004) 12130-12135. [328] M. Nousiainen, H.H.W. Sillje, G. Sauer, E.A. Nigg, R. Korner, Phosphoproteome analysis of the human mitotic spindle, PNAS 103 (2006) 5391-5396. [329] H. Molina, D.M. Horn, N. Tang, S. Mathivanan, A. Pandey, Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry, PNAS 104 (2007) 2199-2204. [330] S.C. Kim, Y. Chen, S. Mirza, Y. Xu, J. Lee, P. Liu, Y. Zhao, A Clean, More Efficient Method for In-Solution Digestion of Protein Mixtures without Detergent or Urea, J of Proteome Research 5 (2006) 3446-3452. [331] J.-E. Kim, S.R. Tannenbaum, F.M. White, Global Phosphoproteome of HT-29 Human Colon Adenocarcinoma Cells, J of Proteome Research 4 (2005) 1339-1346. [332] A. Grupe, R. Abraham, Y. Li, C. Rowland, P. Hollingworth, A. Morgan, L. Jehu, R. Segurado, D. Stone, E. Schadt, M. Karnoub, P. Nowotny, K. Tacey, J. Catanese, J. Sninsky, C. Brayne, D. Rubinsztein, M. Gill, B. Lawlor, S. Lovestone, P. Holmans, M. O'Donovan, J.C. Morris, L. Thal, A. Goate, M.J. Owen, J. Williams, Evidence for novel susceptibility genes for late-onset Alzheimer's disease from a genome-wide association study of putative functional variants, Hum. Mol. Genet. 16 (2007) 865-873. [333] M.Z. Kounnas, R.D. Moir, G.W. Rebeck, A.I. Bush, W.S. Argraves, R.E. Tanzi, B.T. Hyman, D.K. Strickland, Cell 82 (1995) 331-340. [334] M.F. Knauer, R.A. Orlando, C.G. Glabe, Brain Res 740 (1996) 6-14. [335] P.G. Ulery, J. Beers, I. Mikhailenko, R.E. Tanzi, G.W. Rebeck, B.T. Hyman, D.K. Strickland, Modulation of β- Amyloid Precursor Protein Processing by the Low Density Lipoprotein Receptor-related Protein (LRP). EVIDENCE THAT LRP CONTRIBUTES TO THE PATHOGENESIS OF ALZHEIMER'S DISEASE, J. Biol. Chem. 275 (2000) 7410-7415. [336] Y. Li, P. van Kerkhof, M.P. Marzolo, G.J. Strous, G. Bu, Identification of a Major Cyclic AMP-Dependent Protein Kinase A Phosphorylation Site within the Cytoplasmic Tail of the Low-Density Lipoprotein Receptor-Related Protein: Implication for Receptor-Mediated Endocytosis, Mol. Cell. Biol. 21 (2001) 1185-1195. [337] S. Ranganathan, C.-X. Liu, M.M. Migliorini, C.A.F. von Arnim, I.D. Peltan, I. Mikhailenko, B.T. Hyman, D.K. Strickland, Serine and Threonine Phosphorylation of the Low Density Lipoprotein Receptor-related Protein by Protein Kinase C{alpha} Regulates Endocytosis and Association with Adaptor Molecules, J. Biol. Chem. 279 (2004) 40536-40544. [338] A.E. Roher, T.C. Kasunic, A.S. Woods, R.J. Cotter, M.J. Ball, R. Fridman, Proteolysis of Aβ Peptide from Alzheimer Disease Brain by Gelatinase A, BBRC 205 (1994) 1755-1761.

219 References

[339] A.R. White, T. Du, K.M. Laughton, I. Volitakis, R.A. Sharples, M.E. Xilinas, D.E. Hoke, R.M.D. Holsinger, G. Evin, R.A. Cherny, A.F. Hill, K.J. Barnham, Q.-X. Li, A.I. Bush, C.L. Masters, Degradation of the Alzheimer Disease Amyloid beta-Peptide by Metal-dependent Up-regulation of Metalloprotease Activity, J. Biol. Chem. 281 (2006) 17670-17680. [340] M. Sariahmetoglu, B.D. Crawford, H. Leon, J. Sawicka, L. Li, B.J. Ballermann, C. Holmes, L.G. Berthiaume, A. Holt, G. Sawicki, R. Schulz, Regulation of matrix metalloproteinase-2 (MMP-2) activity by phosphorylation, FASEB J. 21 (2007) 2486-2495. [341] M.P. Murphy, P. Das, A.C. Nyborg, M.J. Rochette, M.W. Dodson, N.M. Loosbrock, T.M. Souder, C. McLendon, S.L. Merit, S.C. Piper, K.R. Jansen, T.E. Golde, Overexpression of Nicastrin increases Aβ production, FASEB J. 17 (2003) 1138-1140. [342] S.-K. Kim, H.-J. Park, H.S. Hong, E.J. Baik, M.W. Jung, I. Mook-Jung, ERK1/2 is an endogenous negative regulator of the g-secretase activity, FASEB J. 20 (2006) 157-159. [343] J. Xie, Q. Guo, PAR-4 Is Involved in Regulation of β-Secretase Cleavage of the Alzheimer Amyloid Precursor Protein, J. Biol. Chem. 280 (2005) 13824-13832. [344] M. Boosen, S. Vetterkind, J. Kubicek, K.-H. Scheidtmann, S. Illenberger, U. Preuss, Par-4 Is an Essential Downstream Target of DAP-like Kinase (Dlk) in Dlk/Par-4-mediated Apoptosis, Mol. Biol. Cell 20 (2009) 4010- 4020. [345] A. Goswami, R. Burikhanov, A. de Thonel, N. Fujita, M. Goswami, Y. Zhao, J.E. Eriksson, T. Tsuruo, V.M. Rangnekar, Binding and Phosphorylation of Par-4 by Akt Is Essential for Cancer Cell Survival, Molecular Cell 20 (2005) 33-44. [346] G. Hamilton, P. Proitsi, L. Jehu, A. Morgan, J. Williams, M.C. O'Donovan, M.J. Owen, J.F. Powell, S. Lovestone, Candidate gene association study of insulin signaling genes and Alzheimer's disease: Evidence for SOS2, PCK1, and PPARgamma as susceptibility loci, American J of Medical Genetics Part B: Neuropsychiatric Genetics 144B (2007) 508-516. [347] J. Villen, S.A. Beausoleil, S.A. Gerber, S.P. Gygi, Large-scale phosphorylation analysis of mouse liver, PNAS 104 (2007) 1488-1493. [348] L. Pastorino, A. Sun, P.-J. Lu, X.Z. Zhou, M. Balastik, G. Finn, G. Wulf, J. Lim, S.-H. Li, X. Li, W. Xia, L.K. Nicholson, K.P. Lu, The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-β production, Nature 440 (2006) 528-534. [349] P.-J. Lu, X.Z. Zhou, Y.-C. Liou, J.P. Noel, K.P. Lu, Critical Role of WW Domain Phosphorylation in Regulating Phosphoserine Binding Activity and Pin1 Function, J. Biol. Chem. 277 (2002) 2381-2384. [350] F. Eckerdt, J. Yuan, K. Saxena, B. Martin, S. Kappel, C. Lindenau, A. Kramer, S. Naumann, S. Daum, G. Fischer, I. Dikic, M. Kaufmann, K. Strebhardt, Polo-like Kinase 1-mediated Phosphorylation Stabilizes Pin1 by Inhibiting Its Ubiquitination in Human Cells, J. Biol. Chem. 280 (2005) 36575-36583. [351] M. Sastre, I. Dewachter, S. Rossner, N. Bogdanovic, E. Rosen, P. Borghgraef, B.O. Evert, L. Dumitrescu-Ozimek, D.R. Thal, G. Landreth, J. Walter, T. Klockgether, F. van Leuven, M.T. Heneka, Nonsteroidal anti-inflammatory drugs repress β-secretase gene promoter activity by the activation of PPAR{gamma}, PNAS 103 (2006) 443-448. [352] H.S. Camp, S.R. Tafuri, Regulation of Peroxisome Proliferator-activated Receptor gamma Activity by Mitogen- activated Protein Kinase, J. Biol. Chem. 272 (1997) 10811-10816. [353] M. Adams, M.J. Reginato, D. Shao, M.A. Lazar, V.K. Chatterjee, Transcriptional Activation by Peroxisome Proliferator-activated Receptor gamma Is Inhibited by Phosphorylation at a Consensus Mitogen-activated Protein Kinase Site, J. Biol. Chem. 272 (1997) 5128-5132. [354] R. Fluhrer, A. Friedlein, C. Haass, J. Walter, Phosphorylation of Presenilin 1 at the Caspase Recognition Site Regulates Its Proteolytic Processing and the Progression of Apoptosis, J. Biol. Chem. 279 (2004) 1585-1593. [355] F. Kirschenbaum, S.-C. Hsu, B. Cordell, J.V. McCarthy, Substitution of a Glycogen Synthase Kinase-3β Phosphorylation Site in Presenilin 1 Separates Presenilin Function from β-Catenin Signaling, J. Biol. Chem. 276 (2001) 7366-7375. [356] K.-F. Lau, D.R. Howlett, S. Kesavapany, C.L. Standen, C. Dingwall, D.M. McLoughlin, C.C.J. Miller, Cyclin- Dependent Kinase-5/p35 Phosphorylates Presenilin 1 to Regulate Carboxy-Terminal Fragment Stability, Mol Cell Neurosc 20 (2002) 13-20. [357] K. Uemura, A. Kuzuya, Y. Shimozono, N. Aoyagi, K. Ando, S. Shimohama, A. Kinoshita, GSK3β Activity Modifies the Localization and Function of Presenilin 1, J. Biol. Chem. 282 (2007) 15823-15832. [358] Y. Wang, R.M. Baron, G. Zhu, M. Joo, J.W. Christman, E.S. Silverman, M.A. Perrella, R.J. Riese, M. Cernadas, PU.1 Regulates Cathepsin S Expression in Professional APCs, J Immunol 176 (2006) 275-283. [359] J.S. Munger, C. Haass, C.A. Lemere, G.P. Shi, W.S. Wong, D.B. Teplow, D.J. Selkoe, H.A. Chapman, Lysosomal processing of amyloid precursor protein to A beta peptides: a distinct role for cathepsin S, Biochem. J. 311 (1995) 299-305. [360] J.-M. Wang, M.-Z. Lai, H.-F. Yang-Yen, Interleukin-3 Stimulation of mcl-1 Gene Transcription Involves Activation of the PU.1 Transcription Factor through a p38 Mitogen-Activated Protein Kinase-Dependent Pathway, Mol. Cell. Biol. 23 (2003) 1896-1909. [361] J.M. Pongubala, C. Van Beveren, S. Nagulapalli, M.J. Klemsz, S.R. McKercher, R.A. Maki, M.L. Atchison, Effect of PU.1 phosphorylation on interaction with NF-EM5 and transcriptional activation, Science 259 (1993) 1622-1625. [362] W. Huang, E. Horvath, E.A. Eklund, PU.1, Interferon Regulatory Factor (IRF) 2, and the Interferon Consensus Sequence-binding Protein (ICSBP/IRF8) Cooperate to Activate NF1 Transcription in Differentiating Myeloid Cells, J. Biol. Chem. 282 (2007) 6629-6643. [363] M. Joo, G.Y. Park, J.G. Wright, T.S. Blackwell, M.L. Atchison, J.W. Christman, Transcriptional Regulation of the Cyclooxygenase-2 Gene in Macrophages by PU.1, J. Biol. Chem. 279 (2004) 6658-6665. [364] F. Liu, Y. Su, B. Li, Y. Zhou, J. Ryder, P. Gonzalez-DeWhitt, P.C. May, B. Ni, Regulation of amyloid precursor protein (APP) phosphorylation and processing by p35/Cdk5 and p25/Cdk5, FEBS Letters 547 (2003) 193-196. 220 References

[365] T. Saito, R. Onuki, Y. Fujita, G.-i. Kusakawa, K. Ishiguro, J.A. Bibb, T. Kishimoto, S.-i. Hisanaga, Developmental Regulation of the Proteolysis of the p35 Cyclin-Dependent Kinase 5 Activator by Phosphorylation, J. Neurosci. 23 (2003) 1189-1197. [366] H. Kamei, T. Saito, M. Ozawa, Y. Fujita, A. Asada, J.A. Bibb, T.C. Saido, H. Sorimachi, S.-i. Hisanaga, Suppression of Calpain-dependent Cleavage of the CDK5 Activator p35 to p25 by Site-specific Phosphorylation, J. Biol. Chem. 282 (2007) 1687-1694. [367] G.N. Patrick, P. Zhou, Y.T. Kwon, P.M. Howley, L.-H. Tsai, p35, the Neuronal-specific Activator of Cyclin- dependent Kinase 5 (Cdk5) Is Degraded by the Ubiquitin-Proteasome Pathway, J Biol Chem 273 (1998) 24057- 24064. [368] O.M. Grbovic, P.M. Mathews, Y. Jiang, S.D. Schmidt, R. Dinakar, N.B. Summers-Terio, B.P. Ceresa, R.A. Nixon, A.M. Cataldo, Rab5-stimulated up-regulation of the endocytic pathway increases intracellular levels of betaCTFs and Aβ production, J. Biol. Chem. (2003) M304122200. [369] M. Chiariello, C.B. Bruni, C. Bucci, The small GTPases Rab5a, Rab5b and Rab5c are differentially phosphorylated in vitro, FEBS Letters 453 (1999) 20-24. [370] O.M. Andersen, J. Reiche, V. Schmidt, M. Gotthardt, R. Spoelgen, J. Behlke, C.A.F. von Arnim, T. Breiderhoff, P. Jansen, X. Wu, K.R. Bales, R. Cappai, C.L. Masters, J. Gliemann, E.J. Mufson, B.T. Hyman, S.M. Paul, A. Nykjaer, T.E. Willnow, Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein, PNAS 102 (2005) 13461-13466. [371] E. Rogaeva, Y. Meng, J.H. Lee, Y. Gu, T. Kawarai, F. Zou, T. Katayama, C.T. Baldwin, R. Cheng, H. Hasegawa, F. Chen, N. Shibata, K.L. Lunetta, R. Pardossi-Piquard, C. Bohm, Y. Wakutani, L.A. Cupples, K.T. Cuenco, R.C. Green, L. Pinessi, I. Rainero, S. Sorbi, A. Bruni, R. Duara, R.P. Friedland, R. Inzelberg, W. Hampe, H. Bujo, Y.-Q. Song, O.M. Andersen, T.E. Willnow, N. Graff-Radford, R.C. Petersen, D. Dickson, S.D. Der, P.E. Fraser, G. Schmitt-Ulms, S. Younkin, R. Mayeux, L.A. Farrer, P. St George-Hyslop, The neuronal sortilin-related receptor SORL1 is genetically associated with Alzheimer disease, Nat Genet 39 (2007) 168-177. [372] W. Hampe (2008), Universitätsklinikum Hamburg-Eppendorf, Institut für Biochemie und Molekularbiologie II - Molekulare Zellbiologie. [373] Y. Li, H. Wang, S. Wang, D. Quon, Y.-W. Liu, B. Cordell, Positive and negative regulation of APP amyloidogenesis by sumoylation, PNAS 100 (2003) 259-264. [374] G. Bossis, F. Melchior, SUMO: regulating the regulator, Cell Division 1 (2006) 13. [375] V. Dorval, M.J. Mazzella, P.M. Mathews, R.T. Hay, P.E. Fraser, Modulation of Ab generation by small ubiquitin- like modifiers does not require conjugation to target proteins, Biochem J 404 (2007) 309-316. [376] S.A. Armstrong, D.A. Barry, R.W. Leggett, C.R. Mueller, Casein Kinase II-mediated Phosphorylation of the C Terminus of Sp1 Decreases Its DNA Binding Activity, J. Biol. Chem. 272 (1997) 13489-13495. [377] M.R. Bonello, L.M. Khachigian, Fibroblast Growth Factor-2 Represses Platelet-derived Growth Factor Receptor- {alpha} (PDGFR-{alpha}) Transcription via ERK1/2-dependent Sp1 Phosphorylation and an Atypical cis-Acting Element in the Proximal PDGFR-{alpha} Promoter, J. Biol. Chem. 279 (2004) 2377-2382. [378] A. De Siervi, M. Marinissen, J. Diggs, X.-F. Wang, G. Pages, A. Senderowicz, Transcriptional Activation of p21waf1/cip1 by Alkylphospholipids: Role of the Mitogen-Activated Protein Kinase Pathway in the Transactivation of the Human p21waf1/cip1 Promoter by Sp1, Cancer Res 64 (2004) 743-750. [379] J. Milanini-Mongiat, J. Pouyssegur, G. Pages, Identification of Two Sp1 Phosphorylation Sites for p42/p44 Mitogen-activated Protein Kinases. THEIR IMPLICATION IN VASCULAR ENDOTHELIAL GROWTH FACTOR GENE TRANSCRIPTION, J. Biol. Chem. 277 (2002) 20631-20639. [380] Y.-C. Chang, S. Illenye, N.H. Heintz, Cooperation of E2F-p130 and Sp1-pRb Complexes in Repression of the Chinese Hamster dhfr Gene, Mol. Cell. Biol. 21 (2001) 1121-1131. [381] P.K. Datta, P. Raychaudhuri, S. Bagchi, Association of p107 with Sp1: genetically separable regions of p107 are involved in regulation of E2F- and Sp1-dependent transcription, Mol. Cell. Biol. 15 (1995) 5444-5452. [382] Y. Zhang, M. Liao, M.L. Dufau, Phosphatidylinositol 3-Kinase/Protein Kinase Cz-Induced Phosphorylation of Sp1 and p107 Repressor Release Have a Critical Role in Histone Deacetylase Inhibitor-Mediated Depression of Transcription of the Luteinizing Hormone Receptor Gene, Mol. Cell. Biol. 26 (2006) 6748-6761. [383] S. Kitazume-Kawaguchi, N. Dohmae, K. Takio, S. Tsuji, K.J. Colley, The relationship between ST6Gal I Golgi retention and its cleavage-secretion, Glycobiology 9 (1999) 1397-1406. [384] S. Kitazume, Y. Tachida, R. Oka, K. Shirotani, T.C. Saido, Y. Hashimoto, Alzheimer‟s b-secretase, b-site amyloid precursor protein-cleaving enzyme, is responsible for cleavage secretion of a Golgi-resident sialyltransferase, PNAS 98 (2001) 13554–13559. [385] I. Sugimoto, S. Futakawa, R. Oka, K. Ogawa, J.D. Marth, E. Miyoshi, N. Taniguchi, Y. Hashimoto, S. Kitazume, ST6Gal I cleavage by BACE1 enhances the sialylation of soluble glycoproteins : A novel regulatory mechanism for alpha 2,6-sialylation, J. Biol. Chem. (2007) M704766200. [386] S. Kitazume, Y. Tachida, R. Oka, K. Nakagawa, S. Takashima, Y.-C. Lee, Y. Hashimoto, Screening a series of sialyltransferases for possible BACE1 substrates, Glycoconjugate J 23 (2006) 437-441. [387] S.F. Lichtenthaler, D.-i. Dominguez, G.G. Westmeyer, K. Reiss, C. Haass, P. Saftig, B. De Strooper, B. Seed, The Cell Adhesion Protein P-selectin Glycoprotein Ligand-1 Is a Substrate for the Aspartyl Protease BACE1, J. Biol. Chem. 278 (2003) 48713-48719. [388] H.-K. Wong, T. Sakurai, F. Oyama, K. Kaneko, K. Wada, H. Miyazak, M. Kurosawa, B. De Strooper, P. Saftig, N. Nukina, b-Subunits of Voltage-gated Sodium Channels Are Novel Substrates of b-Site Amyloid Precursor Protein- cleaving Enzyme (BACE1) and g-Secretase, J Biol Chem 280 (2004) 23009–23017. [389] D.Y. Kim, L.A. MacKenzie Ingano, B.W. Carey, W.H. Pettingell, D.M. Kovacs, Presenilin/g-Secretase-mediated Cleavage of the Voltage-gated Sodium Channel b2-Subunit Regulates Cell Adhesion and Migration, J Biol Chem 280 (2005) 23251–23261.

221 References

[390] H. Miyazaki, F. Oyama, H.-K. Wong, K. Kaneko, T. Sakurai, A. Tamaoka, N. Nukina, BACE1 modulates filopodia- like protrusions induced by sodium channel b4 subunit, BBRC 361 (2007) 43-48. [391] C.A.F. von Arnim, A. Kinoshita, I.D. Peltan, M.M. Tangredi, L. Herl, B.M. Lee, R. Spoelgen, T.T. Hshieh, S. Ranganathan, F.D. Battey, C.-X. Liu, B.J. Bacskai, S. Sever, M.C. Irizarry, D.K. Strickland, B.T. Hyman, The LDL- receptor related protein (LRP) is a novel β-secretase (BACE 1) substrate, J. Biol. Chem. (2005) M414248200. [392] P.-H. Kuhn, E. Marjaux, A. Imhof, B. De Strooper, C. Haass, S.F. Lichtenthaler, REGULATED INTRAMEMBRANE PROTEOLYSIS OF THE INTERLEUKIN-1 RECEPTOR II BY α-, β-, AND γ-SECRETASE, J Biol Chem (2007) [393] M. Willem, A.N. Garratt, B. Novak, M. Citron, S. Kaufmann, A. Rittger, B. DeStrooper, P. Saftig, C. Birchmeier, C. Haass, Control of Peripheral Nerve Myelination by the β-Secretase BACE1, Science 314 (2006) 664-666. [394] G. Strobel, BACE Found to Have Big Job in Wrapping Motoneurons, 10th ICAD meeting, Madrid, (2006) on www.alzforum.org. [395] X. Hu, C.W. Hicks, W. He, P. Wong, W.B. Macklin, B.D. Trapp, R. Yan, Bace1 modulates myelination in the central and peripheral nervous system, Nat Neurosci 9 (2006) 1520-1525. [396] A.V. Savonenko, T. Melnikova, F.M. Laird, K.A. Stewart, D.L. Price, P.C. Wong, Alteration of BACE1-dependent NRG1/ErbB4 signaling and schizophrenia-like phenotypes in BACE1-null mice, PNAS 105 (2008) 5585-5590. [397] G.T. Wong, D. Manfra, F.M. Poulet, Q. Zhang, H. Josien, T. Bara, L. Engstrom, M. Pinzon-Ortiz, J.S. Fine, H.-J.J. Lee, L. Zhang, G.A. Higgins, E.M. Parker, Chronic Treatment with the g-Secretase Inhibitor LY-411,575 Inhibits β- Amyloid Peptide Production and Alters Lymphopoiesis and Intestinal Cell Differentiation, J. Biol. Chem. 279 (2004) 12876-12882. [398] S.J. Stachel, C.A. Coburn, T.G. Steele, K.G. Jones, E.F. Loutzenhiser, A.R. Gregro, H.A. Rajapakse, M.T. Lai, M.C. Crouthamel, M. Xu, K. Tugusheva, J.E. Lineberger, B.L. Pietrak, A.S. Espeseth, X.P. Shi, E. Chen-Dodson, M.K. Holloway, S. Munshi, A.J. Simon, L. Kuo, J.P. Vacca, Structure-Based Design of Potent and Selective Cell- Permeable Inhibitors of Human β-Secretase (BACE-1), J. Med. Chem. 47 (2004) 6447-6450. [399] E.W. Baxter, K.A. Conway, L. Kennis, F. Bischoff, M.H. Mercken, H.L. DeWinter, C.H. Reynolds, B.A. Tounge, C. Luo, M.K. Scott, Y. Huang, M. Braeken, S.M.A. Pieters, D.J.C. Berthelot, S. Masure, W.D. Bruinzeel, A.D. Jordan, M.H. Parker, R.E. Boyd, J. Qu, R.S. Alexander, D.E. Brenneman, A.B. Reitz, 2-Amino-3,4-dihydroquinazolines as Inhibitors of BACE-1 (β-Site APP Cleaving Enzyme): Use of Structure Based Design to Convert a Micromolar Hit into a Nanomolar Lead, J. Med. Chem. 50 (2007) 4261-4264. [400] S.R. Stauffer, M.G. Stanton, A.R. Gregro, M.A. Steinbeiser, J.R. Shaffer, P.G. Nantermet, J.C. Barrow, K.E. Rittle, D. Collusi, A.S. Espeseth, M.-T. Lai, B.L. Pietrak, M.K. Holloway, G.B. McGaughey, S.K. Munshi, J.H. Hochman, A.J. Simon, H.G. Selnick, S.L. Graham, J.P. Vacca, Discovery and SAR of isonicotinamide BACE-1 inhibitors that bind b-secretase in a N-terminal 10s-loop down conformation, Bioorganic & Medicinal Chemistry Letters 17 (2007) 1788-1792. [401] M. Fardilha, S.I. Vieira, A. Barros, M. Sousa, O.A.B. da CRUZ e SILVA, E.F. da CRUZ e SILVA, Differential Distribution of Alzheimer's Amyloid Precursor Protein Family Variants in Human Sperm, Annals of the New York Academy of Sciences 1096 (2007) 196-206. [402] O.L. Serov, B. Chowdhary, J.E. Womack, J.A. Marshall Graves, Comparative Gene Mapping, Chromosome Painting and the Reconstruction of the Ancestral Mammalian Karyotype, in: A. Ruvinsky,J. Marshall Graves (Eds.) Mammalian genomics, CABI Pub., Wallingford, UK; Cambridge, USA, 2004, pp. [403] R. Stanyon, G. Stone, M. Garcia, L. Froenicke, Reciprocal chromosome painting shows that squirrels, unlike murid rodents, have a highly conserved genome organization, Genomics 82 (2003) 245-249. [404] S. Ohno, Evolution by , Springer-Verlag, New York, Heidelberg, Berlin, 1970 [405] Y. Van de Peer, S. Maere and A. Meyer, 2R or not 2R is not the question anymore, Nature Reviews Genetics 11, 166 (February 2010) [406] A.B. Butler, R.S. Larry, Evolution of Vertebrate Brains, in: Encyclopedia of Neuroscience, Academic Press, Oxford, 2009, pp. 57-66. [407] J. Osório, S. Rétaux, The lamprey in evolutionary studies, Development Genes and Evolution 218 (2008) 221- 235. [408] E.J. Coulson, K. Paliga, K. Beyreuther, C.L. Masters, What the evolution of the amyloid protein precursor supergene family tells us about its function, Neurochemistry International 36 (2000) 175-184. [409] R.W.J. Collin, D. van Strien, J.A.M. Leunissen, G.J.M. Martens, Identification and expression of the first nonmammalian amyloid-β precursor-like protein APLP2 in the amphibian Xenopus laevis, Eur J Biochemistry 271 (2004) 1906-1912. [410] E.F. Vanin, Processed Pseudogenes: Characterstics and Evolution, Ann. Rev. Genet. 19 (1985) 253-272 [411] A. Pavlicek, J. Paces, R. Zika and J. Hejnar, Length distribution of long interspersed nuclear elements (LINEs) and processed pseudogenes of human endogenous retroviruses: implications for retrotransposition and pseudogene detection, Gene 300 (2002) 189-194 [412] H.A.F. Booth and P.W.H. Holland, Eleven daughters of NANOG, Genomics 84 (2004) 229-238 [413] Z. Zhang, P.M. Harrison, Y. Liu and M. Gerstein, Millions of Years of Evolution Preserved: A Comprehensive Catalog of the Processed Pseudogenes in the Human Genome, Genome Res. 13 (2003) 2541-2558 [414] D. Graur, Y. Shuali and W.H. Li, Deletions in processed pseudogenes accumulate faster in rodents than in humans, J Mol Evol. 28(4) (1989) 279-285 [415] J.T. Weir and D. Schluter, Calibrating the avian molecular clock, Mol Ecol 17 (2008) 2321-2328 [416] W.H. Li, Molecular Evolution, Sinauer Associates, Sunderland, USA, 1997 [417] W.H. Li, T. Gojobori and M. Nei, Pseudogenes as a paradigm of neutral evolution, Nature 292 (1981) 237pp [418] G. Abrusan, H.J. Krambeck, T. Junier, J. Giordano and P.E. Warburton, Biased distributions and decay of LINEs in the chicken genome, Genetics 178(1) (2008) 573-81 [419] C. Esnault, J. Maestre and T. Heidmann, Human LINE retrotransposons generate processed pseudogenes, Nat. Genet. 24 (2000) 363–367 222 References

[420] D. Torrents, M. Suyama, E. Zdobnov and P. Boork, A Genome-Wide Survey of Human Pseudogenes, Genome Res 13 (2003) 2559-2567 [421] H. Nishihara, M. Hasegawa and N. Okada, Pegasoferae, an unexpected mammalian clade revealed by tracking ancient retroposon insertions, PNAS 103(26) (2006) 9929-9934 [422] T. Dagan, R. Sorek, E. Sharon, G. Ast and D. Graur, AluGene: a database of Alu elements incorporated within protein-coding genes, Nuc Acid Res 32 (2004) 489-492 [423] M. R. Shen, M.A. Batzer and P.L. Deininger, Evolution of the Master Alu Gene(s), J Mol Evol 33 (1991) 311-320 [424] www.nsm.uh.edu/~dgraur/molevol/fall2010/slides/7a_transposable_elements.ppt [425] Q. Ji, Z.X. Luo, C.X. Yuan, J.R. Wible, J.P. Zhang and J.A. Georgi, The earliest known eutherian mammal, Nature 416(6883) (2002) 816–822 [426] A. Messer, The maintenance and identification of mouse cerebellar granule cells in monolayer culture, Brain Res 130 (1977) 1-12. [427] M. Leist, P. Ghezzi, G. Grasso, R. Bianchi, P. Villa, M. Fratelli, C. Savino, M. Bianchi, J. Nielsen, J. Gerwien, P. Kallunki, A.K. Larsen, L. Helboe, S. Christensen, L.O. Pedersen, M. Nielsen, L. Torup, T. Sager, A. Sfacteria, S. Erbayraktar, Z. Erbayraktar, N. Gokmen, O. Yilmaz, C. Cerami-Hand, Q.-w. Xie, T. Coleman, A. Cerami, M. Brines, Derivatives of Erythropoietin That Are Tissue Protective But Not Erythropoietic, Science 305 (2004) 239- 242. [428] C.L. Fisher, G.K. Pei, Modification of a PCR-Based Site-Directed Mutagenesis Method, BioTechniques 23 (1997) 570-574. [429] B.D. Gitter, D.L. Czilli, W. Li, D.K. Dieckman, M.H. Bender, J.S. Nissen, T.E. Mabry, T. Yin, L.N. Boggs, D.B. McClure, S.P. Little, E.M. Johnstone, J.E. Audia, P.C. May, P.A. Hyslop, Stereoselective inhibition of amyloid beta peptide secretion by LY450139, a novel functional gamma secretase inhibitor, Neurobiology of Aging 25 (2004) S571-S571. [430] K.M. Walton, K. Fisher, D. Rubitski, M. Marconi, Q.-J. Meng, M. Sladek, J. Adams, M. Bass, R. Chandrasekaran, T. Butler, M. Griffor, F. Rajamohan, M. Serpa, Y. Chen, M. Claffey, M. Hastings, A. Loudon, E. Maywood, J. Ohren, A. Doran, T.T. Wager, Selective Inhibition of Casein Kinase 1 Epsilon Minimally Alters Circadian Clock Period, J Pharmacol Exp Ther (2009) jpet.109.151415. [431] Joshinari, Controlled release of simvastatin acid, Dental Materials J 26 (2007) 451-456. [432] A.L.C. Cardoso, S. de Almeida, J. Pelisek, C. Culmsee, E. Wagner, M.C. Pedroso de Lima, siRNA delivery by a transferrin-associated lipid-based vector: a non-viral strategy to mediate gene silencing, The J of Gene Medicine 9 (2007) 170-183. [433] A. Khvorova, A. Reynolds, S.D. Jayasena, Functional siRNAs and miRNAs Exhibit Strand Bias, Cell 115 (2003) 209-216. [434] A. Khvorova, A. Reynolds, D. Leake, W. Marshall, S. Scaringe (2007) (U.S.P. Office, Ed.), pp. 58, Dharmacon Inc., Lafayette, CO (US), USA. [435] X. Feng, P. Zhao, Y. He, Z. Zuo, Allele-specific silencing of Alzheimer's disease genes: The amyloid precursor protein genes with Swedish or London mutations, Gene 371 (2006) 68-74. [436] S.-C. Kao, A.M. Krichevsky, K.S. Kosik, L.-H. Tsai, BACE1 Suppression by RNA Interference in Primary Cortical Neurons, J. Biol. Chem. 279 (2004) 1942-1949. [437] O. Singer, R.A. Marr, E.M. Rockenstein, L. Crews, N.G. Coufal, F.H. Gage, I.M. Verma, E. Masliah, Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model, Nature Neuroscience 8 (2005) 1343-1349. [438] C. Liu, Y. Li, M. Semenov, C. Han, G.-H. Baeg, Y. Tan, Z. Zhang, X. Lin, X. He, Control of b-Catenin Phosphorylation/Degradation by a Dual-Kinase Mechanism, Cell 108 (2002) 837-847. [439] J. Berger, J. Hauber, R. Hauber, R. Geiger, B.R. Cullen, Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells, Gene 66 (1988) 1-10. [440] E.J. King, A.R. Armstrong, A convenient method for determining serum and bile phosphatase activity, Can Med Assoc J 31 (1934) 376-381. [441] S. Perrot, H. Dutertre-Catella, C. Martin, J.-M. Warnet, P. Rat, A New Nondestructive Cytometric Assay Based on Resazurin Metabolism and an Organ Culture Model for the Assessment of Corneal Viability, Cytometry Part A 55 (2003) 7-14. [442] C. Volbracht, S. Penzkofer, D. Mansson, K.V. Christensen, K. Fog, S. Schildknecht, M. Leist, J. Nielsen, Measurement of cellular β-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems, Analytical Biochemistry 387 (2009) 208-220. [443] B. Zimmer (2008) in Department of Biology, Vol. Dipl. in Biology, pp. 75, University of Konstanz, Konstanz. [444] G. Shaw, S. Morse, M. Ararat, F.L. Graham, Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells, FASEB J. (2002). [445] D. Paris, N. Patel, A. Quadros, M. Linan, P. Bakshi, G. Ait-Ghezala, M. Mullan, Inhibition of Ab production by NF- kB inhibitors, Neuroscience Letters (2006) [446] S.H.Y. Loh, L. Francescut, P. Lingor, M. Bähr, P. Nicotera, Identification of new kinase clusters required for neurite outgrowth and retraction by a loss-of-function RNA interference screen, Cell Death and Differentiation 15 (2009) 283-298. [447] M. Kishi, Y.A. Pan, J.G. Crump, J.R. Sanes, Mammalian SAD Kinases Are Required for Neuronal Polarization, Science 307 (2005) 929-932. [448] H. Youn, M. Jeoung, Y. Koo, H. Ji, W.R. Markesbery, I. Ji, T.H. Ji, Kalirin is under-expressed in Alzheimer's disease hippocampus, J Alzheimers Dis 11 (2007) 385-397. [449] Verma, Biochem Biophys Acta 473 (1977) [450] Perbal, A practical guide to molecular cloning, Wiley & Sons, New York, 1984.

223 References

[451] U. KRUEGER, T. BERGAUER, B. KAUFMANN, I. WOLTER, S. PILK, M. HEIDER-FABIAN, S. KIRCH, C. ARTZ- OPPITZ, M. ISSELHORST, J. KONRAD, Insights into Effective RNAi Gained from Large-Scale siRNA Validation Screening, Oligonucleotides 17 (2007) [452] X. Zhu, R.J. Castellani, A. Takeda, A. Nunomura, C.S. Atwood, G. Perry, M.A. Smith, Differential activation of neuronal ERK, JNK/SAPK and p38 in Alzheimer disease: the `two hit' hypothesis, Mech Age Dev 123 (2001) 39- 46. [453] M.J. Savage, Y.-G. Lin, J.R. Ciallella, D.G. Flood, R.W. Scott, Activation of c-Jun N-Terminal Kinase and p38 in an Alzheimer's Disease Model Is Associated with Amyloid Deposition, J. Neurosci. 22 (2002) 3376-3385. [454] J. Su, Q. Xie, I. Wilson, L. Li, Differential regulation and role of interleukin-1 receptor associated kinase-M in innate immunity signaling, 19 (2007) 1596-601. [455] C. Popovici, M. Leveugle, D. Birnbaum, F. Coulier, Coparalogy: Physical and Functional Clusterings in the Human Genome, BBRC 288 (2001) 362-370. [456] I. Zucchetti, R. De Santis, S. Grusea, P. Pontarotti, L. Du Pasquier, Origin and evolution of the vertebrate leukocyte receptors: the lesson from tunicates, Immunogenetics 61 (2009) 463-481. [457] C.-Y. Chan, M.R. Salabat, X.-Z. Ding, D.L. Kelly, M.S. Talamonti, J.R.H. Bell, T.E. Adrian, Identification and in silico characterization of a novel gene: TPA induced trans-membrane protein, BBRC 329 (2005) 755-764. [458] A.F.A. Smit, R. Hubley and P. Green, unpublished data. Current Version: open-3.3.0 [459] J.D. Thompson, D.G. Higgins, T.J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice, Nuc Acids Res 22(22) (1994) 4673-4680 [460] S.F. Altschul, W. Gish, W. Miller, E.W. Myers and D.J. Lipman, Basic local alignment search tool, J Mol Biol 215 (1990) 403-10. [461] D. Zheng, A. Frankish, R. Baertsch, P. Kapranov, A. Reymond, S.W. Choo, Y. Lu, F. Denoeud, S.E. Antonarakis, M. Snyder, Y. Ruan, C-L. Wei, T.R. Gingeras, R. Guigo, J. Harrow and M.B. Gerstein, Pseudogenes in the ENCODE Regions: Consensus Annotation, Analysis of Transcription and Evolution, Genome Res 17(6) (2007) 839-851. [462] C. Karreman, AiO, combining DNA/protein programs and oligo-management, Bioinformatics 18(6) (2002) 884-885 [463] C. Koike, M. Uddin, D.E. Wildman, E.A. Gray, M. Trucco, T.E. Starzl and M. Goodman, Functionally important glycosyltransferase gain and loss during catarrhine primate emergence, PNAS 104(2) (2007) 559-564. [464] B. Venkatesh, E.F. Kirkness, Y-H. Loh, A.L. Halpern, A.P. Lee, J. Johnson, N. Dandona, L.D. Viswanathan, A. Tay, J.C. Venter, R.L. Strausberg, S. Brenner, Ancient Noncoding Elements Conserved in the Human Genome, Science 314 (2007) 1892. [465] B. Venkatesh, E.F. Kirkness, Y-H. Loh, A.L. Halpern, A.P. Lee, J. Johnson, N. Dandona, L.D. Viswanathan, A. Tay, J.C. Venter, R.L. Strausberg, S. Brenner, Survey Sequencing and Comparative Analysis of the Elephant Shark (Callorhinchus milii) Genome, PLoS Biol 5(4) (2007) 932-944. [466] X. Messeguer, R. Escudero, D. Farré, O. Nuñez, J. Martínez, M.M. Albà, PROMO: detection of known transcription regulatory elements using species-tailored searches, Bioinformatics, 18(2) (2002) 333-334. [467] D. Farré, R. Roset, M. Huerta, J.E. Adsuara, L. Roselló, M.M. Albà, X. Messeguer, Identification of patterns in biological sequences at the ALGGEN server: PROMO and MALGEN. Nuc Acids Res, 31(13) (2003) 3651-3653. [468] K.A. Lord, B. Hoffman-Liebermann, D.A. Liebermann, Nucleotide sequence and expression of a cDNA encoding MyD88, a novel myeloid differentiation primary response gene induced by IL6, Oncogene 5(7) (1990) 1095-1097 [469] M. Kasahara, K. Naruse, S. Sasaki, Y. Nakatani, W. Qu, B. Ahsan, T. Yamada, Y. Nagayasu, K. Doi, Y. Kasai, T. Jindo, D. Kobayashi, A. Shimada, A. Toyoda, Y. Kuroki, A. Fujiyama, T. Sasaki, A. Shimizu, S. Asakawa, N. Shimizu, S-i. Hashimoto, J. Yang, Y. Lee, K. Matsushima, S. Sugano, M. Sakaizumi, T. Narita, K. Ohishi, S. Haga, F. Ohta, H. Nomoto, K. Nogata, T. Morishita, T. Endo, T. Shin-I, H. Takeda, S. Morishita and Y. Kohara, The medaka draft genome and insights into vertebrate genome evolution, Nature Let 447 (2007) 714-719 [470] Y. Nakatani, H. Takeda, Y. Kohara, et al., Reconstruction of the vertebrate ancestral genome reveals dynamic genome reorganization in early vertebrates, Genome Res 17 (2007) 1254-1265 [471] H.A. Schmidt, K. Strimmer, M. Vingron and A. von Haeseler, TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing, Bioinformatics, 18 (2002) 502-504. [472] S. Guindon, J.F. Dufayard, V. Lefort, M. Anisimova, W. Hordijk, O. Gascuel, New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0, Systematic Biology 59(3) (2010) 307-21 [473] H. Qing, W. Zhou, M.A. Christensen, X. Sun, Y. Tong, W. Song, Degradation of BACE by the ubiquitin- proteasome pathway, FASEB J. (2004) [474] X.-M. Ma, Y. Wang, F. Ferraro, R.E. Mains, B.A. Eipper, Kalirin-7 Is an Essential Component of both Shaft and Spine Excitatory Synapses in Hippocampal Interneurons, J. Neurosci. 28 (2008) 711-724. [475] J. Zhou, C.Y. Liu, S.H. Back, R.L. Clark, D. Peisach, Z. Xu, R.J. Kaufman, The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response, PNAS 103 (2006) 14343-8. [476] A.Y. Hung, C. Haass, R.M. Nitsch, W.Q. Qiu, M. Citron, R.J. Wurtman, J.H. Growdon, D.J. Selkoe, Activation of protein kinase C inhibits cellular production of the amyloid beta-protein, J. Biol. Chem. 268 (1993) 22959-22962. [477] K. HUPPI, S.E. MARTIN, N.J. CAPLEN, Defining and assaying RNAi in mammalian cells, Mol Cell 17 (2005) 1-10. [478] P.E. Visconti, Z. Hao, M.A. Purdon, P. Stein, B.R. Balsara, J.R. Testa, J.C. Herr, S.B. Moss, G.S. Kopf, Cloning and chromosomal localization of a gene encoding a novel serine/threonine kinase belonging to the subfamily of testis specific kinases, Genomics 77 (2001) 163-170.

224 References

[479] Z. Hao, K.N. Jha, Y.-H. Kim, S. Vemuganti, V.A. Westbrook, O. Chertihin, K. Markgraf, C.J. Flickinger, M. Coppola, J.C. Herr, P.E. Visconti, Expression analysis of the human testis-specific serine/threonine kinase (TSSK) homologues. A TSSK member is present in the equatorial segment of human sperm, Mol Hum Reprod 10 (2004) 433-444. [480] R.I. Skotheim, K.S. Korkmaz, T.I. Klokk, V.M. Abeler, C.G. Korkmaz, J.M. Nesland, S.D. Fossa, R.A. Lothe, F. Saatcioglu, NKX3.1 Expression Is Lost in Testicular Germ Cell Tumors, Am J Pathol 163 (2003) 2149-2154. [481] B. Xu, Z. Hao, K.N. Jha, Z. Zhang, C. Urekar, L. Digilio, S. Pulido, J. Strauss III, F.C.J. Flickinger, J.C. Herr, Targeted deletion of Tssk1 and 2 causes male infertility due to haploinsufficiency, Develop Biol 319 (2008) 211- 222. [482] Y.C. YAN, L.F. WANG, S.S. KOIDE, Properties of a monoclonal antibody interacting with human sperm, Arch Androl 18 (1987) [483] Y.C. YAN, Characterization of cDNA encoding a human sperm membrane protein related to A4 amyloid protein, PNAS 87 (1990) 2504-8. [484] G. VANAGE, Infertility induced in rats by immunization with synthetic peptide segments of a sperm protein, Biochem Biophys Res Com 183 (1992). [485] X.Y. TIAN, Extracellular domain of YWK-II, a human spermtransmembrane protein, interacts with rat Mullerian- inhibiting substance, Reproduction 121 (2001) 873-880. [486] P. HUANG, Expression and characterization of the human YWK-II gene, encoding a sperm membrane protein related to the alzheimer betaA4-amyloid precursorprotein, Mol Hum Reprod 6 (2000). [487] D.G. Wansink, W. Peters, I. Schaafsma, R.P. Sutmuller, F. Oerlemans, G.J. Adema, B. Wieringa, C.E. van der Zee, W. Hendriks, Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity, Physiol Genomics 19 (2004) 50-60. [488] M. Dörner and S. Pääbo, Nucleotide Sequence of a Marsupial LINE-l Element and the Evolution of Placental Mammals, Mol Biol Evol 12(5) (1995) 944-948 [489] V. Moucadel, F. Lopez, T. Ara, P. Benech and D. Gautheret, Beyond the 3′ end: experimental validation of extended transcript isoforms, Nuc Acids Res 35(6) (2007) 1947–1957. [490] www.lifl.fr/~touzet/Arena/gautheret05.ppt [491] L. Barella, M. Loetscher, A. Tobler, M. Baggiolini and B. Moser, Sequence variation of a novel heptahelical leucocyte receptor through alternative transcript formation, Biochem J 309(3) (1995) 773-779. [492] M.D. Gunn,V.N. Ngo,K.M. Ansel,E.H. Ekland, J.G. Cyster and L.T. Williams,A B-cell-homing chemokine made in lymphoid follicles activates Burkitt's lymphoma receptor-1, Nature 391(6669) (1998) 799-803. [493] D.F. Legler, M. Loetscher, R.S. Roos, I. Clark-Lewis and M. Baggiolini, B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/CXCR5, J Exp Med 187(4) (1998) 655-660. [494] P.J. Talmud, F. Drenos, S. Shah, T. Shah, J. Palmen, C. Verzilli, T.R. Gaunt, J. Pallas, R. Lovering, K. Li, J.P. Casas, R. Sofat, M. Kumari, S. Rodriguez, T. Johnson, S.J. Newhouse, A. Dominiczak, N.J. Samani, M. Caulfield, P. Sever, A. Stanton, D.C. Shields, on behalf of the ASCOT investigators, S. Padmanabhan, O. Melander, C. Hastie, C. Delles, on behalf of the NORDIL investigators, S. Ebrahim, M.G. Marmot, G.D. Smith, D.A. Lawlor, P.B. Munroe, for the BRIGHT Consortium, I.N. Day, M. Kivimaki, J. Whittaker, S.E. Humphries and A.D. Hingorani, Gene-centric Association Signals for Lipids and Apolipoproteins Identified via the HumanCVD BeadChip, Am J Hum Genet 85(5) (2009) 628-642. [495] C.P. El Haibi, P.K. Sharma, R. Singh, P.R. Johnson, J. Suttles, S. Singh and J.W. Lillard Jr., PI3Kp110-, Src-, FAK-dependent and DOCK2-independent migration and invasion of CXCL13-stimulated prostate cancer cells, Mol Cancer 9 (2010) 85. [496] S. Singh, R. Singh, U.P. Singh, S.N. Rai, K.R. Novakovic, L.W. Chung, P.J. Didier, W.E. Grizzle and J.W. Lillard Jr., Clinical and biological significance of CXCR5 expressed by prostate cancer specimens and cell lines, Int J Cancer 125(10) (2009) 2288-2295. [497] T. Dobner, I. Wolf, T. Emrich and M. Lipp, Differentiation-specific expression of a novel -coupled receptor from Burkitt's lymphoma, Eur J Immunol 22(11) (1992) 2795-2799. [498] H.T. Lee, Y.M. Shiao, T.H. Wu, W.S. Chen, Y.H. Hsu, S.F. Tsai and C.Y. Tsai, Serum BLC/CXCL13 concentrations and renal expression of CXCL13/CXCR5 in patients with systemic lupus erythematosus and lupus nephritis, J Rheumatol 37(1) (2010) 45-52. [499] H. Wei, R. Sun, W. Xiao, J. Feng, C. Zhen, X. Xu and Z. Tian, Type Two Cytokines Predominance of Human Lung Cancer and Its Reverse by Traditional Chinese Medicine TTMP, Cell Mol Immunol 1(1) (2004) 63-69.

225 Acknowledgements

15 Acknowledgements I want to thank Prof. Marcel Leist for countless excellent scientific and presentation-related advice with the interesting main project and for giving me the possibility to finish the side project(s). Prof. Alexander Bürkle and Prof. Daniel Dietrich for helpful advice in the thesis committee meetings and Prof. Dietrich especially for revision of the thesis manuscript.

Dr. Stefan Schildknecht for various support, especially with the publication. Dr. Yana Chernishova for various support, especially with the kinome screen. Dr. Christiaan Karreman, Dr. Suzanne Kadereit, Diana Scholz, Florian Matt, Brigitte Schanze, Philipp Kügler, Dr. Anja Henn, Bastian Zimmer, Bettina Baumann, Dr. Regina Pape, Marion Kapitza, Bettina Schimmelpfennig, Alberto Salvo-Vargas for various support, discussions and fun in lab, office and seminar room. Prof. Dr. Tancred Frickey for alignment of chromosomal regions. Marina Lehmann, Daniel Nagel, Maria Buske, Nina Blatter, Manuela Gfell, Benedikt Klauser, Harald Schuwerk for valuable assistance.

Dr. Christiane Volbracht, Dr. Jacob Nielsen, Dr. Kenneth Vielsted Christensen, Dr. Karina Fog, Agnete Kirkeby from Lundbeck and Prof. Pierluigi Nicotera from DZNE for precious material.

Prof. Albrecht Wendel, Prof. Marcus Gröttrup, Prof. Christof Hauck, Prof. Klaus-Peter Schäfer, Prof. Thomas Hartung, PD Jutta Schlepper-Schäfer, PD Sonja von Aulock, PD Corinna Hermann, Dr. Mardas Daneshian, Dr. Mathias Schmidt, Dr. Stefan Röpcke, Josepha Ittner, Dr. Daniel Feuerstein, Dr. Christine Hoffmann, Dr. Tobias Speicher, Dr. Christopher Schliehe, Dr. Jens Selige, Dr. Andrea Kunzmann, Dr. Christine Strasser, Dr. Isabelle Pochic, Dr. Stefanie Sigel, Dr. Aswin Mangerich, Dr. Tobias Eltze, and the other IRTG1331 members for various support, discussions and fun on tour.

Klaus Hermann, Birgitt Planitz, Andrea Schurhammer, Heidi Henseleit, Dr. Gerald Mende, PD Dieter Schopper from the TFA; Oliver Bahm, Simone Duda, Jörg-Uwe Kunze, Andrea Neuwihler, Armin Schauren from the chemical store; Prof. Jörg Hartig, Dr. Markus Wieland, Dr. Armin Benz, Astrid Joachimi from the Hartig group; Gabriele Sims, Miriam Lienhard, Petra Keller, Patricia Deicher from the procurement; Georg Ritzi, Peter Nietsch, Peter Romer, Ekkehard Moser, Adrian Müller, Guido Gius, Roger Koslowski, Heinz-Günter Möllenhoff from the mechanicians and electricians unit; Klaus Schrodin, Georg Wehowsky, Björn Thomar, Klaus Krasny, Frank Teuchert, Harald Deicher from the facility management; Prof. Elisabeth Gross, Stefan Florian, Ulrike Beck, Elvira Weber, Dr. Birte Kalveram, Dr. Diane Egger-Adam, Ms Varga, Mr von Bodman from the department of biology; Ms Matzner from the thesis exams office and others from the University of Konstanz for their cooperativeness.

Prof. Jim Langeland from the Kalamazoo College, Prof. Hans Robert Kalbitzer from the University of Regensburg, Prof. Byrappa Venkatesh from IMCB, Dr. Karl Frei from the Inn-Salzach-Klinikum; Dr. Peter Oehlen from Nycomed, Dr. Marc Rawer from Qiagen, Sylvia Lang from Integra, Andreas Brugger from VWR.

Alfred especially for the laptop, André, Anne, Chris, Claus, Cornelius, Cordu, David, Doreen, Erik, Evi, Flo, Hafdiz, Helmut, Jochen, Judith, Julian, Juliane, Jürgen, Karén, Karin, Linda, Lisa, Manu, Markus, Martin, Matthias, Max, Micha, Michael, Miriam, Peter, Phillip, Romy, Rüdiger, Sandra, Thorsten, Tom, Ulli, Vera, Zsom, my relatives, especially my parents, grandparents and aunts, the www, the forest, the sun, the lake for emotional support. 226 Some parts of this thesis have already been published:

Poster: Identification of BACE1- and APP-regulating kinases Stephan Penzkofer*, Christiane Volbracht&, Karina Fog&, Kenneth Vielsted& and Marcel Leist*! *:Doerenkamp-Zbinden Chair for alternative in vitro methods, University of Konstanz, Konstanz, Germany; !:corresponding author &:H. Lundbeck A/S, Valby, Denmark Presented at PENS Summer School: “Novel molecular strategies to treat neurodegenerative diseases”, Ofir, Portugal, 7. July 2007

Christiane Volbracht, Stephan Penzkofer, David Mansson, Kenneth Vielsted Christensen, Karina Fog, Stefan Schildknecht, Marcel Leist, Jacob Nielsen, Measurement of cellular β-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems, Analytical Biochemistry 387 (2009) 208-220.

Curriculum vitae of Stephan Penzkofer

06.06.1978 born in Munich, Germany 09/1984 – 06/1998 attendance of schools in Munich and Erding 07/1998 – 04/1999 compulsory military service 05/1999 – 09/1999 newspaper catering and car rental at the Munich Airport 10/1999 – 09/2000 studies in civil engineering at the Technical University of Munich

10/2000 – 09/2002 basic study period in biology at the University of Regensburg, Germany, with intermediate examinations in physics (oral), chemistry (oral) and biology (written; best result of the semester) 10/2002 – 03/2004 laboratory research courses in genetics, organic chemistry, biophysics and biochemistry followed by diploma examinations in organic chemistry, biochemistry and biophysics 04/2004 – 04/2005 diploma thesis with the Institute of Biophysics and physical Biochemistry at the University of Regensburg: “Characterisation of the PDZ2-PIP-Interaction with NMR-titration studies”

10/2005 – 05/2006 research attachment in cell culture, proteomics with mass spectrometry, capillary electrophoresis and flow cytometry with the Institute of Bioengineering and Nanotechnology, Biopolis, Singapore

09/2006 – 09/2011 doctoral thesis with the Doerenkamp-Zbinden-Chair of alternative in vitro methods at the University of Konstanz, Germany: “Screen for kinases affecting amyloidogenic cleavage by BACE1”