Trehalose-Mediated Enhancement of Glycosaminoglycan Degradation in the Lysosomal Storage Disorder Mucopolysaccharidosis III

Home , Enzyme Commission number, Trehalase

Aus dem Institut für Humangenetik der Universität zu Köln Direktorin: Frau Universitätsprofessor Dr. rer. nat. B. Wirth

Trehalose-mediated enhancement of glycosaminoglycan degradation in the lysosomal storage disorder Mucopolysaccharidosis III

Trehalose vermittelte Steigerung des Glykosaminoglykan-Abbaus in der lysosomalen Speichererkrankung Mukopolysaccharidose III

Inaugural-Dissertation zur Erlangung der Doktorwürde der Hohen Medizinischen Fakultät der Universität zu Köln

vorgelegt von Victor Mauri aus Stuttgart

promoviert am 29. Januar 2014

Gedruckt mit Genehmigung der Medizinischen Fakultät der Universität zu Köln, 2014

Dekan: Universitätsprofessor Dr. med. Dr. h.c. Th. Krieg

1. Berichterstatterin: Frau Universitätsprofessor Dr. rer. nat. B. Wirth

2. Berichterstatter: Professor Dr. rer. nat. F.-G. Hanisch

Erklärung

Ich erkläre hiermit, dass ich die vorliegende Dissertationsschrift ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht.

Bei der Auswahl und Auswertung des Materials sowie bei der Herstellung des Manuskriptes habe ich Unterstützungsleistungen von folgenden Personen erhalten:

Univ.-Prof. Dr. rer. nat. Brunhilde Wirth Marco Sardiello, PhD, Assistant Professor BCM Christian Schaaf, MD, PhD, Assistant Professor BCM

Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit nicht beteiligt. Insbesondere habe ich nicht die Hilfe einer Promotionsberaterin/eines Promotionsberaters in Anspruch genommen. Dritte haben von mir weder unmittelbar noch mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertationsschrift stehen.

Die Dissertationsschrift wurde von mir bisher weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.

Köln, den 19. August 2013 ______Unterschrift

Die in dieser Arbeit präsentierten Untersuchungen wurden zwischen März 2011 und Oktober 2011 im Department of Human and Molecular Genetics des Baylor College of Medicine am Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, Texas, USA, in der Arbeitsgruppe von Assistant Professor Marco Sardiello, PhD, durchgeführt.

Die Mehrzahl der dieser Arbeit zugrunde liegenden Experimente sind nach entsprechender Anleitung durch Herrn Dr. Marco Sardiello von mir selbstständig durchgeführt worden.

Bei den Cloning-Experimenten habe ich Unterstützung von Herrn Dr. Alberto di Ronza bekommen.

Das Experiment zur zeitlichen Linearität des NAG-Assays sowie die Experimente zur Z‘-Faktor-Berechnung wurden von Frau Dr. Parisa Lotfi durchgeführt.

Bei der Messung der intestinalen Trehalase-Aktivität bin ich von der labor- technischen Assistentin Frau Sanagasetti unterstützt worden.

Acknowledgment

This thesis would not have been possible without the help, the guidance and the encouragement of several persons who in one way or another contributed to the completion of the studies and the preparation of this thesis.

First, I wish to express my sincere gratitude to Dr. Marco Sardiello, my principal investigator and supervisor at Baylor College of Medicine, for giving me the opportunity to work in his lab on this highly interesting project, for being an excellent scientific mentor, for sharing his great knowledge, curiosity and experience, for valuable discussions, encouragement and for many good moments inside and outside the lab.

I also owe my deepest gratitude to Professor Brunhilde Wirth, director of the Institute of Human Genetics and my supervisor in Köln, Germany, for her helpfulness and great support to make this German-American thesis happen, for her time, for valuable conversations and suggestions while writing the manuscript. Without her support it would not have been possible to write in Germany about the experiments performed in Houston.

Many thanks go also to the other members of the Sardiello-Lab for providing me such a stimulating and friendly working environment, Dr. Alberto di Ronza and Dr. Michela Palmieri for teaching me basic and not so basic laboratory techniques and for their endless help with almost endless problems. Dr. Parisa Lotfi for performing experiments during the review process of our paper. Deepthi Sanagasetti for technical support.

I thank Alberto di Ronza, not just for being a great teacher and lab mate and for his helpfulness at any time, but mostly for his friendship, for being such an invaluable friend inside and outside the lab.

I would like to thank Dr. Christian Schaaf for his constant support and help during my time in the USA, for initially introducing me to Dr. Sardiello, for his invaluable feedback on the manuscript, for his great book “Mit Vollgas zum Doktor” and for his advice on scientific and personal issues.

I would also like to thank the Team Sanfilippo Foundation as well as the Beyond Batten Disease Foundation for their generous funding of this and many other projects to eventually help children with Sanfilippo Syndrome, Batten Disease and other rare, but devastating diseases.

I also owe my deepest gratitude to my parents and my sister, for their everlasting support and encouragement, their confidence in me and my ideas, and for everything they taught me, which seems to be an excellent foundation for life.

Last, but not least, I owe my warmest thanks to Isabel, my “better half”. Thank you for your support at any time and your love!

Table of contents

Table of contents ...... I

Glossary ...... VI

1 INTRODUCTION ...... 1

1.1. Lysosomes and Lysosomal Storage Disorders ...... 1 1.1.1. The lysosome ...... 1 1.1.2. TFEB and the CLEAR network...... 3 1.1.3. Lysosomal storage disorders ...... 5 1.2. Mucopolysaccharidosis III/Sanfilippo Syndrome ...... 11 1.2.1. The mucopolysaccharidoses ...... 11 1.2.2. MPSIII: history, biochemistry and genetic aspects ...... 13 1.2.3. MPSIII: epidemiology, clinical presentation and diagnosis ...... 15 1.2.3.1. Epidemiology ...... 15 1.2.3.2. Clinical picture ...... 15 1.2.3.3. Diagnosis ...... 16 1.2.4. Therapeutic strategies for MPS III (and other LSDs) ...... 17 1.2.4.1. Enzyme replacement therapy ...... 17 1.2.4.2. Gene therapy ...... 18 1.2.4.3. Cell-based therapy ...... 18 1.2.4.4. Substrate reduction therapy ...... 19 1.2.4.5. Pharmaceutical chaperon therapy (PCT) ...... 19 1.2.4.6. Stop codon read-through (SCRT) ...... 20 1.3. Trehalose ...... 20 1.3.1. Chemical properties and natural occurrence ...... 20 1.3.2. Metabolism ...... 21 1.3.3. Safety and toxicity ...... 22 1.3.4. Functions of trehalose ...... 22 1.3.4.1. Protection from various stress conditions ...... 22 1.3.4.2. Induction of autophagy and degradation of macromolecules ...... 24

Table of contents

2 AIMS ...... 26

3 MATERIAL AND METHODS ...... 28

3.1. Cell lines ...... 28 3.1.1. Fibroblast cell lines derived from MPS III patients and control subjects 28 3.1.2. HEK 293T and HeLa cells ...... 29 3.2. Equipment, Chemicals, Reagents and Kits ...... 29 3.2.1. Equipment ...... 29 3.2.2. Mouse dissection equipment ...... 30 3.2.3. Chemicals ...... 30 3.2.4. Reagents and other materials ...... 31 3.2.5. Kits ...... 32 3.3. Solutions and Media ...... 32 3.3.1. Frequently used buffers and solutions ...... 32 3.3.2. Cell culture media ...... 34 3.4. Primers ...... 35

3.5. Software, online tools and databases ...... 37

3.6. Eukaryotic cell culture procedures ...... 38 3.6.1. Cell culture of primary fibroblasts ...... 38 3.6.2. Cell culture of HeLa cells ...... 38 3.6.3. Cell culture of HEK 293T cells ...... 39 3.6.4. Treatment of fibroblast cell lines with chemical substances ...... 39 3.7. DNA techniques ...... 39 3.7.1. Determination of DNA concentration ...... 39 3.7.2. Agarose gel electrophoresis ...... 40 3.7.3. Extraction and purification of DNA from agarose gels ...... 40 3.7.4. Polymerase chain reaction (PCR) ...... 41 3.7.5. Gene expression analysis by quantitative real time PCR ...... 42 3.8. RNA techniques ...... 43 3.8.1. Isolation of total RNA from cells and tissues ...... 43 3.8.2. Determination of RNA concentration ...... 43 3.8.3. Reverse transcription of RNA into cDNA ...... 43

Table of contents

3.9. Cloning procedures and retrovirus preparation ...... 44 3.9.1. TOPO Cloning ...... 45 3.9.2. Transformation ...... 46 3.9.3. Mini preparation of plasmids ...... 47 3.9.4. Enzymatic restriction digestion ...... 47 3.9.5. Identification of correct clones and sequencing ...... 48 3.9.6. Midi preparation of plasmids ...... 48 3.9.7. Ligation and cloning into the pMSCVneo vector ...... 49 3.9.8. Retrovirus construction and transduction of primary fibroblasts ...... 49 3.10. Other molecular biology methods ...... 51 3.10.1. Cell Imaging ...... 51 3.10.1.1. Immunofluorescence ...... 51 3.10.1.2. Confocal microscopy ...... 52 3.10.2. Protein quantification ...... 52 3.11. Working with mice...... 52 3.11.1. Mouse strains and housing conditions ...... 52 3.11.2. Preparation of mouse tissues ...... 53 3.12. Specific methods ...... 53 3.12.1. Determination of glycosaminoglycans in cells ...... 53 3.12.1.1. Cell culture and treatment of fibroblasts ...... 53 3.12.1.2. Extraction of glycosaminoglycans from cells...... 54 3.12.1.3. Measurement of glycosaminoglycans ...... 54 3.12.1.4. Normalization on DNA content ...... 55 3.12.1.5. Calculation of GAG-levels ...... 56 3.12.2. Determination of N-acetylglucosaminidase activity ...... 56 3.12.3. Determination of trehalose in cells and tissues ...... 58 3.12.3.1. Sample preparation of cells ...... 58 3.12.3.2. Sample preparation of mice tissues and removal of free glucose ...... 58 3.12.3.3. Trehalose assay ...... 59 3.12.3.4. Calculation of trehalose levels ...... 59 3.12.4. Determination of murine intestinal trehalase activity ...... 60 3.12.5. Mutation mapping and energetic analysis ...... 62 3.13. Statistical methods...... 63

III

Table of contents

4 RESULTS ...... 64

4.1. Intracellular measurement of trehalose ...... 64

4.2. Determination of intracellular HS levels ...... 65 4.2.1. GAG levels in MPS III patient-derived fibroblasts and WT fibroblasts ... 65 4.2.2. GAG levels under trehalose treatment ...... 67 4.3. GAG levels in trehalose-synthesizing MPS IIIB fibroblasts ...... 73 4.3.1. Cloning of Tps1 into the pMSCVneo vector ...... 73 4.3.1.1. Addition of restriction overhangs and subcloning of Tps1 into pCR-Blunt II-TOPO ...... 74 4.3.1.2. Insertion of Tps1 into the pMSCVneo destination vector ...... 75 4.3.2. Retroviral transduction of fibroblasts with Tps1 ...... 76 4.3.3. GAG levels in trehalose-synthesizing MPS IIIB fibroblasts ...... 77 4.4. Subcellular location of TFEB under trehalose treatment ...... 79

4.5. Expression profile of lysosomal genes in MPS IIIB fibroblasts ...... 81

4.6. N-acetylglucosaminidase activity ...... 88 4.6.1. Development and test of a N-acetylglucosaminidase assay ...... 88 4.6.1.1. Experimental set-up ...... 89 4.6.1.2. NAG activity in MPS IIIB fibroblasts and WT cells ...... 90 4.6.1.3. Assessment of sensitivity: lysosomal enhancement by sucrose treatment ...... 91 4.6.1.4. Assessment of sensitivity: lysosomal enhancement by TFEB overexpression ...... 92 4.6.1.5. Assessment of reproducibility ...... 93 4.6.2. Behavior of fibroblasts under trehalose treatment ...... 94 4.7. Mutation mapping and energetic analysis ...... 95

4.8. Trehalose metabolism in mice ...... 100 4.8.1. Intestinal trehalase activity ...... 100 4.8.2. Expression of Treh ...... 101 4.8.3. Trehalose concentrations in liver and brain ...... 102 4.8.3.1. Trehalose concentrations in livers and brains over time ...... 102 4.8.3.2. Trehalose concentrations in liver under additional treatment with the trehalase inhibitor Validamycin A...... 103 4.8.3.3. Trehalose concentrations in mouse chow ...... 105 4.9. Summary of results ...... 106

Table of contents

5 DISCUSSION ...... 107

5.1. Trehalose can permeate human cell membranes ...... 107

5.2. Trehalose reduces HS levels in MPS III patient-derived fibroblasts .. 108

5.3. HS levels are reduced in MPS IIIB fibroblasts expressing Tps1 ...... 110

5.4. Trehalose induces nuclear translocation of TFEB ...... 111

5.5. Induction of lysosomal genes under trehalose treatment ...... 112

5.6. N-acetylglucosaminidase activity ...... 114 5.6.1. Development and validation of a N-acetylglucosaminidase assay ...... 115 5.6.2. Effects of trehalose on N-acetylglucosaminidase activity in WT and MPS IIIB fibroblasts ...... 119 5.7. Mutation mapping and energetic analysis ...... 120

5.8. Trehalose metabolism in mice ...... 122

5.9. Conclusion ...... 125

5.10. Future directions ...... 126

6 Summary ...... 128

7 Zusammenfassung ...... 130

8 References ...... 132

9 Preliminary Publications ...... IX

10 Appendix ...... X

11 Lebenslauf ...... XV

Glossary

% percent °C degree Celsius µg microgram µl microliter µM micromolar 3D three-dimensional 4MU 4–Methylumbelliferyl AD Alzheimer disease ATP adenosine triphosphate avg average BBB blood-brain barrier BCM Baylor College of Medicine bp base pairs BSA bovine serum albumin c clone cDNA complementary DNA CLEAR Coordinated Lysosomal Expression and Regulation CLN Neuronal ceroid lipofuscinosis cm centimeter CNS central nervous system CS chondroitin sulfate CSF cerebrospinal fluid d days DMB dimethylmethylene blue DMEM Dulbecco’s modified Eagle medium DMSO dimethylsulfoxide DNA deoxyribonucleic acid dNTP deoxynucleotriphosphate DS dermatan sulfate e. g. exempli gratia EC Enzyme Commission number EDTA ethylenediaminetetraacetic acid ER endoplasmic reticulum ERT enzyme replacement therapy et al. et alii FBS fetal bovine serum Fig. Figure FU fluorescence units Fwd forward g gram G6P glucose-6-phosphate G6PDH glucose-6-phosphate dehydrogenase

Glossary

GAG glycosaminoglycan GT gene therapy h hours HCT haematopoietic cell transplantation HD Huntington disease homoz homozygous HS heparan sulfate HTS high-throughput screening i. e. id est ICC immunocytochemistry IV intravenous kb kilobases kDa kilodalton KS keratan sulfate L Liter

LD50 median lethal dose LSD lysosomal storage disorder M Molar M6P mannose-6-phosphate mg milligram min minutes ml milliliter mm millimeter mM millimolar mmol millimol MPR mannose-6-phosphate receptor MPS mucopolysaccharidosis mRNA messenger RNA MSCV murine stem cell virus mTOR mammalian target of rapamycin MUG 4MU-alpha-N-acetyl-D-glucosaminide n. d. not determined n. s. not significant NAD nicotinamide adenine dinucleotide NAG N-acetylglucosaminidase (protein) NAGLU N-acetylglucosaminidase (gene) ng nanogram nM nanomolar nm nanometer nmol nanomol NRI Neurological Research Institute NSF N-ethylmaleimide-sensitive factor OMIM Online Mendelian Inheritance in Man PBS phosphate-buffered saline

VII

Glossary

PCR polymerase chain reaction PCT pharmaceutical chaperon therapy PD Parkinson disease pH power of hydrogen pmol picomol qPCR quantitative real time PCR Rev reverse RNA ribonucleic acid rpm revolutions per minute s second SCRT stop codon read-through SD standard deviation SEM standard error of the mean SNARE soluble NSF attachment protein receptor T 100 treatment with 100 mM trehalose TBE buffers TE buffers TFEB transcription factor EB TGBN Telethon Genetic Biobank Network TGN trans-Golgi network TIGEM Telethon Institute for Genetics and Medicine Tm temperature of melting Tps1 trehalose-6-phosphate synthase 1 U Units UDP uridine diphosphate UT untreated UV ultraviolet V Volt VSV Vesicular stomatitis virus w week w/o without WT wild-type

VIII

Introduction

1 INTRODUCTION The following introduction is subdivided into three parts. The first part (chapter 1.1) starts with an overview of lysosomal functions and recent discoveries in this field. The section will then give an introduction to the concept of lysosomal storage disorders (LSDs).

The second part (chapter 1.2) will focus on the mucopolysaccharidoses (MPS) and more specifically on MPS III/Sanfilippo syndrome, on which is the main focus of this work. The section will highlight biological and clinical aspects of MPS III and close with a brief overview of potential therapeutic strategies for Sanfilippo syndrome and other LSDs.

Finally, the various intriguing properties of the disaccharide trehalose and its involvement as a protectant against various stress conditions will be outlined in the third part of this introduction (chapter 1.3).

1.1. Lysosomes and Lysosomal Storage Disorders 1.1.1. The lysosome Lysosomes, described for the first time by De Duve in 1955 (De Duve et al., 1955), are ubiquitous intracellular organelles whose primary function is the degradation and recycling of macromolecules from secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways (Luzio et al., 2007).

The lysosome is the cell’s main digestive compartment for the degradation of a wide variety of structurally diverse substances, such as proteins, glycosaminoglycans (GAGs), nucleic acids, oligosaccharides, and complex lipids. They are part of the highly dynamic endosome/lysosome system which mediates the internalization, recycling, transport and breakdown of cellular and extracellular components (Lüllmann-Rauch, 2005). An overview of the lysosome/endosome system is provided in Figure 1.

Currently, about 100 proteins have been localized to the lysosome, including hydrolases, soluble accessory proteins and lysosomal membrane proteins. Lysosomes are delimited by a single layer phospholipid membrane which separates

Introduction them from the cytosol and prevents unwanted proteolytic damage to the surroundings. An acidic internal pH of 4.5 – 5 is necessary for the optimal function of various catabolic enzymes and maintained by an ATP-dependent proton pump. Within the lysosome/endosome system there is a pH gradient, with the lysosomes being the most acidic subcompartment (Lübke et al., 2009, Schröder et al., 2010).

Figure 1: The endosomal/lysosomal system The endosomal/lysosomal system mediates the internalization, recycling, transport and breakdown of cellular and extracellular components. There are discrete compartments within this highly dynamic system. Early endosomes are part of the main receptor-recycling pathway, whereas late endosomes are involved in the breakdown of internalized cargo and delivery of molecules to the trans-Golgi network. Intracellular components are sequestered by autophagosomes and fuse with lysosomes giving rise to autolysosomes. (modified from Jeyakumar et al., 2005, Schultz et al., 2011).

Lysosomal proteins are synthesized at the rough endoplasmatic reticulum (ER), modified in the Golgi apparatus and subsequently targeted to lysosomes in a signal- dependent manner. The vast majority of lysosomal hydrolases are imported into lysosomes by a mannose-6-phosphate (M6P) trafficking pathway. In the trans-Golgi

Introduction network (TGN) M6P-residues are recognized and bound to M6P-receptors (MPR) that cycle between the TGN and endosomes. Bound lysosomal hydrolases dissociate from MPRs triggered by the internal acidic pH of endosomes and recycle to the TGN (Lüllmann-Rauch, 2005, Saftig and Klumperman, 2009, Coutinho et al., 2012b). The process of enzyme transfer from late endosomes to lysosomes is not yet fully understood, although several hypotheses exist (Luzio et al., 2007). Lysosomes can be distinguished from endosomes by the lack of MPRs. Several other luminal lysosomal proteins are transported to lysosomes by alternative sorting mechanisms such as the lysosomal integral membrane protein 2 (LIMP2) or sortilin (Lefrancois et al., 2003, Reczek et al., 2007, Coutinho et al., 2012c).

Intracellular components are degraded in lysosomes by autophagy. In this process, complete regions of the cytoplasm, including polyubiquinated proteins, protein aggregates and entire dysfunctional organelles are surrounded by a membrane and form an autophagosome. Autophagosomes fuse with lysosomes to autolysosomes as shown in Figure 1 (Jeyakumar et al., 2005).

Extracellular material is degraded in lysosomes after being internalized into the cell by endocytosis and passing through the different compartments of the lysosome/endosome system: incoming endocytotic vesicles are received by early endosomes which subsequently undergo several steps of maturation into later stage endosomes (Fig. 1). The endocytosed cargo is finally degraded in lysosomes. Molecules that cannot be degraded are retained in the lysosomes (Saftig and Klumperman, 2009).

In addition to their role in catabolism, lysosomes have been shown to be involved in various other physiological processes such as plasma membrane repair, downregulation of cell surface proteins, apoptosis, cholesterol homeostasis, signaling and calcium-dependent secretion in many non-secretory cell types (Schröder et al., 2010, Settembre et al., 2013).

1.1.2. TFEB and the CLEAR network In 2009 it has been discovered that lysosome-mediated degradative pathways are under the common control of a specific regulatory gene network, the CLEAR network

Introduction

(Coordinated Lysosomal Expression and Regulation). Most lysosomal genes show a coordinated transcriptional behavior and share one or more so called CLEAR sites in their promoter. This common motif is the binding site for the transcription factor EB (TFEB), which acts as a master transcriptional regulator of lysosomal biogenesis and function (Sardiello et al., 2009). Under aberrant lysosomal conditions TFEB migrates from the cytoplasm to the nucleus, activates its target genes and thus induces lysosomal biogenesis and improves the capability of the cell to degrade complex molecules. A schematic of the CLEAR network is shown in Figure 2 (Sardiello and Ballabio, 2009).

Figure 2: Schematic of the CLEAR network Intra-lysosomal storage of undegraded material determines the nuclear translocation of TFEB, which in turn enhances cellular clearance (modified from Sardiello and Ballabio, 2009).

Further analysis of the CLEAR gene network revealed that TFEB appears to be involved in the regulation of additional lysosome-associated processes like autophagy, exo- and endocytosis, phagocytosis and immune response. Also non- lysosomal enzymes involved in the degradation of essential proteins are part of the CLEAR network (Palmieri et al., 2011).

A recent study confirmed that TFEB is also involved in the control of autophagy, a cellular catabolic process based on the cooperation of autophagosomes and lysosomes, by increasing the expression of autophagy and lysosomal genes. Several

Introduction crucial steps of the autophagic pathway such as autophagosome formation, autophagosome-lysosome fusion and lysosome-mediated degradation of the autophagosomal content are regulated by TFEB (Settembre and Ballabio, 2011, Settembre et al., 2011).

Another secretory pathway involving lysosomes is lysosomal exocytosis. Here lysosomes are first recruited to the close proximity of the cell surface and then fuse in a second, Ca2+ - regulated step with the plasma membrane, emptying their content outside the cell. TFEB transcriptionally regulates lysosomal exocytosis both by elevating the intracellular Ca2+ level through the induction of its target gene MCOLN1 which codes for a lysosomal nonselective cation channel, and by increasing the population of lysosomes in the proximity of the plasma membrane (Medina et al., 2011).

The regulator of cell growth mTORC1 colocalizes with TFEB on the lysosomal surface and has been reported to play a key role in the regulation of TFEB. The phosphorylation status of TFEB determines its subcellular location and mTORC1- mediated phosphorylation triggers its translocation into the nucleus, thus activating TFEB downstream pathways (Pena-Llopis et al., 2011). In contrast to that, more recent studies concluded that in the presence of nutrients mTORC1 inhibits TFEB activity through phosphorylation. Conversely, inhibition of mTORC1 under aberrant cellular conditions promotes TFEB translocation into the nucleus, thus activating TFEB downstream targets (Martina et al., 2012, Roczniak-Ferguson et al., 2012, Settembre et al., 2012). The activation of lysosomal function is in turn associated with the suppression of mTORC1. mTORC1 suppression together with autophagosome- lysosome fusion are important mechanisms for upregulated lysosomal function during autophagy (Zhou et al., 2013).

1.1.3. Lysosomal storage disorders As described above numerous cellular functions depend on normal lysosomal function. Impaired lysosomal function can lead to lysosomal storage disorders (LSDs), a diverse group of devastating inherited metabolic disorders. Approximately 50 different diseases have been described to date (Bellettato and Scarpa, 2010).

Introduction

However, the number of LSDs is steadily increasing as new disorders are characterized biochemically and genetically.

LSDs are typically inherited in an autosomal recessive manner, with the exception of Fabry disease and mucopolysaccharidosis (MPS) type II, which show X-linked inheritance (Platt and Walkley, 2004). Each LSD results from a mutation in a protein critical for lysosomal function, most commonly mutations in genes encoding for lysosomal hydrolases, but also in genes encoding for lysosomal membrane proteins, proteins involved in post-translational modification and trafficking of lysosomal enzymes or non-lysosomal proteins involved in lysosomal biogenesis (Schultz et al., 2011). All these diseases share as a common biochemical feature the progressive accumulation of undegraded substrates within the lysosome which ultimately leads to cellular dysfunction and death. The particular substrate stored varies depending on the mutated gene, but the substrate type can be used to group LSDs into broad categories, including mucopolysaccharidoses, neuronal ceroid lipofuscinoses, glycogenoses, lipidoses and oligosaccharidoses. However, in most LSDs more than one metabolite accumulates and in some disorders the stored material can be rather heterogeneous (Ballabio and Gieselmann, 2009). Therefore, LSDs are also grouped by the kind of the underlying molecular defect including defects in degradation of glycans, lipids or proteins or defects in lysosomal transporters or trafficking. An overview of LSDs is given in table 1.

The prevalence of LSDs ranges from about 1 in 57 000 live births for Gaucher disease to 1 per 4.2 million live births for sialidosis. Although individually rare, the combined prevalence of LSDs is estimated to be 1 in 7000 – 8000 live births (Meikle et al., 1999, Poorthuis et al., 1999, Dionisi-Vici et al., 2002), making this disease group a major challenge for the health care system. As an increasing number of patients with milder forms are being identified, the above numbers may underestimate the actual frequencies (Greiner-Tollersrud and Berg, 2005, Hemsley and Hopwood, 2011). Particular disorders were reported to have a much higher prevalence in selected populations. For example, the incidence of Gaucher disease and Tay-Sachs disease in the Ashkenazi Jewish population was reported to be 1 per 855 and 1 per 3900, respectively (Meikle et al., 1999).

Introduction

The underlying pathophysiological mechanisms by which the stored metabolites alter cell function have not been completely understood. Suggested mechanisms include alterations of exocytosis, signaling pathways or lysosomal pH regulation, alterations of intracellular calcium homeostasis, oxidative stress, inflammation and others (Jeyakumar et al., 2005, Ballabio and Gieselmann, 2009, Bellettato and Scarpa, 2010, Vitner et al., 2010, Schultz et al., 2011). Based on the observation that patients with LSDs show impaired autophagosome-lysosome fusion resulting in defective autophagy, several recent studies suggest that impaired autophagy pathways may play an important role in the pathogenesis of LSDs (Settembre et al., 2008, Elrick et al., 2012, Lieberman et al., 2012). Also a potential overlap between the pathological mechanisms of LSDs and other, more common neurodegenerative diseases with aggregation of macromolecules, such as Alzheimer disease and Parkinson disease, has been hypothesized (Bahr and Bendiske, 2002, Urbanelli et al., 2011, Osellame et al., 2013, Swan and Saunders-Pullman, 2013). Recently, TFEB has been reported to play a role in the pathogenesis of Parkinson desease and suggested to be a therapeutic target (Decressac and Björklund, 2013, Decressac et al., 2013). A shared feature with Alzheimer disease might be impairment of astrocyte function which triggers neurodegeneration in both AD and LSDs (Di Malta et al., 2012).

Introduction

Table 1: Lysosomal storage disorders Classified according to molecular defect (Platt and Walkley, 2004).

Disease Deficiency

Primary lysosomal hydrolase defect Gaucher disease Glucocerebrosidase GM1 gangliosidosis GM1-β-galactosidase Tay – Sachs disease β-Hexosaminidase A Sandhoff disease β-Hexosaminidase A+B Fabry disease α-Galactosidase A Krabbe disease β-Galactosyl ceramidase Niemann–Pick disease Types A and B Sphingomyelinase Metachromatic leukodystrophy Arylsulphatase A MPS IH (Hurler syndrome) α-Iduronidase MPS IS (Scheie syndrome) α-Iduronidase MPS II (Hunter syndrome) Iduronate sulphatase MPS IIIA (Sanfilippo A syndrome) Heparan sulphamidase MPS IIIB (Sanfilippo B syndrome) N-Acetylglucosaminidase MPS IIIC (Sanfilippo C syndrome) Acetyl CoA:α-glucosaminide N-acetyltransferase MPS IIID (Sanfilippo D syndrome) N-acetyl glucosamine-6-sulphatase MPS IV A (Morquio A disease) Acetyl galactosamine-6-sulphatase MPS IVB (Morquio B disease) β-Galactosidase MPS V (redesignated MPS IS) MPS VI (Maroteaux Lamy Syndrome) Acetyl galactosamine-4-sulphatase (ARSB) MPS VII (Sly Syndrome) β-Glucuronidase MPS IX Hyaluronidase Farber disease Acid ceramidase Cholesteryl ester storage disease Acid lipase Pompe disease (type II) α1,4-glucosidase Aspartylglucosaminuria Glycosylasparaginase Fucosidosis α-Fucosidase α-Mannosidosis α-Mannosidase β-Mannosidosis β-Mannosidase Schindler disease N-acetylgalactosaminidase Sialidosis α-Neuraminidase Infantile neuronal ceroid lipofuscinoses (CLN1) Palmitoyl protein thioesterase Late infantile neuronal ceroid lipofuscinosis (CLN2) Carboxypeptidase

Introduction

Table 1: Lysosomal storage disorders (continued from previous page) Disease Deficiency

Post-translational processing defect in lysosomal enzymes Mucosulphatidosis (MSD) Multiple sulphatases

Trafficking defect in lysosomal enzymes Mucolipidosis type II (I -cell disease) N-acetyl glucosamine phosphoryl transferase Mucolipidosis type IIIA (pseudo-Hurler N-acetyl glucosamine phosphoryl transferase polydystrophy) Mucolipidosis type IIIC

Defect in lysosomal enzyme protection Protective protein cathepsin A (PPCA) Galactosialidosis (β-galactosidase and neuraminidase)

Defect in soluble non-enzymatic lysosomal proteins Niemann–Pick type C NPC2 GM2 activator protein deficiency, Variant AB GM2 activator protein Sphingolipid activator protein (SAP) deficiency Sphingolipid activator protein Neuronal ceroid lipofuscinosis (CLN5)

Transmembrane (non-enzyme) protein defect Lysosome-associated membrane protein 2 Danon disease (LAMP2) Niemann–Pick Type C NPC1

Cystinosis Cystinosin

Infantile free sialic acid storage disease (ISSD) Sialin

Salla disease (free sialic acid storage) Sialin Juvenile neuronal ceroid lipofuscinosis (CLN3,

Batten disease) Neuronal ceroid lipofuscinoses (CLN6 and CLN8) Mucolipidosis type IV Mucolipin

Unclassified Neuronal ceroid lipofuscinoses (CLN4 and CLN7)

LSDs are clinically highly diverse. The age of onset, severity of symptoms, organ manifestations and central nervous manifestations vary significantly between the

Introduction diseases. Some level of genotype-phenotype correlation exists, with clinical phenotypes ranging from severe/infantile to mild/adult-onset forms of disease (Greiner-Tollersrud and Berg, 2005, Urbanelli et al., 2011). Individuals with LSDs can show early symptoms, but many appear clinically normal at birth. Typically, early developmental milestones are met by the children, which implies that lysosomal storage does not affect neuronal function and maturation at early developmental stages (Schultz et al., 2011).

Since most lysosomal enzymes are ubiquitously expressed, LSDs often affect multiple organ systems. Common clinical signs of many LSDs include hepatosplenomegaly, cardiac disease, abnormal skeletal growth (dystosis multiplex), immune defects and facial dysmorphism. Two thirds of LSDs show significant central nervous system (CNS) involvement. Clinical signs associated with CNS disease include progressive neurodegeneration and cognitive impairment, auditory and visual defects, seizures, and peripheral neuropathy (Wraith, 2004, Hawkins-Salsbury et al., 2011). The clinical aspects of Sanfilippo syndrome are described in detail in chapter 1.2.3.2 of this work.

The diagnosis of most LSDs starts with an accurate clinical evaluation including urine and blood analysis since in many cases the undegraded metabolites can be found in excess in the urine of the patients. Most LSDs are then confirmed by the detection of a specific enzyme deficiency. Molecular genetic testing can refine and confirm the enzymatic diagnosis. Since LSDs are untreatable when CNS pathology is present, early detection and therapy is a crucial factor (Wraith, 2004). Recently, a screening method based on the immune-quantification of lysosomal and other related proteins in dried blood spot samples was found to have 99% sensitivity in the detection of several LSDs (Fuller et al., 2011, Reuser et al., 2011).

To date, there is no causal therapy for LSDs. Several potential therapeutic approaches are being tested in vitro and in vivo in animal models including enzyme replacement therapy (ERT), gene therapy (GT), cell-based therapy, substrate reduction therapy, chemical chaperons and others (reviewed in detail by Hawkins- Salsbury et al., 2011, Hemsley and Hopwood, 2011, Urbanelli et al., 2011).

The different therapeutic approaches are described briefly with respect to Sanfilippo syndrome in chapter 1.2.4 of this Introduction.

Introduction

1.2. Mucopolysaccharidosis III/Sanfilippo Syndrome 1.2.1. The mucopolysaccharidoses The mucopolysaccharidoses (MPS) are a group of LSDs caused by the deficiency of enzymes necessary for the degradation of glycosaminoglycans (GAGs) leading to intralysosomal accumulation of GAGs.

GAGs (formerly called mucopolysaccharides) are long, unbranched polysaccharides consisting of a repeating disaccharide unit. GAG chains covalently bound to proteins form proteoglycans which are ubiquitous present both in the cell membrane and in the extracellular matrix and are an important component of connective tissues due to their water-binding capacity (Valstar et al., 2008).

Four different pathways of lysosomal degradation of GAGs are distinguished depending on the molecule to be degraded: dermatan sulfate, heparan sulfate, keratan sulfate, and chondroitin sulfate. Their degradation pathways are shown in Figure 3. Mutations in each one of the involved enzymes have been reported and result in seven different MPS which are listed with further information in table 2. All MPSs show a series of clinical features in variable degrees including organomegaly, dysostis multiplex, decreased growth, recurrent infections and a chronic, progressive course of the disease. Hearing, vision, and cardiovascular function may also be affected. Most MPSs do not show a neuronopathic phenotype and the disorders have thus been considered as potentially treatable with enzyme replacement therapy. MPS III, which is the main focus of this work, does show a neuronopathic phenotype as described in detail below (Greiner-Tollersrud and Berg, 2005, Coutinho et al., 2012a).

Introduction

Figure 3: Glycosaminoglycan degradation pathways Enzymes involved in the degradation of the GAGs heparan sulfate, keratan sulfate, dermatan sulfate and chondrotin sulfate. A deficiency of an enzyme causes the respective disease (modified from KEGG-map00531).

Introduction

Table 2: Classification of the mucopolysaccharidoses (DS: dermatan sulfate; HS: heparan sulfate; CS: chondroitin sulfate; KS: keratin sulfate) (modified from Coutinho et al., 2012a).

Disease Subtype Gene Deficient enzyme Affected GAG

Hurler α-L-iduronidase DS/HS

MPS I Hurler/Scheie IDUA α-L-iduronidase DS/HS

Scheie α-L-iduronidase DS/HS

MPS II Hunter IDS Iduronate sulfatase DS/HS

Sanfilippo A SGSH Heparan N-sulfatase HS

Sanfilippo B NAGLU N-Acetyl-a-glucosaminidase HS MPS III Acetyl CoA:a-glucosaminide N- Sanfilippo C HGSNAT HS acetyltransferase Sanfilippo D GNS N-acetylglucosamine 6-sulfatase HS

Morquio A GALNS Galactose 6-sulfatase KS/CS MPS IV Morquio B GLB1 β-galactosidase KS

MPS V No longer used, since MPS V was found to be a milder form of MPS I (Type Scheie)

MPS VI (Maroteaux-Lamy) ARSB Arylsulfatase B DS

MPS VII (Sly) GUSB β-glucuronidase DS/KS/CS

MPS IX HYAL Hyaluronidase 1

1.2.2. MPSIII: history, biochemistry and genetic aspects Sanfilippo syndrome was named after Sylvester Sanfilippo who reported in 1963 for the first time about patients with mucolpolysaccharides in the urine and severe neurological dysfunction manifesting in loss of learned abilities and behavioral disorder (Sanfilippo et al., 1963).

Sanfilippo syndrome or MPS III is characterized biochemically by the progressive lysosomal accumulation of the glycosaminoglycan heparan sulfate (HS) and the excretion of HS or fragments of it into body fluids. The underlying defect is a deficiency in one of the enzymes in the degradation pathway of heparan sulfate (Valstar et al., 2008). The molecular mechanism by which stored GAGs cause neurodegeneration is not fully understood, suggested mechanisms are described above in the LSD chapter of this work. Interestingly, pathological links between

Introduction

Parkinson disease and Sanfilippo syndrome have been proposed recently (Winder- Rhodes et al., 2012).

The degradation pathway of HS involves several enzymes as shown in Figure 3. Mutations in those enzymes that are only involved in the degradation of HS cause MPS III: Heparan N-sulfatase (mutated in MPS IIIA), N-Acetyl-a-glucosaminidase (mutated in MPS IIIB), Acetyl-CoA:a-glucosaminide N-acetyltransferase (mutated in MPS IIIC) and N-acetylglucosamine-6-sulfatase (mutated in MPS IIID). Multiple mutations, mostly missense point mutations, have been described in each of the four genes coding for these enzymes, forming the four subtypes A, B, C and D of MPS III (Valstar et al., 2008). The disease genes with enzyme code and gene localization are listed in table 3. On the other hand, mutations in enzymes involved in the degradation of HS and dermatan sulfate cause other LSDs: mutations in -L-iduronidase cause MPS I, mutations in iduronate sulfatase cause MPS II, and mutations in - glucuronidase cause MPS VII.

Table 3: Summary Mucopolysaccharidosis III (Sanfilippo syndrome)

Gene Enzyme Characteriza Disease OMIM Gene Enzyme location code tion

(Scott et al., MPS IIIA #252900 SGSH 17q25.3 Heparan N-sulfatase 3.10.1.1 1995)

(Zhao et al., N-Acetyl-a- 1996, Weber MPS IIIB #252920 NAGLU 17q21.1 3.2.1.50 glucosaminidase et al., 1999)

(Fan et al., Acetyl CoA:a- 2006, MPS IIIC #252930 HGSNAT 8p11.1 glucosaminide N- 2.3.1.78 Hrebicek et acetyltransferase al., 2006, Fan et al., 2011)

N-acetylglucosamine (Robertson et MPS IIID #252940 GNS 12q14 3.1.6.14 6-sulfatase al., 1988)

Introduction

1.2.3. MPSIII: epidemiology, clinical presentation and diagnosis 1.2.3.1. Epidemiology MPS III is the most common mucopolysaccharidosis. Birth prevalences of 0.28 – 4.1 per 100 000 live births have been reported (Nelson, 1997, Meikle et al., 1999, Poorthuis et al., 1999, Applegarth et al., 2000, Nelson et al., 2003, Pinto et al., 2004, Baehner et al., 2005), a prevalence of about 1.5 – 1.9 per 100 000 live births is considered to be the most adequate estimate (Valstar et al., 2008). However, these numbers may underestimate the true prevalence given the large phenotypic variability and undiagnosed mild cases. The most common type of MPS III is type A in north-west Europe, while type B is the most frequent one in south-east Europe (Meikle et al., 1999, Poorthuis et al., 1999, Baehner et al., 2005). Type C and D appear to be much rarer.

1.2.3.2. Clinical picture It is generally assumed that the four MPS III subtypes are clinically indistinguishable. However, some studies suggest that type A is more severe, with an earlier onset, more rapid progression and earlier death than the other types of MPS III (Meyer et al., 2007, Ruijter et al., 2008). In general, symptoms and severity of the disease vary widely between patients, even between siblings with the same genotype (Valstar et al., 2008).

Pregnancy and delivery are usually normal and the affected children appear clinically normal at birth. Patients usually present for the first time with developmental delay which starts between 1 – 4 years of age after an initial normal or almost normal development. At diagnosis, speech development is generally much more delayed than motor development (Valstar et al., 2008).

Neuropsychiatric symptoms are predominant features of Sanfilippo disease, including severe behavioral problems which usually start around the age of 3 – 5 years, progressive mental deterioration and developmental regression. Behavioral symptoms include restless, destructive, chaotic, anxious and sometimes aggressive behavior (Valstar et al., 2008, Malcolm et al., 2012). Behavioral problems combined with normal physical strength and mobility make MPS III patients frequently very

Introduction difficult to manage (Cleary and Wraith, 1993, Bax and Colville, 1995, Valstar et al., 2011).

Somatic features are relatively mild in MPS III. Frequent symptoms among MPS III patients include organomegaly, recurrent ear, nose and throat infections in young patients, sleep disturbances ranging from settling difficulties to complete reversal of day – night rhythm, hearing problems and recurrent episodes of diarrhea (Valstar et al., 2008). In most patients mild facial dysmorphisms are noted. Older patients frequently develop epileptic seizures (Valstar et al., 2010). Osteonecrosis of the femoral head was found to be highly prevalent in MPS III patients, also other musculoskeletal manifestations are reported in rare cases (White et al., 2011, de Ruijter et al., 2013).

Due to the progressive neurodegeneration and thus mental deterioration, behavioral symptoms decline with age and disappear eventually, finally leading to severe dementia and complete loss of initiative. At the same time, motor retardation progresses leading to swallowing difficulties and spasticity (Valstar et al., 2008).

MPS IIIA and C patients frequently lose speech before the age of 10 years and 15 years respectively. The ability to walk is lost in the mid-teenage years in MPS IIIA patients, while the majority of MPS IIIC patients lose their ability to walk between 20 and 30 years of age. Patients eventually regress to a fully bedridden and vegetative state and become dependent on care early in their teenage years (Meyer et al., 2007, Ruijter et al., 2008, Valstar et al., 2011).

MPS IIIB patients usually die at the end of the 2nd or beginning of the 3rd decade of life, but longer survival has been reported in patients with an attenuated phenotype (Cleary and Wraith, 1993, Valstar et al., 2008).

1.2.3.3. Diagnosis After accurate clinical evaluation, the biochemical diagnosis of mucopolysaccharidoses including Sanfilippo syndrome starts with the quantification of GAGs in the urine using the DMB test. Here GAGs form a complex with the dye dimethylmethylene blue (DMB), which can then be quantified spectrophotometrically (de Jong et al., 1992). Since the sensitivity of the DMB test in the urine may be less

Introduction than 100%, qualitative electrophoretic separation of GAGs should follow the quantitative measurement. Increased excretion of only heparan sulfate, i.e. without increased excretion of dermatan sulfate, indicates Sanfilippo syndrome (Valstar et al., 2008, Lehman et al., 2011).

For definitive diagnosis of MPS III and classification of the subtype, enzyme assay in leukocytes or cultured fibroblasts is performed. All four enzymes can be assayed using artificial fluorescent substrates (Valstar et al., 2008).

The exact underlying mutation can be assessed by molecular genetic testing, although a genotype – phenotype correlation is not obvious in MPS III.

Since the neurodegeneration cannot be reversed, early diagnosis is crucial for a favorable outcome. However, MPS III patients may be misdiagnosed as having idiopathic developmental delay, attention deficit-hyperactivity or autism spectrum disorders (Wijburg et al., 2013).The measurement of heparan and dermatan sulfate derived disaccharides in dried blood spots may be a suitable newborn screening for MPS III and other mucopolysaccharidoses (de Ruijter et al., 2012a).

1.2.4. Therapeutic strategies for MPS III (and other LSDs) To date there is no established therapy for MPS III and current therapies are largely supportive. In the following chapter common therapeutic approaches for LSDs will be described and evaluated in respect to MPS III. The main focus of treatment is the CNS since MPS III shows mainly neuropsychiatric symptoms.

1.2.4.1. Enzyme replacement therapy Enzyme replacement therapy (ERT) supplies the patient with a recombinant form of the deficient enzyme. Intravenous ERT is in clinical use in a limited number of LSDs, including Gaucher, Fabry and Pompe disease, as well as mucopolysaccharidoses I, II and VI, and shows good effects in ameliorating non-CNS pathology (reviewed by Rohrbach and Clarke, 2007, Desnick and Schuchman, 2012). Intravenously administered recombinant N-acetylglucosaminidase was taken up by liver and spleen in a mouse model of MPS IIIB (Yu et al., 2000). However, the intravenously

Introduction administered enzyme is not able to penetrate the blood-brain barrier (BBB) and therefore does not ameliorate CNS symptomatology (Valstar et al., 2008). In order to enable the transfer of recombinant lysosomal enzymes across the BBB, their structure can be modified. However, this approach was not successful in a mouse model of MPS IIIA (Rozaklis et al., 2011). Another approach to bypass the BBB is the direct injection of recombinant enzyme into the cerebrospinal fluid (CSF), leading to a dose-dependent reduction of HS in the brain and improvement of behavior of MPS IIIA mice (Hemsley et al., 2007) and MPS IIIA dogs (Crawley et al., 2011). This treatment approach for MPS IIIA is currently being evaluated in a clinical trial (# NCT01155778, compare www.clinicaltrials.gov).

1.2.4.2. Gene therapy Gene therapy aims at reconstituting enzyme production by replacement of a mutated disease gene in the patient genome with a wild-type form using a viral vector (reviewed by Tomanin et al., 2012). Ventricular injection of a viral vector carrying sulfaminidase showed a reduction of storage as well as behavioral improvement in MPS IIIA mice (Fraldi et al., 2007). Similar results combined with a prolonged lifespan were shown also in MPS IIIB mice (Cressant et al., 2004, Di Natale et al., 2005, Fu et al., 2007, Fu et al., 2010, Fu et al., 2011, Heldermon et al., 2013) and in a dog model of MPS IIIB (Ellinwood et al., 2011). Recently, also intravascular injection of a viral vector carrying a sulfaminidase modified to be able to cross the BBB was shown to recover a normal behavioral phenotype in MPS IIIA mice (Sorrentino et al., 2013).

1.2.4.3. Cell-based therapy Cells of the monocyte-macrophage lineage are able to cross the BBB, enter the brain and become microglia. After hematopoietic cell transplantation (HCT) or bone marrow transplantation, the microglia of donor origin are believed to secrete the missing lysosomal enzyme which is taken up by the deficient neuronal cells (Krivit et al., 1995). HCT has been used successfully for the treatment of visceral, skeletal and CNS manifestations in MPS I, MPS VI and MPS VII, but was unsuccessful in ameliorating the neurological phenotype in MPS III patients for unknown reasons

Introduction

(reviewed by Boelens et al., 2010). Thus, it is not considered a treatment option for MPS III anymore (Valstar et al., 2008).

1.2.4.4. Substrate reduction therapy While all previously described approaches try to enhance enzyme activity within the brain, substrate reduction therapy seeks to inhibit the synthesis of GAGs and decrease their pathologic storage in that way. The used compounds are small molecules able to cross the BBB and therefore represent a potential approach for the therapy of neuronopathic LSDs (Jakobkiewicz-Banecka et al., 2007).

The soy isoflavone genistein has been shown to influence an epidermal growth factor-dependent pathway and reduces GAG synthesis and storage in vitro (Piotrowska et al., 2006, Jakobkiewicz-Banecka et al., 2009, Arfi et al., 2010) and in vivo in mouse models of MPS IIIB in both peripheral tissues and brain (Malinowska et al., 2009, Malinowska et al., 2010). Some pilot-studies in humans with small patient numbers have been conducted recently with varying results (Piotrowska et al., 2008, Delgadillo et al., 2011, Piotrowska et al., 2011, de Ruijter et al., 2012b).

The efficacy of miglustat (N-butyldeoxynojirimycin, trade name: Zavesca®), a substrate inhibitor which has been in clinical use for the treatment of Gaucher disease for more than 10 years, has recently been evaluated in MPS III patients, but no improvement or stabilization in behavioral problems could be shown after 6 months of treatment (Guffon et al., 2011).

1.2.4.5. Pharmaceutical chaperon therapy (PCT) As many LSDs would have a much more attenuated phenotype or even no phenotype at all if the enzyme activity exceeds 5 -10% of the normal activity, the enhancement of residual enzyme activity may be an effective treatment. Often mutant enzymes with a substantial residual activity are synthesized, but recognized as misfolded by the ER quality control system and rapidly degraded by the ER- associated degradation pathways (Ellgaard and Helenius, 2003). Many chaperons are reversible enzyme inhibitors that protect mutant enzymes from rapid degradation

Introduction through the proofreading stages of synthesis and subsequently increase enzyme activity (Parenti, 2009, Valenzano et al., 2011).

After proof-of-concept studies, pharmaceutical chaperon therapy (PCT) is now being translated into clinical applications for several LSDs, including Fabry, Gaucher and Pompe disease (Fan et al., 1999, Okumiya et al., 2007, Parenti et al., 2007, Yu et al., 2007). To date there is only one report about this approach in MPS III. The competitive HGSNAT inhibitor glucosamine has been shown to be able to partially rescue the HGSNAT activity of several mutants of MPS IIIC (Feldhammer et al., 2009). However, chaperon therapy has been also proposed as a treatment approach for MPS IIIB (Ficko-Blean et al., 2008).

1.2.4.6. Stop codon read-through (SCRT) SCRT takes advantage of drugs such as aminoglycosides that are able to attenuate the termination of translation at the level of a premature STOP codon in the case of non-sense mutations. SCRT is an attractive strategy because premature STOP codons typically lack an appropriate context for an efficient termination of translation in the surrounding sequences, which enhances the selective effects of SCRT drugs leading to little consequences on normal translation while helping complete translation of the mutated protein. Proof-of-concept studies have been published for several genetic diseases including MPS I, but to date there are no reports on MPS III (Linde and Kerem, 2008, Bidou et al., 2012).

1.3. Trehalose 1.3.1. Chemical properties and natural occurrence Trehalose is a nonreducing disaccharide in which two molecules of D-glucose are linked together in an 1,1 – glycosidic linkage (Fig. 4). The molecular formula and weight are C12H22O11 and 342.31 g/mol, respectively. The only naturally occurring isomer is ,trehalose (-D-glucopyranosyl -D-glucopyranoside) which is found in a wide range of organisms including bacteria, fungi, insects, invertebrates and plants (Elbein, 1974). Trehalose was found to be the principal sugar (80-90%) in the hemolymph of several species of insects (Wyatt and Kalf, 1957). High concentrations

Introduction of trehalose are also found in Baker’s and Brewer’s yeast. Interestingly, trehalose is not synthesized by higher species, while the enzyme required to hydrolyze trehalose (trehalase) is expressed in mammals.

Figure 4: Structure of ,1,1-trehalose (Elbein et al., 2003)

1.3.2. Metabolism At least three different pathways for the natural biosynthesis of trehalose exist. The most widely occurring and best understood pathway involves the synthesis of trehalose-6-phosphate out of UDP-glucose and glucose-6-phosphate using the enzyme trehalose-6-phosphate synthase (Tps1 in Sacharomyces cerevisia or OtsA in E. coli). Subsequently, a phosphatase converts trehalose-6-phosphate into free trehalose (Tps2 in S. cerevisiae or OtsB in E. coli) (Elbein, 1974, Elbein et al., 2003). In D. melanogaster there is only one gene (Tps1) that has homology to both Tps1 and Tps2 from S. cerevisiae and catalyzes both reactions of trehalose synthesis (Chen et al., 2002).

The other two pathways have only been reported in few organisms (Elbein, 1974, Elbein et al., 2003). The enzymes of the trehalose synthesis pathway are induced upon a variety of external stimuli (see below).

Another important enzyme in trehalose metabolism is trehalase which catalyses the catabolic conversion of trehalose into two molecules of glucose. It is involved in energy metabolism and plays a regulatory role in controlling the levels of trehalose in trehalose-synthesizing organisms.

Although trehalose does not occur naturally in mammalian cells, the trehalose degrading enzyme trehalase is expressed in humans and other mammals in both intestinal villae membranes and kidney brush border membranes (Welsh et al., 1978,

Introduction

Elbein et al., 2003). Its role in the kidney remains unclear so far, but in the intestine its function is to hydrolyze ingested trehalose into glucose that can be easily absorbed and metabolized. Although ingested trehalose should be converted by trehalase into glucose before absorption, measurable amounts of trehalose could be detected in extracts of brain and liver in a mouse model of Huntington disease orally treated with trehalose (Tanaka et al., 2004). The mechanism of direct absorption of trehalose remains unclear since a specific trehalose transporter has been described to date only in S. cerevisiae and several insects, but is unknown in mammals (Stambuk et al., 1998, Kikawada et al., 2007, Kanamori et al., 2010, Kikuta et al., 2010, Kikuta et al., 2012). While the metabolism of other disaccharides has been studied extensively, to date little is known about absorption, turnover and metabolism of trehalose in mammals.

1.3.3. Safety and toxicity Several safety and toxicity studies are reviewed by Richards et al., 2002. No trehalose related dose-dependent adverse effects were observed in several studies testing trehalose consumption in doses up to 50 g and up to 10% of the total daily diet (Richards et al., 2002). Thus the use of trehalose appears to be safe.

1.3.4. Functions of trehalose 1.3.4.1. Protection from various stress conditions The role of trehalose in nature has been studied extensively and appears to be species dependent (Richards et al., 2002). Many unique properties have been attributed to trehalose setting this molecule apart from other disaccharides.

Several studies suggested that trehalose can act as a protectant against a variety of environmental stress conditions such as desiccation, oxidation, anoxia, heat and cold. It can help cells in retaining their cellular integrity by preventing protein denaturation and stabilizing membranes (Soto et al., 1999, Crowe et al., 2003, Reina- Bueno et al., 2012). Plants, fungi and invertebrates which undergo periods of anhydrobiosis protect their cellular structures by synthesizing large amounts of trehalose prior to the dehydration process (Elbein et al., 2003).

Introduction

More specifically, the larvae of Polypedilum vanderplanki, the highest cryptobiotic invertebrate, tolerate complete desiccation and extreme temperatures due to the massive synthesis of trehalose prior to dehydration (Watanabe et al., 2002). In several E. coli strains intracellular trehalose concentrations were found to be correlated with desiccation resistance (Zhang and Yan, 2012). Also human fibroblasts were shown to exhibit higher desiccation tolerance when artificially expressing a trehalose-synthesizing gene (Guo et al., 2000).

In many organisms an increase of thermotolerance is correlated with the induction of heat-shock proteins following a sudden rise or fall in temperature. Moreover both heat-shock and cold-shock were shown to induce enzymes of trehalose metabolism resulting in subsequent elevation of trehalose levels and increased thermotolerance in several yeasts, Escherichia coli and in the nematode Heterorhabditis bacteriophora (De Virgilio et al., 1990, De Virgilio et al., 1994, Hottiger et al., 1994, Lee and Goldberg, 1998, Jagdale et al., 2005). Cold-shock induced trehalose synthesis is essential for the viability at low temperature in E. coli and yeast (Kandror et al., 2002, Kandror et al., 2004).

Chen and colleagues showed that trehalose synthesis protects Drosophila melanogaster from protein denaturation after hypoxic or anoxic injury. Similar effects were seen in mammalian HEK-293 cells after transfection with the Drosophila trehalose-synthesizing gene Tps1 (Chen et al., 2002, Chen et al., 2003, Chen and Haddad, 2004).

Trehalose accumulation also plays a role in the resistance of cells against free oxygen radicals. It has been shown to protect proteins and amino acids from oxidative damage in vitro (Benaroudj et al., 2001) and in vivo in the corneal epithelium (Cejkova et al., 2011).

Many of these effects are attributed to a chaperon-like effect of trehalose. Trehalose can act as a chemical chaperon itself and prevent proteins from denaturing at high temperatures by stabilizing them directly (Singer and Lindquist, 1998). The underlying mechanism is not completely understood but several hypotheses exist (reviewed by Jain and Roy, 2009).

Introduction

Crowe and colleagues demonstrated successfully that human platelets survive freeze-drying cryopreservation through membrane stabilization after loading with trehalose (Wolkers et al., 2001, Crowe et al., 2003). Also the biochemical preservation of rat lung slices after cold storage was found improved (Bull et al., 2000). Moreover trehalose enhanced the recovery and preserved the function of human pancreatic islets after long-term storage (Beattie et al., 1997). Its unique properties make trehalose also an intriguing molecule for medical application in ophthalmology (Luyckx and Baudouin, 2011).

1.3.4.2. Induction of autophagy and degradation of macromolecules In addition to the properties of trehalose as a stress protectant, several studies have shown its involvement in the clearance of storage macromolecules like they occur in LSDs or other neurodegenerative diseases such as Parkinson disease (PD), Alzheimer disease (AD) or Huntington disease (HD). Recent studies suggest that in addition to the stabilizing effects on protein folding, trehalose is able to activate autophagy pathways and protect cells against pro-apoptotic insults (Sarkar et al., 2007).

Mammalian cell-based studies have shown that trehalose can act as an mTOR- independent activator of autophagy, which in turn enhances the clearance of substrates like expanded huntingtin (associated with HD), mutated alpha-synuclein (associated with PD), total and phosphorylated tau (associated with AD) and other stored molecules (Sarkar et al., 2007, Gomes et al., 2010, Casarejos et al., 2011, Krüger et al., 2011, Lan et al., 2012). Moreover, trehalose has been shown to prevent insulin amyloid formation (Arora et al., 2004) and aggregation of AD-associated beta- amyloid in vitro (Liu et al., 2005). Through induction of autophagy trehalose can counteract cellular prion infection (Aguib et al., 2009) and induce clearance of mutant androgen receptor in cell-based models of spinal and bulbar muscular atrophy (Rusmini et al., 2013). Also the ability of trehalose to extend the life span of the nematode Caenorhabditis elegans by decreasing the accumulation of age-related lipofuscin has been reported (Honda et al., 2010).

In vivo analyses have shown that trehalose ameliorates polyalanin- and polyglutamine-mediated pathology in mouse models of oculopharyngeal muscular

Introduction dystrophy and Huntington disease, respectively (Tanaka et al., 2004, Davies et al., 2006). Also dopaminergic and tau pathology in mice with a deletion in the PD- associated gene Parkin and overexpressing tau has been shown to be alleviated when treated with trehalose as well as a reduction of tau aggregations in a human mutant P301S tauopathy mouse model (Rodriguez-Navarro et al., 2010, Schaeffer et al., 2012). A recent study showed that trehalose treatment increases the life span and delays disease onset in a mouse model of amyotrophic lateral sclerosis by enhancing autophagy in motoneurons (Castillo et al., 2013). Other recent studies suggested antidepressant-like effects of trehalose related to its autophagy enhancing properties (Kara et al., 2013) and the prevention of neural tube defects due to restored autophagy in type 1 diabetic mice (Xu et al., 2013).

Aims

2 AIMS The lysosome is the cell’s main compartment for the degradation of macromolecules. Impaired lysosomal pathways have been associated with numerous diseases including lysosomal storage disorders and other neurodegenerative diseases such as Alzheimer, Parkinson and Huntington diseases. Sanfilippo disease (or MPS III) is a devastating LSD characterized by impaired degradation of the glycosaminoglycan heparan sulfate leading to its progressive lysosomal accumulation. Patients typically show severe signs of neurodegeneration including behavioral symptoms and mental deterioration, which finally leads to severe dementia and early death.

Trehalose is a disaccharide of glucose naturally found in lower eukaryotes. Several reports have suggested that trehalose can act as a protectant against a variety of stress conditions. Moreover trehalose has been shown to be beneficial in models of storage diseases. Based on these result a role of trehalose in stabilization of protein folding and activation of cellular clearance pathways has been proposed. High solubility and lack of toxicity make trehalose a promising molecule for the treatment of neurodegenerative diseases caused by protein misfolding and/or defects in clearance pathways. However, the therapeutic potential of trehalose has never been tested in models of lysosomal storage disorders.

The main aim of the present study was to investigate the effects of trehalose treatment on fibroblasts derived from patients with Sanfilippo syndrome. To this goal, we planned to assess whether trehalose can decrease cellular GAG deposits by measuring levels of HS under trehalose treatment

Moreover we planned to investigate the putative underlying mechanisms of action, both the activation of cellular clearance pathways and the chaperon effects. Since lysosomal processes including autophagy and lysosomal exocytosis have been shown to be under coordinated transcriptional control of the master gene TFEB, and trehalose is known to stimulate clearance of pathologically stored deposits, it was hypothesized that trehalose might enhance lysosomal function through TFEB activation. To determine the involvement in lysosomal pathways we planned to analyze the expression profile of several lysosomal genes upon trehalose treatment.

Aims

Also, we planned to investigate the subcellular localization of TFEB under trehalose treatment.

The analysis of chaperon properties of trehalose was projected to be performed by assaying enzymatic activity of N-acetylglucosaminidase in fibroblasts from Sanfilippo B patients. Since therefore no suitable assay existed, an experimental protocol had to be developed and validated in a first step. Integration of bioinformatic data into the analysis was planned in order to determine the influence of a certain mutation on folding and stability of a protein and to predict the likelihood of a mutation to be retrievable by a chemical chaperon.

Since little is known about trehalose metabolism in mammals, we planned to analyze intestinal trehalase activity as well as trehalose concentrations in liver and brain of C57BL/6 mice upon trehalose administration.

The findings presented in this thesis provide new insights into the mechanisms underlying trehalose-mediated effects. Lysosomal enhancement with trehalose or other small molecules might be a novel therapeutic approach for the treatment of LSDs including Sanfilippo syndrome and other neurodegenerative diseases.

Material and Methods

3 MATERIAL AND METHODS 3.1. Cell lines 3.1.1. Fibroblast cell lines derived from MPS III patients and control subjects Untransformed MPS III patient-derived primary fibroblasts cell lines and control fibroblast cell lines from healthy donors were either purchased from the Coriell Institute for Medical Research or obtained from the Telethon Genetic Biobank Network (TGBN), Italy. Tables 4 and 5 provide a genotypic and phenotypic description of the used MPS III patient-derived fibroblast cell lines and control fibroblast cell lines, respectively. Two fibroblast cell lines from patients with neuronal ceroid lipofuscinosis were kindly provided by Dr. Filippo Santorelli, Pisa, Italy. They were considered as a negative control bearing another storage disorder for the assay of N-acetylglucosaminidase, which is the deficient enzyme in MPS III.

Table 4: Information about used MPS III patient-derived fibroblast cell lines.

Cell line Disease Code Source Age/Sex Genotype

A1 MPS IIIA GM00879 Coriell 3 yr female E447K, R245H A2 MPS IIIA GM01881 Coriell 3 yr male S66W, V131M

B1 MPS IIIB FFF0051996 TGBN female L35F, G292R B2 MPS IIIB FFF0071993 TGBN male Homoz W649C B3 MPS IIIB FFF0242004 TGBN male Homoz Y92H B4 MPS IIIB FFF0331981 TGBN female Homoz E336X B5 MPS IIIB FFF0402004 TGBN male T81A, Y140C B6 MPS IIIB FFF0502006 TGBN male V77G, H414R B7 MPS IIIB FFF0631986 TGBN male (brother of # B8) Homoz V501G B8 MPS IIIB FFF0641986 TGBN female (sister of # B7) Homoz V501G B9 MPS IIIB FFF0821991 TGBN Female Y140C, W156C B10 MPS IIIB GM00156 Coriell 7 yr male Homoz R626X B11 MPS IIIB GM00737 Coriell 7 yr male L682R, Y140C B12 MPS IIIB GM01426 Coriell 1 yr female Homoz E153K B13 MPS IIIB GM02552 A Coriell 7 yr female R297X, R643H B14 MPS IIIB GM02931 Coriell 3 yr female Homoz P358L

C1 MPS IIIC GM05157 Coriell 8 yr male Not genotyped

D1 MPS IIID GM17495 A Coriell 18 yr male Homoz R355X

Material and Methods

Table 5: Information about control fibroblast cell lines.

Cell line Disease Code Source Age/Sex

WT1 wild-type GM03440 Coriell 20 yr male WT2 wild-type GM03651 Coriell 25 yr female WT3 wild-type GM00498 Coriell 3 yr male

NCL1 NCL Dr. Santorelli unknown NCL2 NCL Dr. Santorelli unknown

3.1.2. HEK 293T and HeLa cells Both HEK 293T cells and HeLa cells are available at Dr. Sardiello’s laboratory. Apart from regular HeLa cells there are also HeLa cells available which are stably overexpressing TFEB-3xFLAG.

3.2. Equipment, Chemicals, Reagents and Kits 3.2.1. Equipment . Analytical balance AE 50 Mettler . Cell incubator Autoflow5510 NuAire . Centrifuges Allegra X-22 Beckman Coulter 5810 R Eppendorf Microfuge®16 Beckman Coulter 5415 D Eppendorf Avanti J-26XP Beckman Coulter . Electrophoresis chamber Easycast B2 Thermo Scientific . Gel imaging system Gel Doc™ XR+ Biorad . Microplate reader Synergy 2 BioTek . Microscopes CKX41 Olympus TCS SP5 Leica . Microwave Emerson . pH-meter M240 Corning . Power supply EC-105 E-C Apparatus

Material and Methods

. Real time thermocycler 7300 RT-PCR System Applied Biosystems . Sonicator Sonifier 450 Branson . Spectrophotometer Nanodrop ND-1000 Peqlab Genesys 10 Bio Thermo Scientific . Thermocycler Veriti 96-well Applied Biosystems . Thermomixer Comfort 1.5 ml Eppendorf . Tissue culture hood Labgard NU-425 NuAire . Water purification system Milli-Q Water System Millipore

3.2.2. Mouse dissection equipment . Crile Hemostat Fine Science Tools . Dissector Scissors Fine Science Tools . Dumont #7b Forceps - Curved Fine Science Tools . Dumont #L5 Forceps - Straight Fine Science Tools . Fine Scissors Fine Science Tools . Semken Forceps Fine Science Tools . Standard Pattern Forceps Fine Science Tools . Surgical Scissors Fine Science Tools . Vannas Spring Scissors Fine Science Tools

3.2.3. Chemicals Whenever possible, only chemicals with purity grade “pro analysis” were used for the experiments in this work. For RNA isolation and analysis, only chemicals free of RNAses have been used. All standard chemicals and most reagents were purchased from the following companies:

. life technologies (formerly Invitrogen) Grand Island, NY, USA . Moscerdam Substrates Oegstgeest, Netherlands . New England Biolabs Ipswich, MA, USA . Qiagen Hilden, Germany . Sigma Aldrich St. Louis, MO, USA . Thermo Fisher Scientific Waltham, MA, USA . vwr Radnor, PA, USA

Material and Methods

3.2.4. Reagents and other materials

. 4-MU-N-acetyl-α-D-glucosaminide (# M3B) Moscerdam Substrates . AccuPrime™ Taq DNA Polymerase High Fidelity Invitrogen . Acetic acid Sigma . Alexa-Fluor 488 antibody (goat anti-rabbit) Invitrogen . Anti-FLAG antibody (rabbit) Sigma . Buffer P1 (Resuspension buffer) Qiagen . Buffer P2 (Lysis buffer) Qiagen . Buffer P3 (Neutralization Buffer) Qiagen . Corning 96-well-plates for Flourescence vwr . Corning® Tissue Culture Dishes vwr . D-(+)-Trehalose dihydrate (# T0167) Sigma . Disposable Plastic Cuvettes vwr . DMEM High Glucose Medium (SH30243.FS) HyClone . EcoR I New England Biolabs . Fetal Bovine Serum HyClone . GeneRuler™ 1 kb DNA Ladder Fermentas . Glucose Assay Reagent (# G3293) Sigma . Glycine Sigma . Imidazole Sigma . Isoflurane Baxter . LB Agar Sigma . LB Broth Sigma . L-Cysteine hydrochloride Sigma . L-Glutamine solution Sigma . Lipofectamine™ LTX and Plus Reagent Invitrogen . MicroAmp® Optical 96-Well Reaction Plate Invitrogen . Millex-HV Filter Unit 0.45 µm Millipore . NEBuffer 2 New England Biolabs . Omnipur Agarose VWR . Opti-MEM® Invitrogen . Papain from papaya latex (# P3125) Sigma . Paraformaldehyde Sigma . Penicillin-Streptomycin Sigma . Perfecta Sybr Fastmix Rox Quanta BioSciences . Phosphate Buffered Saline HyClone . Phusion® High-Fidelity DNA Polymerase Finnzymes . Potasium phosphate monobasic Sigma . RNase A Qiagen . S.O.C Medium Invitrogen

Material and Methods

. SapphireAmp Fast PCR MasterMix Takara . Sodium acetate trihydrate Sigma . Sodium borohydride Sigma . Sodium hydroxide Sigma . Sodium phosphate monobasic Sigma . Steriflip-GP Filter Unit 0.22 µm Millipore . Trehalase from porcine kidney (# T8778) Sigma . Tris-HCl 1M, pH8.5 Qiagen . Triton X-100 Sigma . Trypsin HyClone . VECTASHIELD Mounting Medium with DAPI Vector lab . Xho I New England Biolabs

3.2.5. Kits . Blyscan™ Glycosaminoglycan Assay biocolor . miRNeasy Mini Kit Qiagen . Pierce® BCA Protein Assay Kit Thermo Scientific . PureLink™ HiPure Plasmid Filter Midiprep Kit Invitrogen . QuantiTect Reverse Transcription Kit Qiagen . QIAquick Gel Extraction Kit Qiagen . Quick Ligation™ Kit New England Biolabs . Zero Blunt® TOPO® PCR Cloning Kit Invitrogen

3.3. Solutions and Media 3.3.1. Frequently used buffers and solutions

Blocking solution (for ICC): For 100 ml: 0.1% Saponin 100 mg 10% Fetal bovine serum 10 ml PBS to a final volume of 100 ml store in aliquots at – 20° C

Material and Methods

0.2 M Glycine Buffer, pH 10.8: For 1000 ml: Glycine 15.01 g

Deionized H20 to a final volume of 1000 ml 5 M NaOH adjust pH to 10.8 store at room temperature

Homogenization buffer: For 100 ml: 0.1 M NaCl 0.58 g 0.05 M Tris-HCl 0.79 g 0.5% Triton X-100 500 µl

Deionized H20 to a final volume of 100 ml store at room temperature

2 M Imidazole Buffer, pH 7.0: For 100 ml: Imidazole 13.62 g

Deionized H20 to a final volume of 100 ml 5 M NaOH adjust pH to 7.0 store at 4°C

Papain extraction reagent: For 100 ml:

0.1 M Sodium acetate 0.82 g

0.01 M Na2-EDTA 0.37 g 5 mM Cysteine HCL 79 mg 0.2 M Sodium phosphate buffer, pH 6.4 to a final volume of 100 ml Papain from papaya latex 800 µl

Papain is introduced when all other components have dissolved. The reagent can be stored 10 days at 4°C.

Material and Methods

0.2 M Sodium acetate buffer, pH 4.5: For 1000 ml: Sodium acetate trihydrate 9.80 g Glacial acetic acid 7.39 ml

Deionized H20 to a final volume of 1000 ml check pH store at room temperature

0.2 M Sodium phosphate buffer, pH 6.4 For 1000 ml: Sodium phosphate monobasic 20.3 g Sodium phosphate dibasic heptahydrate 14.2 g

Deionized H20 to a final volume of 1000 ml check pH store at room temperature

Substrate solution for NAG assay: For 10 ml: 2 mM 4MU-alpha-N-acetyl-D-glucosaminide 7.588 mg 0.5% Triton X-100 50 µl 50x Protease inhibitor 200 µl Sodium acetate buffer 0.2 M, pH 4.5 to a final volume of 10 ml store in aliquots at – 80° C

3.3.2. Cell culture media Medium for human fibroblasts: For 512 ml: DMEM High Glucose 400 ml 20% FBS 100 ml 100 U/ml Penicillin / 100 µg/ml Streptomycin 6 ml 2 mM L-Glutamin 6 ml

Material and Methods

Medium for HEK 293T cells and HeLa cells: For 512 ml: DMEM High Glucose 450 ml 10% FBS 50 ml 100 U/ml Penicillin / 100 µg/ml Streptomycin 6 ml 2 mM L-Glutamin 6 ml

Transfection medium (without antibiotics): For 506 ml: DMEM High Glucose 450 ml 10% FBS 50 ml 2 mM L-Glutamin 6 ml

Freezing medium: For 10 ml: 90% Fibroblast medium (see above) 9 ml 10% DMSO 1 ml prepared fresh

3.4. Primers

All primers were individually designed and purchased from Sigma-Aldrich. They were used for PCRs, semi-quantitative RT-PCRs or quantitative real timePCRs. Primers were delivered in lyophilized form and subsequently diluted in Milli-Q-water to achieve primer stock solutions with a concentration of 100 pmol/μl. Stock solutions were stored in aliquots at – 20°C and served for the preparation of working solutions with a concentration of 10 pmol/µl for PCRs and 2 pmol/μl for quantitative real-time PCRs. All primer pairs for qPCR were designed exon junction-spanning in order to avoid amplification of potential residues of genomic DNA.

Table 6: Primers used for Tps1 cloning

Primer Name Sequence 5'  3' Tm (°C)

Tps1_EcoRI_F GAATTCATGCCCGACACGGAAATCAT 60 Tps1_XhoI_R CTCGAGTCACACCTCCAGATTGTTCGC

Material and Methods

Table 7: Primers used for Tps1 sequencing

Primer Name Sequence 5'  3' Tm (°C)

Tps1_Scr_1 CCCATTCCCGCCATGGGA 61

Tps1_Scr_2 TCGCGATGGCATGAACCTG 58

Tps1_Scr_3 TCAGTGACCTGCTGAAGGC 57

Tps1_Scr_R CCACTGCCCTTGATCACCA 58

Table 8: Oligonucleotides used for quantitative real-time PCRs in human samples

Tm Amplicon Gene Primer Name Sequence 5'  3' (°C) size (bp) ATP6V0D1_RT_F TGCGGCTTTTTTCCAGGACT ATP6V0D1 57 73 ATP6V0D1_RT_R GGTGTTGCGGATGATCTCGAT GAPDH_RT_F TGCACCACCAACTGCTTAGC GAPDH 58 87 GAPDH_RT_R GGCATGGACTGTGGTCATGAG GBA_RT_F TGGGTACCCGGATGATGTTA GBA 56 110 GBA_RT_R AGATGCTGCTGCTCTCAACA GNS_RT_F CCCATTTTGAGAGGTGCCAGT GNS 58 79 GNS_RT_R TGACGTTACGGCCTTCTCCTT GRN_RT_F TCTGGACAAATGGCCCACA GRN 57 103 GRN_RT_R CCCTGAGACGGTAAAGATGCA HEXA_RT_F CAACCAACACATTCTTCTCCA HEXA 55 91 HEXA_RT_R CGCTATCGTGACCTGCTTTT HPRT_RT_F TGACACTGGCAAAACAATGCA HPRT 58 94 HPRT_RT_R GGTCCTTTTCACCAGCAAGCT LAMP1_RT_F ACGTTACAGCGTCCAGCTCAT LAMP1 58 78 LAMP1_RT_R TCTTTGGAGCTCGCATTGG MCOLN1_RT_F TTGCTCTCTGCCAGCGGTACTA MCOLN1 59 89 MCOLN1_RT_R GCAGTCAGTAACCACCATCGGA NAGLU_RT_F CAGAAGGAAGGAGCAGGAGT NAGLU 57 101 NAGLU_RT_R ATGTTCCCGAGGCTGTCAC STX4_RT_F TTTTCTGGCTACCGAAGTGG STX4 55 142 STX4_RT_R CTTCTTCCTCGCCTTCTTCTG TFEB_RT_F CCAGAAGCGAGAGCTCACAGAT TFEB 58 71 TFEB_RT_R TGTGATTGTCTTTCTTCTGCCG TPP1_RT_F GATCCCAGCTCTCCTCAATACG TPP1 56 100 TPP1_RT_R GCCATTTTTGCACCGTGTG

Material and Methods

Table 9: Oligonucleotides used for quantitative real-time PCRs in murine samples

Tm Amplicon Gene Primer Name Sequence 5'  3' (°C) size (bp) Treh_rt_f ATGCTAATCTGCGTGTCTGG Treh 55 129 Treh_rt_r CAATGAAGGGGTGCTTTGAG Gapdh_rt_f GTATGACTCCACTCACGGCAAA Gapdh 56 54 Gapdh_rt_r TTCCCATTCTCGGCCTTG Hprt_rt_f CCCCAAAATGGTTAAGGTTGC Hprt 55 76 Hprt_rt_r AACAAAGTCTGGCCTGTATCC

3.5. Software, online tools and databases

Software . ApE v2.0.36 (plasmid editor) Wayne Davis . Cn3D v4.3 (macromolecular structure viewer) NCBI . EndNote X5 (reference organization) Thomson Research . Gen5™ v1.09 (plate reader) BioTek . Image Lab 3.0 (gel imaging) Biorad . Leica LCS confocal software Leica . Office 2007 Professional (word processing etc) Microsoft . Photoshop CS4 (image processing) Adobe . SDS software v1.2 (qPCR analysis) Applied Biosystems

Online tools . Oligo Analyzer 3.1 (primer design) . PoPMuSiC v.2.1 (energetic analysis) . Primer3Plus (primer design)

Databases . ENSEMBL www.ensembl.org . Medline www.ncbi.nlm.gov/PubMed . NCBI www.ncbi.nlm.nih.gov . OMIM www.ncbi.nlm.nih.gov/omim . UCSC Genome Browser www.genome.ucsc.edu/

Material and Methods

3.6. Eukaryotic cell culture procedures

In order to avoid any contamination with fungi or bacteria, all cell culture work was carried out under a laminar-flow tissue culture hood using only sterile solutions and materials. To further increase protection of cells from contamination, penicillin and streptomycin were added to the culture medium. Fetal bovine serum served as an important source of essential growth factors.

3.6.1. Cell culture of primary fibroblasts

Fibroblasts were grown as adherent cultures and cultured at 37°C in 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, HyClone), supplemented with 20% FBS, 1% L-Glutamin, 100 U/ml Penicillin and 100 µg/ml Streptomycin. Tissue culture dishes with a diameter of 6 cm, 10 cm or 15 cm were used. Cells were grown to confluence, then washed with PBS and released from the bottom of the dish by incubation with trypsin. Trypsinization was stopped by addition of fresh culture medium. Cells were then centrifuged at 1000 rpm for 3 min and resuspended in fresh medium. Depending on the cell division rate of the respective cell line, the fibroblasts from one culture dish were split in two to three new dishes of the same size or one dish of the next bigger size respectively.

To store the fibroblasts for further experiments, cells were washed with PBS and trypsinized. After addition of culture medium and centrifugation at 1000 rpm for 3 min, the cell pellet was suspended in a sterile mix consisting of 90% normal cell culture media and 10% dimethylsulfoxide (DMSO) (chapter 3.3.2) and frozen at -196°C in liquid nitrogen. At any time, these aliquots can be thawed and cells can be grown again in culture medium using the conditions described above.

3.6.2. Cell culture of HeLa cells HeLa cells were grown as adherent cultures in medium described in chapter 3.3.2.

Cells were kept in an atmosphere with 5% CO2 at 37°C. Tissue culture dishes with a diameter of 10 cm or 15 cm were used.

Material and Methods

Cells were grown to confluence, then washed with PBS and released from the bottom of the dish by incubation with trypsin. Trypsinization was stopped by addition of fresh culture medium. Cells were then centrifuged at 1000 rpm for 3 min in order to get rid of the trypsin and resuspended in fresh medium. HeLa cells from one 10 cm culture dish were split in six to ten new 10 cm dishes.

3.6.3. Cell culture of HEK 293T cells HEK 293T cells were grown in medium described in chapter 3.3.2 in an atmosphere with 5% CO2 at 37°C. Cells were grown to confluence, then carefully washed with PBS and released from the bottom of the dish by washing with PBS. No trypsin was necessary since HEK 293T cells do not adhere firmly to standard cell culture dishes. After centrifugation at 1000 rpm for 3 min, cells were resuspended in fresh medium. Tissue culture dishes with a diameter of 10 cm or 15 cm were used and split into three to four dishes at confluence.

3.6.4. Treatment of fibroblast cell lines with chemical substances Primary fibroblasts from MPS III patients and control donors were treated with trehalose or sucrose. Trehalose and sucrose were dissolved in regular cell culture medium as described in chapter 3.3.2 and filtered through a sterile filter membrane with 0.22 µm pore size before use. Fibroblasts were then incubated with the respective drug at 37° C in 5% CO2 for varying time periods.

For GAG-clearance experiments fibroblasts were treated with a concentration of 100 mM trehalose. This concentration was based on previous experiments of the group and literature findings. For other experiments concentrations from 25 mM to 125 mM sugar were used. Regular medium served as control treatment.

3.7. DNA techniques 3.7.1. Determination of DNA concentration The concentration of DNA samples was measured using the NanoDrop ND-1000 spectrophotometer. 1.5 μl of DNA solution were applied and the absorptions at 260

Material and Methods nm and 280 nm were measured. 1 µl of solvent served as blank. The ratio of the absorbance at 260 nm and 280nm (A260/280) was used to assess automatically the purity of the nucleic acids in the analyzed solution. The optimal ratio for DNA samples is ~1.8, lower ratios can indicate contamination with proteins or organic compounds, higher ratios can indicate contamination with RNA.

3.7.2. Agarose gel electrophoresis Agarose gel electrophoresis is a technique to separate nucleic acids fragments in an electric field depending on their size or charge. The underlying principle is the migration of DNA fragments towards the + – pole in an applied electric field due to the negative charge of the ribose-phosphate backbone of DNA. Agarose gel electrophoresis was used to separate PCR products or identify correct clones from cloning experiments. 1% standard gels were prepared by dissolving 1.5 g agarose in 150 ml TBE buffer under heating in a microwave. After a short cool-down ethidium bromide was added to a final concentration of 1µg/ml. The 1% agarose solution was poured into an appropriate gel tray and after solidifying transferred to a gel electrophoresis chamber. The gel was covered in 1x TBE buffer, DNA samples were mixed with DNA loading buffer and pipetted into the wells of the gel. Subsequently fragments were separated at 120 V for ~20 minutes. A 1 kb DNA ladder (Fermentas) was run together with the samples in a separate lane in order to estimate the size of the DNA fragments.

3.7.3. Extraction and purification of DNA from agarose gels After separating DNA into fragments in a 1% agarose gel (chapter 3.7.2), bands were visualized under UV light (wave length 302 nm). The band containing the respective DNA was cut out with a sharp blade and transferred to a microcentrifuge tube. Extraction and purification was then carried out using the QIAquick Gel Extraction Kit (Qiagen) following the manufacturer’s instructions. DNA was eluted from the column with an appropriate volume of TE buffer.

Material and Methods

3.7.4. Polymerase chain reaction (PCR) Polymerase chain reaction is an enzymatic method to specifically amplify certain regions of a single stranded DNA fragment using site specific oligonucleotides (primers). When the primer recognizes the template and anneals to the recognition sequence, a DNA polymerase synthesizes a new complementary DNA strand beginning from the 3’-end of the primer (elongation). By raising the temperature the new double strand DNA will denature into two single strand DNA templates which are then open again for annealing of the primers (denaturation and annealing).

For PCRs in this work, the Phusion® High-Fidelity DNA Polymerase (Finnzymes) was used on an Applied Biosystems Veriti 96-well thermocycler. A standard cycling sequence is shown in table 10, a typical PCR reaction in table 11. Information about primers is given in chapter 3.4.

Table 10: Standard cycling sequence of a typical PCR reaction

Cycle Step Temperature Duration No. of cycles

Initial denaturation 98 ° C 30 s 1 Denaturation 98 ° C 10 s Annealing 64 ° C 30 s 30 Elongation 72 ° C 2 min 30 s Final elongation 72 ° C 10 min 1

Table 11: Components of a typical PCR reaction

Component Amount (µl)

cDNA template 1 Phusion Taq DNA Polymerase (0.03 U/µl) 0.3 dNTPs (final concentration 200 µM each) 0.4 5x Phusion HF Buffer 4 Fwd primer (final concentration 0.05 µM) 1 Rev primer (final concentration 0.05 µM) 1

H2O 12.3

Total volume 20

Material and Methods

3.7.5. Gene expression analysis by quantitative real time PCR Quantitative real time PCR (qPCR) was used to investigate the expression levels of lysosomal genes under a specific treatment. For this purpose total RNA was isolated from human fibroblasts or HeLa cells and reversely transcribed as explained in chapter 3.8.

When used for qPCR, cDNAs of the samples were diluted in a ratio of 1:4 in Milli-Q water. qPCR was performed on a Applied Biosystems 7300 Real Time PCR System using PerfeCTa® SYBR® Green FastMix®, ROX™. The components of a standard reaction are described in table 12. The standard qPCR conditions are described in table 13. Experiments were always conducted in triplicates. All primers used in qPCR including annealing temperature and amplicon size are given in table 6 and 7.

Expression analysis was performed using SDS v1.2 software (Applied Biosystems). The threshold cycle (TC) was extracted from the PCR amplification plot. The ΔTC value was used to describe the difference between the TC of a target gene and the TC of the housekeeping gene: ΔTC = TC (target gene) - TC (housekeeping gene).

Table 12: Example of a standard qPCR reaction

Component Amount (µl) cDNA template 0.5 H2O 1 PerfeCTa SYBR Green FastMix 7.5 Fwd primer 3 Rev primer 3

Total volume 15

Table 13: Standard qPCR conditions

Step Temperature Duration Replications

Heating 95 ° C 10 min 1 95 ° C 1 s Amplification 40 63 ° C 15 s 95 ° C 15 s 60 ° C 30 s 1 95 ° C 15 s

Material and Methods

3.8. RNA techniques

All RNA work was performed using RNAse free substances and material. Since RNA is less stable compared to DNA, all RNA work was conducted on ice or samples were otherwise stored at – 80°C.

3.8.1. Isolation of total RNA from cells and tissues Isolation of total RNA from cells or tissues was performed using QIAzol and the miRNeasy Kit (Qiagen) according to the manufacturer`s instructions.

3.8.2. Determination of RNA concentration Total RNA isolated from human fibroblasts and HeLa cells was used for quantitative real time PCR analysis (qPCR). The traditional method for assessing RNA concentration and purity is UV spectroscopy. The RNA samples were measured using the NanoDrop ND-1000 spectrophotometer. 1.5 μl of RNA solution were applied and the absorptions at 260 nm and 280 nm were measured. 1 µl of solvent served as blank. The ratio of the absorbance at 260 nm and 280 nm (A260/280) was used to assess automatically the purity of the nucleic acids in the analyzed solution. The optimal ratio for RNA samples is ~2.0, lower ratios can indicate contamination with other molecules like DNA, proteins or organic compounds.

3.8.3. Reverse transcription of RNA into cDNA Since RNA cannot be used as a template in a quantitative real time PCR (qPCR) analysis, single-stranded RNA templates were first reversely transcribed into double- stranded complementary DNA (cDNA). For this purpose, the QuantiTect Reverse Transcription Kit (Qiagen) was used following the manufacturer`s instructions. A DNAse digestion step was included in the kit in order to avoid contamination with genomic DNA. For each individual sample 1000 ng of total RNA were transcribed in a total volume of 20 μl.

Material and Methods

3.9. Cloning procedures and retrovirus preparation

Figure 5: Summary of all cloning and retrovirus production steps

Material and Methods

3.9.1. TOPO Cloning Cloning of plasmids was performed using the Zero Blunt® TOPO® PCR Cloning Kit (Invitrogen). This technique provides an efficient one-step cloning strategy for the direct insertion of blunt-end PCR products into a plasmid vector. It uses the vaccinia virus derived enzyme DNA topoisomerase I that can function as both a restriction enzyme and as a ligase. The plasmid-vectors are supplied linearized with DNA topoisomerase I covalently bound to the 3´ phosphate of the extending 5´-CCCTT-3´ sequence of each strand of the vector. After addition of a Taq-amplified blunt PCR product to the reaction solution, topoisomerase I forms a covalent bound between the overhanging adenosine rest of the PCR-fragment and a tyrosyl residue of the enzyme and cleaves the DNA fragment with the vector. The Topo vector used in this work was pCR®-Blunt II-TOPO®. Figure 6 shows a map of the vector.

Figure 6: pCR®-Blunt II-TOPO vector map

Before Tps1 was inserted into the pCR-Blunt II-TOPO vector, Xho I and EcoR I restriction overhangs were added via HiFi-Taq PCR. The used primers were Tps1_EcoRI_F and Tps1_XhoI_R using as template a wild type Drosophila

Material and Methods melanogaster cDNA. That cDNA was retrotranscribed as described in chapter 3.8.3 from a total Drosophila melanogaster RNA extract kindly provided from the laboratory of Juan Botas at BCM. The PCR reactions were set in 20 µl reaction volume using 1 µl template DNA as described in chapter 3.7.4. The correct size of the PCR product was checked by agarose gel electrophoresis. The TOPO cloning reaction was set as shown in table 14. All components of the cloning reaction were mixed gently and incubated for 25 min at room temperature. Afterwards the TOPO construct was chemically transformed into chemo-competent DH10beta cells as described in chapter 3.9.2.

Table 14: Standard cloning reaction

Reagent Amount (µl)

TOPO® Vector 1

Salt solution 1

Fresh PCR product 2

H2O 2

Total volume 6

3.9.2. Transformation This technique was frequently used to transform TOPO constructs or ligation reactions into DH10beta chemo-competent cells. For this purpose one vial (50 µl) of DH10beta cells was thawed on ice and mixed with the 6 µl cloning reaction or 20 µl ligation reaction respectively. Following incubation on ice for 10 min, cells were transformed by heat shock for 1 min at 42°C. The tubes were kept on ice for another 2 min, then 300 µl S.O.C medium were added. Next, tubes were incubated for at least 1 h at 37°C shaking horizontally in a thermomixer at 800 rpm.

The tubes were then centrifuged for 3 min at 6000 rpm, the supernatant removed by inversion and the pellet subsequently resuspended in the remaining medium. The bacteria were dispersed on agar-plates containing kanamycin or ampicillin in a final concentration of 50 µg/ml and incubated over night at 37°C.

Material and Methods

The next day at least 12 colonies per construct were picked with a pipette tip, put into 3 ml of Luria Broth containing 1µg/ml kanamycin or ampicillin and incubated again over night at 37°C.

3.9.3. Mini preparation of plasmids Plasmids were isolated from bacterial culture by mini preparation. For this purpose 1.5 ml of the overnight incubated bacteria containing medium were transferred into a microcentrifuge tube and centrifuged for 3 min at 6000 rpm. The pellet was resuspended thoroughly in 200 µl P1 resuspension buffer (Qiagen), followed by the addition of 200 µl P2 lysis buffer (Qiagen). The tubes were mixed by inversion, afterwards 200 µl P3 neutralization buffer (Qiagen) was added. After inverting the tubes gently several times, they were centrifuged at 13 000 rpm for 15 min at 4°C. The supernatant was transferred to a new tube and mixed with 800 µl cold ethanol 100%. Then, samples were incubated for 1 h at – 20°C and subsequently centrifuged at 13 000 rpm for 15 min at 4°C. The supernatant was replaced by 500 µl cold ethanol 70% and centrifuged for another 5 min at 13 000 rpm and 4°C. Any trace of ethanol was eliminated, the pellet dried at 37°C and finally resuspended in 50 µl Milli- Q water. DNA concentrations were measured by NanoDrop as described in chapter 3.7.1.

3.9.4. Enzymatic restriction digestion Enzymatic restriction digestion was frequently used for analytical digestion of mini preparations in order to identify correct clones of cloning reactions and for the preparative digestion of site-specific cloning reactions for further procedure. Restriction endonucleases cut double- or single stranded DNA by hydrolyzing phosphodiester bonds between two nucleotides. Each enzyme recognizes specific nucleotide sequences which are called restriction sites. 2 µl of mini preparations were restricted for analytical digestions and 10 µg of DNA for preparative digestions, respectively. For this purpose reactions were incubated for 90 min at 37°C. Examples of typical restriction reactions for analytical and preparative digestions are given in table 15.

Material and Methods

Table 15: Restriction digestion reactions for analytical and preparative digestions

Analytical digestion Preparative digestion

Component Amount

10x NEBuffer 2 2 µl 3 µl

10x BSA 2 µl 3 µl

EcoR I 0.6 µl 1.5 µl

Xho I 0.6 µl 1.5 µl

Plasmid 2 µl 2 µg

H2O 12.8 µl to a total volume of 30 µl

Total volume 20 µl 30 µl

Preparative digestion products were loaded on an agarose gel and extracted as described in chapter 3.7.3. Extracts were subsequently ligated as described in chapter 3.9.7.

3.9.5. Identification of correct clones and sequencing Analytical digestions from mini preparations were loaded on an agarose gel and analyzed as described in chapter 3.7.2. Mini preparations of positive clones were sequenced by Genewiz Inc. Custom made primers used for sequencing are listed in chapter 3.4.

3.9.6. Midi preparation of plasmids To isolate plasmid DNA out of 300 ml of bacterial cultures, the PureLink™ HiPure Plasmid Filter Midiprep Kit (Invitrogen) was used following strictly the manufacturer’s protocol.

Material and Methods

3.9.7. Ligation and cloning into the pMSCVneo vector After the enzymatic restriction digestion of plasmids (chapter 3.9.4) and subsequent extraction and purification from agarose gels (chapter 3.7.3), the concentration of the eluates was determined on a NanoDrop (chapter 3.7.1). Then the ligation reaction was performed using the Quick Ligation Kit (New England Biolabs). pMSCVneo vector and insert were incubated in a vector:insert ratio of 1:3 for 30 min at room temperature. After ligation plasmids were transformed into DH10beta chemo-competent cells Figure 7: pMSCVneo vector map as described in chapter 3.9.2.

3.9.8. Retrovirus construction and transduction of primary fibroblasts To insert genes of interest into the genome of primary fibroblast cell lines, a system to produce VSV-G-pseudotyped MSCV based retroviruses was used. HEK 293T cells were co-transfected with three plasmids: the pMSCVneo vector containing the gene of interest, Gag/pol and VSV-G. Gag and pol encode for two major proteins in the retroviral genome, gag for core proteins and pol for reverse transcriptase and integrase functions. VSV-G (Vesicular stomatitis virus) codes for an envelope protein and forms the coat of the virion. Only cells which integrate all three vectors into their genome are able to produce retroviruses.

The triple transfection was performed with Lipofectamine™ LTX and Plus Reagent (Invitrogen). For each transfection reaction conducted in a 10 cm tissue culture dish 3 ml Opti-MEM medium were mixed with 30 µl Plus Reagent and 5 µg DNA of each of the three plasmids. The mixture was incubated for 5 minutes at room temperature,

Material and Methods afterwards 90 µl of Lipofectamine LTX were added and the mix incubated for another 30 minutes at room temperature. A control reaction was always carried out transfecting HEK 293T cells with gag/pol, VSV-G and the pMSCVNneo vector without any insert. Table 16 shows the components of a standard triple transfection reaction.

Table 16: Components of standard triple transfection reaction

Transfection Control transfection

Component Conc. Amount

Opti-MEM® 3 ml 3 ml Plus Reagent 30 µl 30 µl Incubated for 5 minutes at room temperature

Gag/pol 1190 ng/µl 4.2 µl 4.2 µl VSV-G 1670 ng/µl 3.0 µl 3.0 µl pMSCVneo_Tps1 1390 ng/µl 3.6 µl - pMSCVneo 2080 ng/µl - 2.4 µl Incubated for 30 minutes at room temperature

In the meanwhile, HEK 293T cells were collected from their tissue culture dishes, counted and resuspended in transfection medium (chapter 3.3.2). After 30 minutes 2 x 106 HEK 293T cells in 8 ml transfection medium were added to the mix in the 10 cm transfection dishes. Now the triple transfection took place and HEK 293T cells started to produce retroviruses while incubated at 37° C in 5% CO2.

After 48 h of incubation, the virion-containing supernatant was collected and filtered through a sterile filter membrane with 0.45 µm pore size in order to remove cells from the medium. Then the virus containing medium was added to the target cells cultured in 6 cm culture dishes, plates were sealed carefully with parafilm and centrifuged for one hour at 3 000 rpm. Afterwards the transduced fibroblasts were taken back to the incubator and cultured under normal conditions. 6 h after the transduction the virus- containing medium was replaced by normal cell culture medium.

All steps were carried out with high caution since the retroviral particles are highly infectious to any mammalian cell.

Material and Methods

3.10. Other molecular biology methods 3.10.1. Cell Imaging 3.10.1.1. Immunofluorescence Immunohistochemistry is a technique used to assess the presence of a specific protein or antigen in cells by using a specific antibody, which binds to it, thereby allowing visualization and examination under a microscope.

HeLa cells overexpressing TFEB-3xFLAG were grown as adherent cultures on coverslips in a 24-well plate at 37 °C in 5% CO2. At 40-60 % confluence cells were washed three times with PBS. To ensure free access for the antibody to its antigen cells need to be fixed and permeabilized. Therefore coverslips with cells were incubated with 4% paraformaldehyde for 10 min at room temperature. The cells were then washed two times with a solution consisting of 0.1% saponin in PBS. For permeabilization and for blocking of unspecific antigen binding sites, the cells were then incubated with blocking solution (0.1% Saponin, 10% FBS, in PBS, chapter 3.3.1) for one hour at room temperature.

In a next step cells were incubated with the primary antibody, which binds to its specific antigen diluted 1:200 in blocking solution for three hours at room temperature. Unbound antibody was afterwards removed by washing three times with 0.1% saponin in PBS, 10 min each wash.

The coverslips were then incubated with a secondary antibody that is an antibody against the primary antibody. This antibody is labeled with a fluorophore that can be made visible by laser excitation under a microscope. The secondary antibody was diluted 1:1000 in 0.1% saponin in PBS, the coverslips were incubated for one hour at room temperature in the dark.

Finally unbound secondary antibody was removed by washing the coverslips in 0.1% saponin in PBS, subsequently they were mounted on microscope slides with a mounting media containing DAPI.

Finally, cells were examined under a confocal microscope.

Material and Methods

3.10.1.2. Confocal microscopy Confocal laser scanning microscopy is a valuable tool for obtaining high-resolution images. The key feature of confocal microscopy is its ability to produce high- resolution images of thick specimens by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. Images are taken point-by-point and reconstructed with a computer.

Confocal images were taken with an inverted Leica TCS SP5 laser-scanning microscope. Fluorochrome Alexa 488 was excited with the 488 nm Argon laser and emission fluorescence collected in the green channel.

3.10.2. Protein quantification The quantitative determination of the protein concentration was carried out using the Pierce® BCA Protein Assay Kit, that is working according to the method described by Smith (Smith et al., 1985). This method is based on the reduction of Cu2+ ions from cupric sulfate to Cu1+ by the peptide bonds. The amount of developing Cu1+ ions is proportional to the amount of protein present in the solution. Subsequently two molecules of bicinchoninic acid react with each Cu1+ ion, forming a purple-colored product. That product strongly absorbs light at a wavelength of 562 nm.

5 μl of each protein sample to be analyzed was pipetted in triplicates in a 96-well plate; then 200 μl of BCA working solution was added to each well. After incubation for 30 min at 37°C, absorption of the samples was measured against a BCA working solution blank at a wavelength of 562 nm. To calculate the protein concentrations in the unknown samples, the obtained values were compared to the values obtained from a standard curve, which was prepared with a dilution series of bovine serum albumin (BSA) of known concentrations.

3.11. Working with mice 3.11.1. Mouse strains and housing conditions C57BL/6 wild-type mice, between four and six weeks of age, were purchased from the main animal facility of BCM. Animals were housed with three animals per cage in

Material and Methods the animal facility of the Neurological Research Institute. Mice were maintained on a 12-h light/12-h dark cycle under a standard rodent diet (2920 Teklad, Harlan) and water ad libitum.

All research and animal care procedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee (protocol number AN-5691).

3.11.2. Preparation of mouse tissues For the preparation of mouse organs, the animals were euthanized with Isoflurane. Mice were decapitated with surgical scissors and brains extracted from the cranium. Then, mice were placed on their dorsal side and upper and lower limbs were fixed using needles. A longitudinal section was cut through the abdominal wall and the rib cage to open the animal. To measure intestinal trehalase activity, the upper part of the jejunum was dissected. 5 cm of small intestine were isolated, beginning approximately 2 cm aboral of the stomach. The intestinal pieces were cleaned from remaining feces. All tissues (brain, liver and small intestine) were put into 1.5 ml tubes, snap frozen in liquid nitrogen and placed at -80°C for extended storage.

A little piece of small intestine was used for RNA extraction. For preparation, it was homogenized in QIAzol reagent using a cordless motor and disposable pestles and subsequently snap frozen and stored at -80°C. Total RNA was extracted as described in chapter 3.8.1.

3.12. Specific methods 3.12.1. Determination of glycosaminoglycans in cells 3.12.1.1. Cell culture and treatment of fibroblasts MPS III patient-derived fibroblasts were cultured in 10 cm tissue culture dishes. For these experiments the cells of one 10 cm dish were split and plated on three 10 cm dishes. One of them was treated with trehalose 100 mM in the medium, another one – treated with medium without trehalose – served as a control. The cells of these 10 cm dishes were split and plated on three 6 cm dishes each at day four and incubated

Material and Methods with or without trehalose containing media for another four days. In this way, three biological replicates for each condition were acquired.

3.12.1.2. Extraction of glycosaminoglycans from cells After the treatment with trehalose, cells were removed from the incubator and washed once with PBS. Then 600 µl Papain extraction reagent (chapter 3.3.1) were added to each 6 cm dish. The plates were sealed with parafilm to avoid extensive evaporation and incubated for three hours at 65°C with occasional mixing. The reagent detaches the cells, extracts the GAGs and makes them soluble.

After three hours the remaining reagent from each 6 cm dish was transferred to a microcentrifuge tube and centrifuged for 10 min at 10 000 rpm. The intracellular GAGs were now dissolved in the supernatant and ready for further analysis.

3.12.1.3. Measurement of glycosaminoglycans For the measurement of heparan sulfate a slightly modified protocol of the Blyscan™ Glycosaminoglycan Assay (biocolor) was used. Principle of this assay is the specific binding of 1,9-dimethylmethylene blue to glycosaminoglycans. During the procedure soluble GAGs form a complex with the 1,9-dimethylmethylene blue dye that precipitates under centrifugation. The bound dye can be released by dissolving the pellet in a dissociation reagent. The developing color is proportional to the amount of GAGs in the sample and can be measured spectrophotometrically at 656 nm.

100 µl of each sample to be analyzed were pipetted into a microcentrifuge tube, then 1 ml of Blyscan Dye Reagent was added. The samples were mixed thoroughly by inverting the tubes and placed on a gentle mechanical shaker for 30 min. During this time period a glycosaminoglycan-dye complex formed and precipitated from the soluble unbound dye. 100 µl Papain extraction reagent served as blank, a dilution series with GAGs of known concentrations was prepared as a standard. All samples, blanks and standards were run in duplicates.

After 30 min the tubes were centrifuged for at least 15 min at 14 000 rpm (maximum speed) at 4°C in order to firmly pack the GAG-dye complex at the bottom of the tube.

Material and Methods

Subsequently the unbound dye reagent was drained, first with a pipette with 1000 µl tip, the remaining 100 µl then with a 1 ml syringe with needle. The complex gets back in solution very easily, so spinning and immediate draining is important in order to avoid loss of pellet. Repetitive centrifugation can be necessary.

Afterwards 250 µl Blyscan Dissociation Reagent were added to the dry pellet in each tube. The tubes were then vortexed until all bound dye had been dissolved. 200 µl of each sample were then transferred to individual wells of a 96-well plate and read on a microplate reader at 656 nm. To calculate the GAG concentrations in the unknown samples, the obtained values were compared to the values obtained from a standard curve, which is shown in Figure 8.

3,500

3,000

y = 0,5422x + 0,1764 2,500

2,000

1,500

1,000 Absorbance (656 nm) (656 Absorbance 0,500

0,000 0,0 1,0 2,0 3,0 4,0 5,0 6,0 GAGs (in µg) Figure 8: Glycosaminoglycan standard curve Each data point represents the mean ±SD of 8 independent measurements.

3.12.1.4. Normalization on DNA content Because of differences in the growth rate of treated and untreated cells and individual growth rate of each culture dish, the obtained results had to be normalized. Normalization on protein content was not suitable in this case for two reasons: papain, the integral part of the extraction reagent, is a protease, so significant amounts of non-cellular protein were already present in the buffer. In addition to that, both EDTA and cysteine which are both necessary for stabilizing the papain in the extraction buffer, are known to interfere with standard protein assays like the used

Material and Methods

Pierce BCA protein assay. Thus, obtained measurements were normalized to DNA content.

The remaining sample from the extraction step was used for normalization. The remaining amount of liquid in each tube was determined by weighing each microcentrifuge tube on a micro scale. The tubes were vortexed to resuspend the formed pellet, then 1 ml of 100% ethanol was added and mixed by inversion. After centrifugation for 10 min at 13 000 rpm at 4°C the ethanol 100% was drained and 1 ml of 70% Ethanol added. After another spin for 5 min at 13 000 rpm at 4°C the pellet was dried and finally resuspended in 100 µl Milli-Q-water. The DNA concentration was then measured spectrophotometrically on a NanoDrop as described in chapter 3.7.1.

3.12.1.5. Calculation of GAG-levels The concentration of glycosaminoglycans can be calculated as follows:

cGAG = concentration of GAGs [in ng GAGs/ng DNA] GAG = concentration of GAGs measured in extracted sample [in µg/100 µl] DNA = DNA concentration in 100 µl determined by NanoDrop [in ng/µl]

Vr = remaining volume after GAG assay [in µl]

3.12.2. Determination of N-acetylglucosaminidase activity Several spectrophotometrical and flourometrical methods are available for the measurement of the activity of the N-acetylglucosaminidase (NAG) in fibroblasts. Principle of all assays is the incubation of a cell extract with a substrate specific for the enzyme to be measured. The enzyme digests the substrate effecting a change in color that can be measured spectrophotometrically or flourometrically.

Material and Methods

All currently available methods are not suitable for high-throughput screenings since a large amount of cells is needed for each tested condition. Moreover all current methods require cell disruption by sonication or freeze-thawing which is difficult to standardize. The normalization on protein content introduces another source of error. For these reasons a protocol for a rapid and sensitive NAG assay was developed which can be carried out completely in a 96-well plate.

4MU-alpha-N-acetyl-D-glucosaminide (Moscerdam substrates) served as the specific substrate for the NAG releasing the fluorescent 4–Methylumbelliferyl (4MU). While the NAG as a lysosomal enzyme works best at an acidic pH, the fluorescence of 4MU can be measured best at pH 10.8. A substrate buffer with pH 4.5 carries the substrate but also serves to permeabilize the cells. A protease inhibitor was added in order to prevent degradation of the (mutated) enzyme during the 17 h incubation.

Experimental procedure: Sample fibroblasts were trypsinized, counted with a Neubauer hemocytometer and diluted in their standard medium (chapter 3.3.2) to a concentration of 105 cells/ml. Then 100 µl of this suspension were plated in each well of a 96-well plate (104 fibroblasts per well) and incubated at 37°C and 5% CO2 over night to achieve cell attachment. Special white plates for fluorescence were used. 4 wells were plated for each condition to be tested. In order to prevent edge effects, no fibroblasts were plated in columns 1 and 12, instead they were filled with 100 µl PBS. Rows A and H served as background controls (as described below).

The next day, the medium was replaced by 100 µl of medium containing the drug to be tested or 100 µl control medium. The cells were then treated according to the respective protocol while incubated at standard conditions (37°C and 5% CO2)

After treatment, the medium was drained and the cells were washed three times with PBS. Then 50 µl of substrate solution (2 mM 4MU-alpha-N-acetyl-D-glucosaminide in 0.2 M Na-acetate buffer pH 4.5 with 0.5% Triton X-100) and 1x protease inhibitor (chapter 3.3.1) were added. Rows A and H only served as background controls. For this purpose, they were incubated with 50 µl buffer without substrate. Plates were sealed with plastic foil to prevent evaporation, covered in aluminum foil in order to protect them from light and incubated for 17 h at 37°C.

Material and Methods

Next, 150 µl 0.2 M glycine buffer pH 10.8 were added to each individual well and the released fluorescence was measured on a plate reader using an excitation wavelength of 360 nm and measuring the emission at 460 nm.

NAG-activity was then expressed as a fluorescence ratio between treated and untreated and compared to the activity of fibroblast wild type controls respectively.

3.12.3. Determination of trehalose in cells and tissues 3.12.3.1. Sample preparation of cells HeLa cells were incubated with trehalose containing medium or control medium respectively. After incubation the cell culture medium was removed and cells were washed three times with PBS. Cells were then trypsinized, collected and centrifuged for 3 min at 1000 rpm. The pellet was resuspended in 900 µl homogenization buffer (chapter 3.3.1), and subsequently sonicated two times for ten seconds in ice water. 15 µl of this solution were used to determine protein content as described in chapter 3.10.2. The remaining solution was incubated at 99°C for 10 min to denature proteins. The mixture was then centrifuged for 3 min at 10 000 rpm.

3.12.3.2. Sample preparation of mice tissues and removal of free glucose ~100 mg liver tissue or one hemisphere of a brain were put in 400 µl of homogenization buffer (chapter 3.3.1) and homogenated by sonication in ice water.

Since glucose is the main sugar in mammalian tissues, free glucose had to be removed before assaying levels of trehalose. Therefore, 200 µl of tissue homogenate were mixed with 200 µl of alkaline borohydrate (10 mg/ml sodium borohydrate in 50 mM NaOH). The mixture was stirred vigorously and incubated shaking for 30 min at 40°C to effect complete reduction of reducing-sugars to sugar alcohols. Subsequently, excess borohydrate was removed by adding 500 µl 200 mM acetic acid. Finally, the pH was adjusted to approximately 7 with 200 µl of 2 M imidazole buffer.

The samples were incubated at 99°C for 10 min to denature proteins and afterwards centrifuged for 3 min at 10 000 rpm.

Material and Methods

3.12.3.3. Trehalose assay 430 µl aliquots of the supernatant from the centrifugation step of either cell or tissue preparation were placed into two tubes. To one of the tubes 5 µl trehalase (Sigma) were added to a final concentration of 0.05 U/ml in order to digest the present trehalose into two molecules of glucose. To the second tube 5µl of the carrier of trehalase were added (a buffer consisting of 50% glycerol, 1% Triton X-100 and 25 mM potassium phosphate pH 6.5, according to the manufacturer’s data sheet). This reaction served as a background reaction in order to determine the preexisting glucose in the mixture. Both aliquots were mixed thoroughly and incubated over night (15 h) at 37°C.

The next day, glucose was measured in both aliquots using a glucose assay reagent (Sigma). Principle of this assay is the phosphorylation of glucose by ATP catalyzed by hexokinase. Glucose-6-phosphate (G6P) is then oxidized to 6-phosphogluconate in the presence of oxidized nicotinamide adenine dinucleotide (NAD) in a reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). An equimolar amount of NAD is reduced to NADH during this oxidation. This results in an increase in absorbance at 340 nm that is directly proportional to the glucose concentration in the analyzed sample. (Bondar and Mead, 1974).

200 µl of each sample where incubated in cuvettes with a path length of 1 cm with either 1 ml glucose assay reagent or 1 ml deionized water for 20 min at room temperature. The cuvette with glucose assay reagent served as the test cuvette, the one with water served as a sample blank. Also one reagent blank was measured with 1 ml glucose assay reagent and 200 µl deionized water. After 20 min, the absorbance was measured at 340 nm against deionized water.

3.12.3.4. Calculation of trehalose levels The trehalose concentration is calculated as the difference of glucose between the sample incubated with trehalase (present trehalose was digested to glucose) and the sample without trehalase (only glucose background is measured), divided by two, since trehalose is a disaccharide of two glucose molecules.

Material and Methods

The glucose concentration in each sample is calculated as follows:

A = Spectrophotometrically measured absorbance at 340 nm

ATotal Blank = ASample Blank + AReagent Blank

A = ATest – ATotal Blank D = Dilution factor: D = 1.1/0.2 = 5.5 for tissue samples (from the glucose- removal step); no dilution for cell samples (D = 1) TV = Total Assay Volume [in ml] = 1.2 ml SV = Sample Volume [in ml] = 0.2 ml

MWglucose = Molecular weight glucose = 180.2 g/mole ε = Millimolar Extinction Coefficient for NADH at 340 nm = 6.22 d = Light path (cm) = 1 cm

The measured trehalose concentrations were normalized to protein content. Protein concentrations were measured as described in chapter 3.10.2.

The specificity of the used trehalase for trehalose was confirmed through the incubation with other disaccharides. The used trehalase did not hydrolyze sucrose, maltose and lactose.

3.12.4. Determination of murine intestinal trehalase activity Trehalase is an intestinal enzyme which catalyzes the catabolic conversion of trehalose into two molecules of glucose. Principle of the assay is the incubation of an intestine sample with or without trehalose for a certain period of time. Afterwards, the released glucose is measured and the activity can be calculated out of the glucose difference of reaction and background reaction.

Material and Methods

For this purpose, mice were euthanized and 5 cm of jejunum were dissected as described in chapter 3.11.2. The 5 cm of mouse intestine were homogenized by 10 s sonication in homogenization buffer (chapter 3.3.1).

200 µl aliquots of homogenate were placed into two tubes. To one of the tubes 28 µl

1.64M trehalose in H20 were added to a final concentration of 200 mM trehalose. To the second tube 28µl of H20 were added. In the first tube, the added trehalose is converted to glucose over time. The second tube serves as control reaction to determine the glucose background. Both aliquots were mixed thoroughly and incubated exactly 30 min at 37°C.

To stop the reaction immediately, the reaction tubes were snap frozen in liquid nitrogen and stored at -80°C until further processing. The frozen samples were incubated at 99°C for 10 min to denature proteins and afterwards centrifuged for 3 min at 10 000 rpm.

Glucose levels were measured with a glucose assay reagent (Sigma) as described in chapter 3.12.3.3. However, only 10 µl of sample were used instead of 200 µl in the trehalose concentration assay.

Protein concentrations were measured as described in chapter 3.10.2. The measured values were normalized on protein content and trehalase activity expressed as µmol trehalose/mg protein/h.

The linearity of the assay was tested. The assay was linear over a time span of incubation from 4 min up to 2 h in both of the tested samples (Fig. 9).

Material and Methods

8,00

7,00 y = 0,1034x - 0,0099 6,00 5,00 A 4,00 y = 0,0577x - 0,0095 B 3,00 Linear (A) 2,00 Linear (B)

Released glucose (in mg/ml) (in glucose Released 1,00 0,00 0 20 40 60 80 100 120 140 Incubation time (in min) . Figure 9: Standard curve for intestinal trehalase activity assay The linearity of the assay over time was comfirmed with two samples.

3.12.5. Mutation mapping and energetic analysis For the mapping of mutations on a three-dimensional model of the NAG enzyme, the conservation of the particular amino acid was determined in a first step by aligning the primary protein structure of the human NAG enzyme with the primary protein structure of the same enzyme of mouse, zebrafish, Xenopus, Drosophila melanogaster and Clostridium perfringens.

Using this alignment, the corresponding mutation in clostridium perfringens was determined for each human mutation of interest. Since to date no 3D structure of the human NAG enzyme has been published, all mutations were mapped in the 3D structure of the NAG enzyme of C. perfringens (Ficko-Blean et al., 2008) in order to determine whether they fall in residues likely involved in the enzymatic reaction or in structural parts of the proteins such as alpha-helices or beta-sheets.

On the base of the same three-dimensional structure of the NAG enzyme of C. perfringens (PDB ID: 2VCC) the changes in stability of the NAG protein depending on each mutation were predicted using the online tool PoPMuSiC v2.1 (http://babylone.ulb.ac.be/PoPV2a/).

Material and Methods

3.13. Statistical methods

Data are presented as mean ± standard deviation (SD) or standard error of the mean (SEM) as given in the text. Student’s t-test or Mann-Whitney U test were used to test whether two groups are statistically different from each other. Two levels of statistical significance were distinguished: p < 0.05 and p < 0.01. Significant differences are indicated with different numbers of asterisks within the respective Figures and/or are given within the text.

For assessment of reproducibility of the NAG-assay, Z’-factors were calculated according to the following equation:

‘SD’ and ‘Mean’ represent the standard deviation and the average of data points of samples, respectively, comparing two different levels of treatment (Zhang et al., 1999).

Results

4 RESULTS 4.1. Intracellular measurement of trehalose Although there are several studies that show effects of trehalose both in vitro and in vivo, it is not understood how trehalose is delivered into the cell. So far no trehalose specific transporters have been described in mammalian cells. To assess in a first step whether trehalose is able to penetrate human cell membranes and potentially cause an intracellular effect afterwards, intracellular trehalose was measured after treatment with trehalose.

For this purpose, one 15 cm tissue culture dish with HeLa cells was incubated for 72 h with medium containing trehalose in a final concentration of 100 mM. Another 15 cm tissue culture dish with HeLa cells served as a control and was incubated with normal medium without trehalose.

After incubation for 72 h at 37° C in 5% CO2, cells were washed three times with PBS in order to remove completely cell culture media containing trehalose. Subsequently, trehalose present in the cells was determined as described in chapter 3.12.3. Measured trehalose levels are shown in Figure 10. In trehalose-treated HeLa cells, 29.94 nmol trehalose/mg protein were detected, while no trehalose was detectable in the control cells.

35,00

30,00

25,00 20,00

15,00

10,00

5,00 Under limit trehalose (nmol/mg protein) (nmol/mg trehalose of sensitivity 0,00 Untreated 100 mM trehalose

Figure 10: Intracellular trehalose levels in HeLa cells Significant amounts of trehalose were found in HeLa cells after 72 h of treatment with 100 mM trehalose, while no trehalose could be detected in untreated cells.

Results

4.2. Determination of intracellular HS levels

Patients with Sanfilippo syndrome have mutations in genes coding for enzymes needed for the degradation of heparan sulfate. The intracellular accumulation of glycosaminoglycans leads finally to the destruction of the cell. One therapeutic strategy for MPS III is to reduce intracellular HS deposits.

To evaluate whether the treatment with trehalose can have an impact on intracellular HS deposits, MPS III patient-derived fibroblasts and control fibroblasts were treated with trehalose. Afterwards, intracellular levels of HS were analyzed.

4.2.1. GAG levels in MPS III patient-derived fibroblasts and WT fibroblasts MPS III patient-derived fibroblasts and fibroblasts from healthy control donors were cultured in 10 cm tissue culture dishes at 37° C in 5% CO2. For the determination of intracellular GAG levels, 4 days after the last splitting each 10 cm dish was split into three 6 cm tissue culture dishes and incubated for another 4 days. GAG levels were then determined in each of the three 6 cm dishes as described in chapter 3.12.1.

GAG levels were analyzed in fibroblasts derived from two MPS IIIA patients, 14 MPS IIIB patients, one MPS IIIC patient and one MPS IIID patient. Three cell lines from control donors served as wild type controls.

The average intracellular GAG levels of the three wild type controls were 14.22 (SEM: ±4.16) ng GAGs/ng DNA. In MPS III patients measured GAG levels were significantly higher: 52.66 and 53.28 ng GAGs/ng DNA in the MPS IIIA patients, 66.79 ng GAGs/ng DNA in the MPS IIIC patient and 37.87 ng GAGs/ng DNA in the MPS IIID patient (Fig. 11A). The average GAG levels in MPS IIIB patients were 35.07 (SEM: ±2.88) ng GAGs/ng DNA (Fig. 11B and 11C). A summary of the results is given in Figure 11 and table 17.

Results

A B

80 ** 45

** ) 70 ) 40

60 35 30 50 25

40 ng GAG/ng DNA GAG/ng ng ng GAG/ng DNA GAG/ng ng 20 30 15 20 10 10

GAG level (in (in level GAG 5 GAG level (in (in level GAG 0 0 A1 A2 C1 D1 Avg MPS IIIB WT MPS IIIA MPS IIIC MPS IIID WT

C 70

60 )

40 ng GAG/ng DNA GAG/ng ng 30

20 GAG level (in (in level GAG 10

0 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 WT 1 WT 2 WT 3 MPS IIIB WT

Figure 11: Heparan sulfate levels in MPS III fibroblasts and control fibroblasts A: HS levels in MPS IIIA, C and D fibroblasts and average HS levels of control fibroblasts. B: Average HS levels in MPS IIIB fibroblasts compared to WT controls.C: HS levels of each single MPS IIIB fibroblast cell line and WT fibroblast cell line. All values are the mean of at least three experiments ±SEM, as shown in table 17.

Results

Table 17: Heparan sulfate levels in MPS fibroblasts and control fibroblasts Mean HS levels for each cell line ±SEM corresponding to Figure 11. n gives the number of experiments done for each cell line.

Mean HS level Disease Cell line Mutation n SD SEM Avg SEM (in ng GAGs/ng DNA) A1 E447K, R245H 3 52.66 2.45 1.42 MPS IIIA 52.97 0.31 A2 S66W, V131M 3 53.28 3.77 2.18 B1 L35F, G292R 3 43.63 4.97 2.87 B2 Homoz W649C 3 21.69 5.62 3.25 B3 Homoz Y92H 6 49.59 8.97 3.66 B4 Homoz E336X 3 25.37 3.58 2.07 B5 T81A, Y140C 6 32.76 6.42 2.62 B6 V77G, H414R 9 27.79 4.98 1.66 B7 Homoz V501G 6 22.27 1.96 0.80 MPS IIIB 35.07 2.88 B8 Homoz V501G 3 38.43 1.26 0.72 B9 Y140C, W156C 3 32.41 1.55 0.89 B10 Homoz R626X 3 34.71 2.29 1.32 B11 L682R, Y140C 6 57.54 20.61 8.42 B12 Homoz E153K 6 32.25 5.11 2.08 B13 R297X, R643H 3 26.32 1.51 0.87 B14 Homoz P358L 5 46.19 9.92 4.44 MPS IIIC C1 Not genotyped 6 66.79 4.65 1.90 MPS IIID D1 Homoz R355X 3 37.87 1.80 1.04 WT1 6 21.11 1.65 0.67 WT WT2 3 14.80 1.59 0.92 14.22 4.16 WT3 3 6.75 1.18 0.68

4.2.2. GAG levels under trehalose treatment To investigate a potential trehalose-mediated reduction of intracellular GAGs, MPS III patient-derived fibroblasts and control fibroblasts were treated with trehalose. Subsequently, GAG levels were measured.

For this purpose patient and wild-type control fibroblasts – cultured in 10 cm tissue culture dishes – were split into three dishes. Two of them were then used for the experiment, i.e. one of them was incubated with medium containing trehalose in a concentration of 100 mM, and the other one was incubated with standard medium without trehalose. These 10 cm dishes were split into three 6 cm dishes each at day

Results four and the incubation with or without trehalose containing media was continued for another four days. The concentration of 100 mM trehalose in the culture medium was chosen based on published results of other research groups (Liu et al., 2005, Davies et al., 2006, Sarkar et al., 2007, Aguib et al., 2009, Casarejos et al., 2011) and unpublished results of our laboratory.

After a total of eight days of treatment intracellular GAG levels were measured in both trehalose treated and untreated fibroblasts as described in chapter 3.12.1. GAG levels were also analyzed after different periods of incubation in order to determine the optimal treatment time. With shorter incubation periods no significant effect on GAG levels could be observed (data not shown). Consequently, 8 days of treatment with trehalose in a concentration of 100 mM was selected as the condition for all further experiments.

The average GAG levels of wild type fibroblasts decreased under trehalose treatment from 14.22 (SEM: ±4.16) ng GAGs/ng DNA to 8.02 (SEM: ±4.47) ng GAGs/ng DNA (43.60% decrease on average).

GAG levels in trehalose treated fibroblasts decreased by 73.64% and 36.63% in the MPS IIIA cell line, 39.54% in the MPS IIIC cell line and 69.90% in the MPS IIID cell line.

In the MPS IIIB patient fibroblast samples a decrease of GAG levels after trehalose treatment was observed in 11 of the 14 samples. In three of the 14 MPS IIIB samples no difference was observed between treated and untreated cells (cell lines B2, B3 and B4). The average GAG levels in trehalose treated MPS IIIB patient fibroblasts were 27.48 (SEM: ±2.59) ng GAGs/ng DNA including the three non-responsive cell lines. On average, the GAG levels decreased 21.64% in MPS IIIB fibroblasts from 35.07 (SEM: ±2.88) ng GAGs/ng DNA to 27.48 (SEM: ±2.59) ng GAGs/ng DNA. When excluding the three non-responsive cell lines, HS levels were reduced by 28.55 % from 35.85 (SEM: ±3.05) ng GAGs/ng DNA to 25.61 (SEM: ±2.33) ng GAGs/ng DNA. All data are shown in detail in table 18. A summary of the experiment is given in Figures 12 – 14.

Results

A 80 n. s.

** ** ** ** ) 70 60 50 40 ng GAG/ng DNA GAG/ng ng Untreated 30 trehalose 100 mM 20

10 GAG level (in (in level GAG 0 A1 A2 C1 D1 Avg

MPS IIIA MPS IIIC MPS IIID WT B 120

** ** ** ** n. s.

100

60 GAG levels GAG 40

(in % compared to untreated) to compared % (in 20

0 A1 A2 C1 D1 Avg MPS IIIA MPS IIIC MPS IIID WT

Figure 12: HS levels under trehalose treatment in MPS IIIA, C and D fibroblasts HS levels in MPS IIIA, C and D fibroblasts under treatment with trehalose 100 mM compared to untreated cells. A: HS levels in absolute numbers. B: HS levels in % compared to untreated cells (**: p < 0.01; n.s. = not significant).

Results

A 80

* n. s. n. s. n. s. * ** ** ** * ** * * ** * n. s. ** **

) 70

40 ng GAG/ng DNA GAG/ng ng

GAG level (in (in level GAG 10

0 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 WT 1 WT 2 WT 3 MPS IIIB WT

untreated trehalose 100 mM

B 140

* n. s. n. s. n. s. * ** ** ** * ** * * ** * n. s. ** **

120

100

20 GAG levels (in % compared to untreated) to compared % (in levels GAG 0 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 WT 1 WT 2 WT 3 MPS IIIB WT

Figure 13: HS levels under trehalose treatment in MPS IIIB fibroblasts HS levels in MPS IIIB fibroblasts under treatment with trehalose 100 mM compared to untreated cells. A: HS levels in absolute numbers. B: HS levels in % compared to untreated cells. (*: p < 0.05; **: p < 0.01; n.s. = not significant)

Results

45 p=0.061 p=0.015 p=0.367

) 40 * 35 30 25

ng GAG/ng DNA GAG/ng ng 20 untreated trehalose 100 mM 15 10

GAG level (in (in level GAG 5 0 MPS IIIB MPS IIIB WT (w/o B2,B3,B4)

Figure 14: Average HS levels under trehalose treatment in MPS IIIB fibroblasts and WT Average HS levels in MPS IIIB fibroblasts and WT fibroblasts under treatment with trehalose 100 mM compared to untreated cells. The average HS levels in MPS IIIB fibroblasts is shown in the first column, while the second column gives the average HS levels in MPS IIIB fibroblasts, excluding the non-responsive cell lines B2, B3 and B4. The third column shows the average HS levels of WT controls.

Results

Table 18: HS levels in treated and untreated MPS III fibroblasts and WT controls Mean HS levels for each cell line ±SEM corresponding to Figures 12 – 14.

Untreated Trehalose 100 mM Cell line Mutation % of UT Change (%) p-value Mean SEM Mean SEM MPS IIIA A1 E447K, R245H 52.66 1.42 13.88 2.87 26 -74% < 0.001 A2 S66W, V131M 53.28 2.18 33.76 2.93 63 -37% 0.006 MPS IIIB B1 L35F, G292R 43.63 2.87 30.99 1.57 71 -29% 0.018 B2 Homoz W649C 21.69 3.25 21.64 1.67 100 0% 0.989 B3 Homoz Y92H 49.59 3.66 50.61 3.08 102 +2% 0.836 B4 Homoz E336X 25.37 2.07 30.73 1.2 121 +21% 0.088 B5 T81A, Y140C 32.76 2.62 25.09 2.00 77 -23% 0.042 B6 V77G, H414R 27.79 1.66 16.58 2.02 60 -40% 0.001 B7 Homoz V501G 22.27 0.80 12.68 0.61 57 -43% < 0.001 B8 Homoz V501G 38.43 0.72 34.00 0.30 88 -12% 0.005 B9 Y140C, W156C 32.41 0.89 27.09 1.43 84 -16% 0.034 B10 Homoz R626X 34.71 1.32 23.27 1.76 67 -33% 0.007 B11 L682R, Y140C 57.54 8.42 35.56 2.58 62 -38% 0.032 B12 Homoz E153K 32.25 2.08 28.02 1.66 87 -13% 0.022 B13 R297X, R643H 26.32 0.87 16.03 1.26 61 -39% 0.003 B14 Homoz P358L 46.19 4.44 32.42 2.69 70 -30% 0.029

Average : 35.07 2.88 27.48 2.59 78 -22% 0.061 Avg w/o B2/B3/B4 : 35.85 3.05 25.61 2.33 71 -29% 0.015 MPS IIIC C1 Not genotyped 66.79 1.90 40.38 1.51 60 -40% <0.001 MPS IIID D1 Homoz R355X 37.87 1.04 11.4 2.95 30 -70% 0.001 WT WT1 21.11 0.67 16.47 1.64 78 -22% 0.259 WT2 14.80 0.92 6.36 0.43 43 -57% 0.001 WT3 6.75 0.68 1.24 0.18 18 -82% 0.001 Average: 14.22 4.16 8.02 4.47 56 -44% 0.367

Results

4.3. GAG levels in trehalose-synthesizing MPS IIIB fibroblasts As described in chapter 4.2, a significant reduction of intracellular GAG deposits was only observed after 8 days of treatment with trehalose in a rather high concentration. To ascertain whether this long treatment is necessary due to slow intracellular metabolic processes or because of slow uptake of trehalose into the cell, Tps1, a gene from D. melanogaster coding for the enzyme trehalose-6-phosphate synthase 1, was cloned and transduced into one MPS IIIA and one MPS IIIB fibroblast cell line using a retroviral vector. Afterwards, the intracellular GAG levels were determined as described in chapter 3.12.1.

The experiment was also carried out as a proof of principle: according to the hypothesis that trehalose can reduce intracellular GAG deposits, trehalose- synthesizing cells should have lower GAG levels than cells that are not producing trehalose.

4.3.1. Cloning of Tps1 into the pMSCVneo vector While the main sugar used in the metabolism of mammals is glucose, trehalose is the main sugar in the hemolymph of several insects (Wyatt and Kalf, 1957). Drosophila melanogaster expresses the gene Tps1 which codes for the enzyme trehalose-6- phosphate synthase 1. With this enzyme the insect is able to synthesize trehalose out of two molecules of glucose. No homologous enzyme is described in mammals.

The Tps1 transcript is 2430 bp long and is located on the 2L arm of chromosome 2 of the Drosophila genome. For this work, the gene was subcloned into the pCR-Blunt II- TOPO vector and afterwards inserted into the pMSCVneo destination vector (Fig. 15).

Results

Figure 15: Steps of Tps1 cloning into the destination vector Step A: Amplification of Tps1 using EcoR I and Xho I restriction overhang primers and subcloning into pCR-Blunt II-TOPO. Step B: Enzymatic restriction with EcoR I and Xho I and ligation into the pMSCVneo destination vector. (Abbreviations: Ampr = ampicillin resistance, LTR = long terminal repeat, Ψ+ = extended packaging signal, MCS = multiple cloning site, r PPGK = phosphoglycerate kinase promoter, which controls expression of Neo = neomycin resistance.

4.3.1.1. Addition of restriction overhangs and subcloning of Tps1 into pCR- Blunt II-TOPO For inserting Tps1 into the pMSCVneo destination vector, Xho I and EcoR I restriction sites had to be added to the ends of Tps1. For this purpose, Tps1 was HiFi Taq-amplified from a Drosophila melanogaster cDNA with Xho I and EcoR I restriction-overhang primers (Tps1_EcoRI_F and Tps1_XhoI_R ) and subcloned into the pCR-Blunt II-TOPO vector. The construct was transformed into DH10beta chemocompetent cells and selected against kanamycin. Colonies were picked the next morning, grown for another 24 h and plasmids isolated by mini preparation. Tps1 positive clones were detected through analytical restriction digestion and afterwards confirmed by sequencing of the insert (Fig. 16). All mentioned techniques are described in detail in chapters 3.7 and 3.9 of the Material and Methods section of this work.

Results

Figure 16: Identification of correct TOPO clones by analytical restriction digestion Correct clones from TOPO cloning were identified by analytical restriction digestion of mini preparations with EcoR I and XhoI. Clones C1 – C9 were positive, clones C10 – C12 negative. Expected bands: Tps1: 2.4 kb; pCR-Blunt II-TOPO: 3.5 kb. Clones C1 and C2 were sequenced and selected for subcloning into the destination vector (Abbreviations: C = clone).

4.3.1.2. Insertion of Tps1 into the pMSCVneo destination vector To insert Tps1 into the pMSCVneo destination vector, 10 µg of the Tps1-TOPO- plasmid and the empty destination vector were enzymatically digested using Xho I and EcoR I. After restriction digest was completed, the Tps1 band (~ 2.4 kb) and the linearized pMSCVneo vector (~ 6.5 kb) were cut out of the gel and purified as described in chapter 3.7.3. After ligation the construct was again transformed into DH10beta chemocompetent cells and selected against ampicillin. Colonies were picked the next morning and grown in LB medium for another 24 h. Subsequently plasmids were isolated by minipreparation and Tps1 positive clones detected through analytical restriction digestion (Fig. 17). After observation of the correct band pattern, clones # 1’ and # 2’ were analyzed by sequencing (see page X, Figure I in the appendix). Since larger amounts of the destination plasmid were needed for the preparation of the retrovirus, new growth medium was inoculated with clone # 1’ and # 2’ and midi preparation of the plasmids was performed using the PureLink™ HiPure Plasmid Filter Midiprep Kit (Invitrogen).

Results

Figure 17: Subcloning of Tps1 into the pMSCVneo destination vector A: Preparative enzymatic restriction digestion of Tps1 in pCR-Blunt II-TOPO with EcoR I and Xho I (expected bands: Tps1: 2.4 kb; TOPO - vector: 3.5 kb). B: Enzymatic restriction digestion of the pMSCVneo destination vector (band size: 6.5 kb) with EcoR I and Xho I for linearization. C: Analytical restriction digestions of mini preparations for verifying of correct cloning. Sequences of both Clone 1’ and 2’ were checked (see Figure I, page X of the appendix) (Expected bands: Tps1 and pMSCVneo).

4.3.2. Retroviral transduction of fibroblasts with Tps1 Fibroblasts derived from MPS IIIA patient # A1 (mutation: E447K, R245H) and MPS IIIB patient # B14 (mutation: homozygous P358L) were transduced with Tps1 using a VSV-G pseudotyped MSCV based retrovirus. The retrovirus was produced in HEK 293T cells after co-transfection with pMSCVneo containing Tps1 and two other plasmids coding for integral parts of the virion (gag/pol and VSV-G). Tps1 had been cloned before into the pMSCVneo vector as described above. Fibroblasts were transduced by centrifugation with virus containing medium from HEK 293T cells. A control transduction was performed with medium from HEK 293T cells transfected with the pMSCVneo vector without Tps1 insert.

To control the transduction efficiency 24 h after transduction total RNA from one transduced 6 cm tissue culture dish was extracted and retrotranscribed into cDNA. PCR was performed using the primers Tps1_EcoRI_F and Tps1_XhoI_R and the products were visualized by gel electrophoresis. A bright band at ~2.4 kb was visible in the Tps1 transduced sample as shown in Figure 18, indicating a mRNA transcript of Tps1. In the control transduced samples the respective band had the same brightness as in random cDNAs that were run as additional negative controls in the

Results same PCR. All mentioned techniques are described in detail in the Material and Methods section of this work.

Figure 18: PCR on Tps1 for transduction control PCR of Tps1 on transduced and control transduced cell lines # A1 and # B14 (expected size: 2.4 kb). Two random human cDNAs served as negative control. (Abbreviations: ctrl td = control transduced with empty vector, td = transduced with vector carrying Tps1).

4.3.3. GAG levels in trehalose-synthesizing MPS IIIB fibroblasts Intracellular GAG levels were determined in fibroblasts from patient # B14 24 h and 48 h after transduction with Tps1. The cell number in the cultures of patient # A1 was too little after transduction for assessment of HS levels.

After 24 h, no significant difference could be observed between Tps1-transduced and control-transduced fibroblasts. After 48 h, GAG levels in Tps1-transduced fibroblasts were considerably lower than in control-transduced fibroblasts as shown in Figure 19. GAG levels in Tps1-transduced fibroblasts were at 78.44% of the levels in control- transduced fibroblasts (47.45 ng GAGs/ng DNA compared to 60.49 ng GAG/ng DNA).

Results

70 110

60 100

50 90

40 80

30 70

20 60

10 50 GAG levels (ng GAG/ng DNA) GAG/ng (ng levels GAG

0 40

24 h 48 h control)to compared % in ( levels GAG 24 h 48 h Time after treatment Time after transduction

Control TPS1-transduced

Figure 19: Heparan sulfate levels in MPS IIIB fibroblasts after Tps1 transduction MPS IIIB cell line # B14 was transduced with a retroviral vector carrying Tps1. HS levels were measured 24 h and 48 h after transduction. A: HS levels in absolute numbers. B: HS levels in % compared to cells transduced with the empty vector.

Results

4.4. Subcellular location of TFEB under trehalose treatment Following our hypothesis, trehalose induces the transcription of lysosomal genes through the activation of TFEB. To induce the transcription of lysosomal genes TFEB has to migrate into the nucleus and bind to the promoter sequence on its target genes.

To assess the subcellular location of TFEB under trehalose treatment, HeLa cells expressing TFEB - 3xFLAG were cultured on coverslips in a 24-well plate at 37°C and 5% CO2 with either medium containing trehalose in a final concentration of 100 mM or control medium without trehalose. The cells were treated for 0 h, 3 h, 6 h, 12 h or 24 h. Subsequently, the intracellular distribution of TFEB was analyzed by immunohistochemistry as described in chapter 3.10.1. At the time of the experiment there was no good antibody against endogenous TFEB suitable for immunohistochemistry commercially available. Hence, HeLa cells expressing TFEB tagged with a 3x-FLAG tag were used instead of primary fibroblasts and TFEB visualized indirectly with an anti-FLAG antibody.

Figure 20 shows the intracellular distribution of TFEB under trehalose treatment over time. TFEB was located completely in the cytoplasm in untreated cells. Under trehalose treatment TFEB migrated gradually into the nucleus over time. After 6 h TFEB was located partially in the cytoplasm and partially inside the nucleus. After 24 h TFEB was almost completely located inside the nucleus.

Results

Figure 20: Subcellular localization of TFEB under trehalose treatment Under 100 mM trehalose treatment TFEB gradually migrates from the cytoplasm into the nucleus over 24 h in HeLa cells overexpressing TFEB - 3xFLAG.

Results

4.5. Expression profile of lysosomal genes in MPS IIIB fibroblasts To investigate the underlying mechanism of the observed reduction of stored GAGs in MPS III fibroblasts under trehalose treatment further, the expression of a set of lysosomal genes was analyzed by quantitative real-time PCR in trehalose treated MPS IIIB fibroblasts and compared to the expression in untreated cells of the same cell line.

Fibroblasts from MPS IIIB patients # B1 (mutation: L35F, G292R), # B6 (V77G, H414R), # B11 (L682R, Y140C) and # B14 (Homozygous P358L) were analyzed. These cell lines were selected for analysis since they all showed a significant reduction of GAGs under trehalose treatment as presented in chapter 4.2. Additionally, the expression profile of one control cell line was analyzed (# WT3).

The expression of the genes TFEB, LAMP1, MCOLN1, STX4, TPP1, ATP6V0D1, SGSH, NAGLU, HEXA and GRN after 24 h, 48 h, 96 h and 1 w of trehalose treatment was analyzed. The expression of each gene was normalized to the expression of GAPDH. A short description of the function of each analyzed gene is given in table 19.

The fibroblasts were cultured with medium containing trehalose in a concentration of 100 mM or control medium without trehalose in an individual 6 cm tissue culture dish for each time point and condition. Since the induction of the lysosomal system may vary in confluent cells versus subconfluent cells (Okada et al., 1971), it was assured that fibroblasts were at ~80% confluence on the day of analysis. Total RNA was extracted from the cells and retrotranscribed into cDNA as described in chapter 3.8. Quantitative real-time PCR was then performed as described in chapter 3.7.5.

In general, lysosomal genes were found to have higher expression levels under trehalose treatment compared to the untreated controls. Expression patterns and fold-changes varied between the different cell lines. The highest expression of most genes was observed after one week of treatment, smaller increases were observed already after 24 h. The results for all cell lines are shown in Figures 21 – 25 and tables 20 – 24.

Results

In all cell lines MCOLN1 and STX4, two genes involved in lysosomal exocytosis pathways, were highly upregulated after one week of trehalose treatment. TFEB showed an undulating expression pattern in cell lines #B6, #B14 and WT3. In cell line #B1 and #B11 TFEB expression peaked after 1 week of incubation after an initial low upregulation followed by a stable course.

Table 19: Biological functions of genes tested by qPCR

Gene Encoded protein Biological function

Regulates lysosomal biogenesis and function and TFEB Transcription factor EB other lysosome-related processes including autophagy Lysosomal-associated LAMP1 Lysosomal membrane glycoprotein membrane protein 1 Unspecific cation channel involved in the late endocytic MCOLN1 Mucolipin 1 pathway and in the regulation of lysosomal exocytosis SNARE protein involved in vesicular transport, STX4 Syntaxin 4 endocytosis, exocytosis and phagocytosis Lysosomal serine protease, involved in tripeptide TPP1 Tripeptidyl peptidase I cleavage. Deficiency causes CLN2 Codes for a subunit of a V-ATPase necessary for ATP6V0D1 V-ATPase, subunit d1 lysosomal acidification N-sulfoglucosamine Lysosomal hydrolase involved in HS degradation. SGSH sulfohydrolase Deficiency causes MPS IIIA Lysosomal hydrolase involved in HS degradation. NAGLU N-acetylglucosaminidase Deficiency causes MPS IIIB Lysosomal hydrolase involved in ganglioside GM2 HEXA Hexosaminidase A degradation. Deficiency causes GM2 gangliosidoses Secreted peptide important for normal development, GRN Granulin wound healing and tumorigenesis.

Results

L35F, G292R (# B1) 12 TFEB 10 LAMP1

8 MCOLN1 STX4 6 TPP1

Fold change Fold 4 ATP6VOD1 SGSH 2 NAGLU 0 HEXA 0h 24h 48h 96h 1 w Treatment time GRN

Figure 21: Expression profile of cell line # B1 under trehalose treatment Fibroblasts from MPS IIIB patient # B1 were treated with 100 mM trehalose. The expression of a set of lysosomal genes was measured by qPCR after 24 h, 48 h, 96 h and 1 w of treatment. Data are expressed as fold increase compared to untreated cells.

Table 20: Expression profile of cell line # B1 under trehalose treatment Corresponding data table to Figure 21. Fibroblasts from MPS IIIB patient # B1 were treated with 100 mM trehalose. The expression of a set of lysosomal genes was measured by qPCR after 24 h, 48 h, 96 h and 1 w of treatment. Data are expressed as fold induction compared to untreated cells. P-value expresses the level of statistical significance of the fold-change.

Gene 24h 48h 96h 1 w Fold p Fold p Fold p Fold p TFEB 1.71 0.17 1.59 0.08 1.56 0.27 4.52 0.01 LAMP1 1.15 0.42 1.35 0.05 1.51 0.10 2.81 0.01 MCOLN1 2.39 0.03 1.89 0.04 1.69 0.11 10.69 <0.01 STX4 1.11 0.29 2.18 0.02 1.47 0.01 10.15 <0.01 TPP1 1.30 0.14 1.32 0.12 1.54 0.19 2.21 0.07 ATP6VOD1 0.93 0.54 1.51 0.04 1.64 0.08 1.19 0.22 SGSH n. d. n. d. n. d. n. d. n. d. n. d. 3.16 0.02 NAGLU 1.73 0.05 1.28 0.18 1.59 0.10 1.75 0.02 HEXA 1.67 0.04 1.31 0.08 1.27 0.25 1.87 0.01 GRN 1.12 0.47 1.95 0.07 0.80 0.04 2.57 0.01

Results

V77G, H414R (# B6) 10 9 TFEB 8 LAMP1

7 MCOLN1 6 STX4 5 TPP1 4 Fold change Fold 3 ATP6VOD1 2 SGSH 1 NAGLU 0 HEXA 0h 24h 48h 96h 1 w Treatment time GRN

Figure 22: Expression profile of cell line # B6 under trehalose treatment Fibroblasts from MPS IIIB patient # B6 were treated with 100 mM trehalose. The expression of a set of lysosomal genes was measured by qPCR after 24 h, 48 h, 96 h and 1 w of treatment. Data are expressed as fold increase compared to untreated cells.

Table 21: Expression profile of cell line # B6 under trehalose treatment Corresponding data table to Figure 22. Fibroblasts from MPS IIIB patient # B6 were treated with 100 mM trehalose. The expression of a set of lysosomal genes was measured by qPCR after 24 h, 48 h, 96 h and 1 w of treatment. Data are expressed as fold induction compared to untreated cells. P-value expresses the level of statistical significance of the fold-change.

Gene 24h 48h 96h 1 w Fold p Fold p Fold p Fold p TFEB 1.82 0.07 0.75 0.18 1.16 0.54 2.67 0.04 LAMP1 1.53 0.14 1.47 0.11 7.33 0.15 3.26 0.03 MCOLN1 3.33 0.04 1.72 0.09 4.63 0.07 8.24 0.02 STX4 1.10 0.30 2.59 0.03 1.47 0.15 3.88 0.02 TPP1 2.69 0.03 1.86 0.06 2.64 0.24 7.00 0.02 ATP6VOD1 1.90 0.07 1.75 0.07 2.02 0.23 2.43 0.03 SGSH 1.12 0.24 0.58 0.03 1.23 0.40 n. d NAGLU 2.08 0.04 1.61 0.11 1.93 0.19 1.37 0.38 HEXA 1.55 0.08 1.20 0.30 2.11 0.11 5.11 0.01 GRN 1.13 0.22 0.71 0.10 1.44 0.19 6.76 0.02

Results

L682R, Y140C (# B11) 5 TFEB 4 LAMP1

MCOLN1 3 STX4 TPP1 2

Fold change Fold ATP6VOD1 1 SGSH NAGLU 0 HEXA 0h 24h 48h 96h 1w Treatment time GRN

Figure 23: Expression profile of cell line # B11 under trehalose treatment Fibroblasts from MPS IIIB patient # B11 were treated with 100 mM trehalose. The expression of a set of lysosomal genes was measured by qPCR after 24 h, 48 h, 96 h and 1 w of treatment. Data are expressed as fold increase compared to untreated cells.

Table 22: Expression profile of cell line # B11 under trehalose treatment Corresponding data table to Figure 23. Fibroblasts from MPS IIIB patient # B11 were treated with 100 mM trehalose. The expression of a set of lysosomal genes was measured by qPCR after 24 h, 48 h, 96 h and 1 w of treatment. Data are expressed as fold induction compared to untreated cells. P-value expresses the level of statistical significance of the fold-change.

Gene 24h 48h 96h 1 w Fold p Fold p Fold p Fold p TFEB 1.05 0.83 1.09 0.89 1.16 0.10 2.67 0.03 LAMP1 1.67 0.15 1.92 0.38 3.28 0.17 4.20 0.01 MCOLN1 2.37 0.03 1.94 0.39 3.09 0.02 4.04 0.06 STX4 2.02 0.06 2.81 0.01 1.29 0.05 2.94 0.01 TPP1 1.68 0.07 1.95 0.37 2.82 0.10 3.53 0.01 ATP6VOD1 1.20 0.25 2.50 0.30 1.53 0.16 2.37 0.02 SGSH n. d. n. d. n. d. 1.95 0.01 NAGLU 1.92 0.04 1.63 0.47 2.56 0.05 2.64 0.02 HEXA 1.79 0.04 1.14 0.87 2.69 0.11 2.45 0.02 GRN 1.49 0.22 1.73 0.02 4.15 0.01 2.47 0.03

Results

Homozygous P358L (# B14) 4 TFEB LAMP1 3

MCOLN1 STX4 2 TPP1

Fold change Fold ATP6VOD1

1 SGSH NAGLU HEXA 0 0h 24h 48h 96h GRN Treatment time

Figure 24: Expression profile of cell line # B14 under trehalose treatment Fibroblasts from MPS IIIB patient # B14 were treated with 100 mM trehalose. The expression of a set of lysosomal genes was measured by qPCR after 24 h, 48 h and 96 h of treatment. Data are expressed as fold increase compared to untreated cells.

Table 23: Expression profile of cell line # B14 under trehalose treatment Corresponding data table to Figure 24. Fibroblasts from MPS IIIB patient # B14 were treated with 100 mM trehalose. The expression of a set of lysosomal genes was measured by qPCR after 24 h, 48 h and 96 h of treatment. Data are expressed as fold induction compared to untreated cells. P-value expresses the level of statistical significance of the fold-change.

Gene 24h 48h 96h Fold p Fold p Fold p TFEB 0.44 0.03 3.41 0.01 1.16 0.81 LAMP1 0.84 0.16 1.00 0.96 1.61 0.03 MCOLN1 1.68 0.05 2.26 0.03 2.13 0.02 STX4 1.12 0.43 1.44 0.01 3.56 0.00 TPP1 1.41 0.21 1.90 0.13 1.22 0.17 ATP6VOD1 1.16 0.31 1.18 0.15 0.92 0.27 SGSH 1.09 0.47 1.14 0.26 1.56 0.02 NAGLU 1.68 0.11 1.53 0.05 0.91 0.22 HEXA 1.18 0.16 1.61 0.02 0.88 0.14 GRN 1.80 0.03 1.30 0.02 1.28 0.05

Results

# WT3 5 TFEB 4 LAMP1

MCOLN1 3 STX4 TPP1 2

Fold change Fold ATP6VOD1 1 SGSH NAGLU 0 HEXA 0h 24h 48h 96h 1w Treatment time trehalose 100mM GRN

Figure 25: Expression profile of cell line # WT3 under trehalose treatment Fibroblasts from control patient # WT3 were treated with 100 mM trehalose. The expression of a set of lysosomal genes was measured by qPCR after 24 h, 48 h, 96 h and 1 w of treatment. Data are expressed as fold increase compared to untreated cells.

Table 24: Expression profile of cell line # WT3 under trehalose treatment Corresponding data table to Figure 25. Fibroblasts from control patient # WT3 were treated with 100 mM trehalose. The expression of a set of lysosomal genes was measured by qPCR after 24 h, 48 h, 96 h and 1 w of treatment. Data are expressed as fold induction compared to untreated cells. P-value expresses the level of statistical significance of the fold-change.

Gene 24h 48h 96h 1 w Fold p Fold p Fold p Fold p TFEB 1.06 0.68 0.56 0.03 2.11 0.04 2.10 0.03 LAMP1 1.18 0.35 0.91 0.25 1.96 0.04 1.29 0.09 MCOLN1 2.59 0.05 2.03 0.14 2.80 0.03 4.06 0.07 STX4 1.73 0.04 1.39 0.15 2.78 0.01 2.35 0.06 TPP1 1.74 0.05 1.95 0.04 3.26 0.01 2.27 0.02 ATP6VOD1 1.27 0.17 1.69 0.02 1.33 0.09 2.84 0.01 SGSH 2.10 0.18 1.81 0.04 1.57 0.04 1.45 0.12 NAGLU 1.71 0.10 1.21 0.15 2.87 0.01 1.47 0.05 HEXA 1.13 0.35 1.31 0.07 4.08 0.03 1.48 0.04 GRN 2.28 0.16 1.98 0.03 4.63 <0.01 2.97 0.01

Results

4.6. N-acetylglucosaminidase activity

Patients with Sanfilippo syndrome have mutations in one of the genes coding for enzymes involved in the degradation of heparan sulfate. In particular, MPS IIIB patients have mutations in the NAGLU gene, resulting in a defective N- acetylglucosaminidase (EC 3.2.1.50). Some mutations can cause misfolding of the protein, which would result in rapid degradation, even though the molecular function of the protein would not necessarily be severely impaired

Among many others, chaperon-like properties have also been attributed to trehalose. To investigate whether trehalose can have an impact on the correct folding of mutant proteins, the activity of N-acetylglucosaminidase (NAG) under treatment with trehalose was analyzed. It was also investigated whether the overexpression of TFEB in an artificial system could increase the activity of the N- acetylglucosaminidase. In a first step, an assay for the measurement of N- acetylglucosaminidase activity was developed since no suitable method was available.

4.6.1. Development and test of a N-acetylglucosaminidase assay Several spectrophotometrical and fluorometrical methods are available for the measurement of the activity of the N-acetylglucosaminidase (NAG) in fibroblasts. Principle of all assays is the incubation of a cell extract with a substrate specific for the enzyme to be measured. The enzyme digests the substrate effecting a change in color that can be measured spectrophotometrically or fluorometrically.

All currently available methods are not suitable for high-throughput screenings since a large amount of cells is needed for each tested condition. Moreover, all current methods require cell disruption by sonication or freeze-thawing which is difficult to standardize. The normalization on protein content introduces an additional source of error. For these reasons a protocol for a rapid and sensitive NAG assay was developed which can be carried out completely in a 96-well plate (NAG one-step cell assay).

Briefly, fibroblasts were plated in one well of a 96-well plate. After overnight attachment, treatment was started on the next day. After treatment, cells were

Results incubated with the specific fluorescent substrate 4MU-alpha-N-acetyl-D- glucosaminide in an acidic buffer for 17 h at 37° C. Finally, the released fluorescence was measured on a plate reader.

4.6.1.1. Experimental set-up To determine the best assay conditions, NAG activity was assessed incubating different cell numbers per well with various substrate concentrations. 5 x 103 or 10 x 103 wild type fibroblasts were plated in wells of a 96-well plate and incubated with the substrate 4MU-alpha-N-acetyl-D-glucosaminide (MUG) in the concentrations 1 mM, 2 mM or 2.5 mM. Incubating 5 x 103 fibroblasts with MUG at a final concentration of 1, 2 and 2.5 mM resulted in 1051±39 FU, 1600±59 FU and 1518±65 FU, respectively. Incubating 104 fibroblasts per well with MUG at a final concentration of 1, 2 and 2.5 mM resulted in 1747±172 FU, 2984±137 FU and 2923±136 FU, respectively. As shown in Figure 26A, a correlation between number of plated cells and released fluorescence was observed. The measured fluorescence was higher in cells incubated with 2 mM substrate concentration than in cells incubated with substrate in a concentration of 1 mM, but did not increase further when incubated with substrate in a concentration of 2.5 mM, suggesting saturation of the enzymatic reaction. Hence, plating of 10 x 103 fibroblasts per well and incubation with substrate in a final concentration of 2 mM was selected as standard condition for all following experiments.

To assess whether the assay was conducted within a linear range with respect to time, 104 fibroblasts were incubated with 2 mM MUG for 5, 9, 13, 17 or 24 h and NAG activity was measured. The measured fluorescence increased linearly with time along the entire time interval tested, with an average hourly increment of 176±13 FU (Pearson correlation coefficient = 0.998; P<0.001) (Fig. 26B).

The experimental procedure of the assay under these final conditions is described in detail in chapter 3.12.2 in the Material and Methods section.

Results

A B

4000 5000

p = 0.002 p < 0.001 p < 0.001 3500 ** ** ** 4500 4000 3000 y = 175,9x 3500 2500 3000 2000 2500 1500 2000 1500 1000 1000 500

Flourescence 360/460 nm (FU) nm 360/460 Flourescence 500 Flourescence 360/460 nm (FU) nm 360/460 Flourescence 0 0 1 mM 2 mM 2.5 mM 0 10 20 30 Substrate concentration Time (hrs)

5 000 cells/well 10 000 cells/well

Figure 26: Set-up conditions A: In order to determine the best assay conditions, NAG activity of # WT3 cells was measured using two different numbers of plated cells per well of a 96-well plate and three different substrate concentrations. B: To assess linearity over time, NAG activity was measured over a time course from 5 to 24 h. Values are expressed as mean ±SD.

4.6.1.2. NAG activity in MPS IIIB fibroblasts and WT cells To assess the ability of the assay to distinguish mutated cells from wild type cells, the N-acetylglucosaminidase activity in fibroblasts from 13 MPS IIIB patients and fibroblasts from five control donors were analyzed under the conditions mentioned above.

The released fluorescence of MPS IIIB patient-derived fibroblasts was significantly lower (p = 0.002) than in wild type controls. MPS IIIB fibroblasts released in average 131 FU (SD: ±28; SEM: ±7.9) and wild type fibroblasts 2468 FU (SD: ±342; SEM: ±152.9), respectively. The released fluorescence was ~18.8 times lower in MPS IIIB fibroblasts than in wild type fibroblasts. Thus, MPS IIIB patients may be easily screened with the developed assay (Fig. 27).

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0 B1 B2 B3 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 WT WT WT NCL NCL #1 #2 #3 1 2 MPS IIIB WT

Figure 27: NAG activity in MPS IIIB fibroblasts and control fibroblasts Comparison of N-acetylglucosaminidase activity between MPS IIIB patients and wild-type controls. The measured flourescence is significantly higher in the controls. Thus, this assay can easily detect MPS IIIB patients. Values are expressed as mean ±SD.

4.6.1.3. Assessment of sensitivity: lysosomal enhancement by sucrose treatment In order to use the assay as a screening tool, it was necessary to assess its sensitivity for measuring differences in activity upon stimulation. Since sucrose is a known enhancer of the lysosomal system, it was used to assess the sensitivity of the developed assay. For this purpose wild type fibroblasts # WT1 and # WT3 were incubated four days with medium containing sucrose in the concentrations 25 mM, 50 mM, 75 mM, 100 mM or 125 mM. Then N-acetylglucosaminidase activity was determined. Also the mRNA levels of the lysosomal genes TFEB, NAGLU, GNS and GBA were measured by qPCR in cell line # WT1 after incubation under the same conditions. GNS and GBA are genes coding for other lysosomal hydrolases involved in the degradation of macromolecules.

Upon treatment with sucrose, an increase of both NAGLU expression and NAG activity could be observed. The observed effects were in a dose-dependent manner (Fig. 28).

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A 2

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activity (fold) activity

- 1 WT3

0,8 NAG NAG 0,6 0,4 0 mM 25 mM 50 mM 75 mM 100 mM 125 mM Sucrose concentration B 2 1,8

1,6 1,4 NAGLU 1,2 TFEB 1 GNS Fold expression Fold 0,8 GBA 0,6 0,4 0 mM 25 mM 50 mM 75 mM 100 mM 125 mM Sucrose concentration

Figure 28: NAG-activity and lysosomal gene expression under sucrose treatment In order to asses the sensitivty of the assay to report differences in NAG activity, cells were treated with the known lysosomal enhancer sucrose. Then NAG-activity (A) and epxression of the lysosomal genes (B) TFEB, NAGLU, GNS and GBA were measured (cell line # WT1 only).

4.6.1.4. Assessment of sensitivity: lysosomal enhancement by TFEB overexpression To determine the impact of TFEB-mediated lysosomal enhancement, the N- acetylglucosaminidase activity in HeLa cells was compared with the activity of HeLa cells artificially overexpressing TFEB - 3xFLAG. Also the mRNA levels of NAGLU, SGSH, GNS, GBA and TFEB (as a control) were determined by qPCR.

As expected, TFEB was highly expressed in HeLa-TFEB-3xFLAG cells compared to wild type HeLa cells (~ 63 – fold). The expression of NAGLU was ~ 3 times higher in

Results

HeLa cells overexpressing TFEB. The mRNA levels of the other lysosomal enzymes (SGSH, GNS and GBA) were higher than in wild-type HeLa cells as shown in Figure 29A.

In agreement with the higher NAGLU mRNA levels, the NAG activity of HeLa-TFEB- cells was ~ 3 times higher than in WT HeLa cells as shown in Figure 29B.

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Figure 29: Relative expression of lysosomal genes and relative NAG-activity in HeLa- TFEB-3xFLAG cells Relative expression levels (A) of the genes TFEB, NAGLU, SGSH, GNS and GBA and relative NAG-activity (B) in HeLa cells overexpressing TFEB-3xFLAG compared with HeLa – WT cells

4.6.1.5. Assessment of reproducibility To verify the signal reproducibility of the assay, a Z’-factor test was performed. The Z’-factor is a statistical parameter used for evaluation and validation of high throughput screening applications (Zhang et al., 1999). Therefore, several replicates of the same condition were acquired in two independent experiments using three 96- well plates per experiment. Cells were either untreated (U) or treated with 25 mM sucrose (low sucrose concentration, L) or 100 mM sucrose (high sucrose concentration, H), respectively. Columns were organized in [H-L-U], [L-U-H], and [U- H-L] schemes in plates 1, 2, and 3, respectively, to avoid any biases in the distribution of treatments. NAG activity was measured as described above. The Z’- factors generated in the two experiments were 0.73 and 0.63 comparing H vs. L

Results signals. The average fold change associated with the H vs. U comparison was 1.7±0.04. The comparison of L vs. U signals generated Z’-factors equal to 0.37 and 0.38 in the two experiments, respectively, with an average fold change of 1.4±0.05 (Fig. 30).

6000

5000 100 mM 4000 25 mM 3000 untreated 2000

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Figure 30: Z′-factor test to evaluate assay reproducibility Relative NAG activity in wild-type fibroblasts treated with 100 mM sucrose (blue dots), 25 mM sucrose (red dots), or left untreated (green dots). The graph reports the results from two independent experiments (n = 3 plates per experiment).

4.6.2. Behavior of fibroblasts under trehalose treatment Since chaperon-like properties of trehalose are described in the literature, the developed N-acetylglucosaminidase activity assay was finally used to investigate the impact of trehalose on the proper folding of mutant proteins. For this purpose, 13 MPS IIIB fibroblast cell lines and two wild-type cell lines (# WT1 and # WT3) were plated in a 96-well plate and incubated for 4 days with medium containing trehalose in the concentrations 20 mM, 100 mM and 250 mM. Subsequently, N- acetylglucosaminidase activity was determined as described in chapter 3.12.2.

The wild-type fibroblasts showed an increase of the N-acetylglucosaminidase activity in a dose-dependent manner up to a concentration of 100 mM trehalose. The activity decreased again at a concentration of 250 mM trehalose in the medium as shown in Figure 31.

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Although an increase of activity could be shown in WT fibroblasts, no difference in N- acetylglucosaminidase activity was detected between treated and untreated MPS IIIB patient-derived fibroblasts (data not shown).

2,5

1,5

WT1 1 WT3

0,5

acetylglucosamindays activty (fold) activty acetylglucosamindays -

N 0 0 50 100 150 200 250 300 trehalose concentration (mM)

Figure 31: NAG-activity under trehalose treatment N-acetylglucosaminidase activity in WT fibroblasts under treatment with trehalose in the concentrations 20 mM, 100 mM and 250 mM.

4.7. Mutation mapping and energetic analysis

How much a certain mutation influences the function of a protein depends on several criteria. If the mutation codes for an amino acid in the active site of the protein, the enzymatic function is more likely altered than by mutations in structural parts of the protein. Moreover, structural mutations are more likely rescuable by chemical chaperons than mutations affecting the active site of the protein. In addition to that the importance of a certain mutated amino acid can be estimated by investigating the conservation of this amino acid across different species.

For the determination of the conservation of a certain amino acid, the primary protein structure of the human N-acetylglucosaminidase was aligned with the primary protein structure of the same enzyme of mouse (Mus musculus), zebrafish, Xenopus, Drosophila melanogaster and Clostridium perfringens. The alignment of the protein sequence of N-acetylglucosaminidase through different species is shown on page XII, Figure II in the appendix.

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Using this alignment, the corresponding amino acid in Clostridium perfringens could be determined for each human mutation. Since no three-dimensional model of the human enzyme has been published to date, all mutations were then mapped in the 3D structure of the N-acetylglucosaminidase enzyme of C. perfringens (PDB ID: 2VCC, Ficko-Blean et al., 2008) in order to determine whether they affect residues likely involved in the enzymatic reaction or in structural parts of the proteins such as alpha-helices or beta-sheets. An example of this analysis is given in Figure 32. All results of the analysis are shown in table 25.

Out of 14 analyzed MPS IIIB patients, eight had homozygous mutations, while the other six patients were compound heterozygous. Out of 17 analyzed mutations, 12 fell in structural parts of the protein and two mutations fell in the active site of the protein as shown in table 25. Three patients had truncating mutations destroying the protein. However, no obvious correlation could be determined between the type of mutation and the rescue of GAG clearance observed upon trehalose administration.

Whether the misfolding of a certain mutation is retrievable by a chemical chaperon also depends on the change in folding free energy upon mutation (ΔΔG). On the base of the three-dimensional model of the NAG enzyme of C. perfringens (PDB ID: 2VCC), changes in stability of the NAG protein depending on each mutation were predicted using the online tool PoPMuSiC v2.1 (http://babylone.ulb.ac.be/PoPV2a/). The results of this analysis are shown along with the results of the mutation mapping in table 25.

Results

Figure 32: Mutation mapping and energetic analysis of the mutation V501G A: Alignment of NAG-protein sequence through different species to determine mutation in C. perfringens and determination of conservation. B: Mutation mapping on a 3D – model of NAG of C. perfringens. C: Calculation of change in folding free energy upon mutation with the PoPMuSiC tool.

Table 25: Structural and energetic analysis of all used Sanfilippo B cell lines The mutations of all used MPS IIIB cell lines were mapped on a 3D model of NAG and analyzed regarding type, location and conservation of the mutations. The change in folding free energy was determined.

Energetic Patient No. Mutation Structural analysis analysis in in Type of Rescue  G in Details H. sapiens C. perfringens mutation possible? kcal/mol Substitution of a conserved amino acid in an a-helix at the B1 L35F E202F structural yes 0,36 external site of the protein Substitution of a non-conserved amino acid in an a-helix at G292R A458R structural yes 0,63 the external site of the protein

Homoz Substitution of a conserved amino acid facing the active site B2 W811C functional no 3,56 W649C of the protein, not likely to be rescuable

Substitution of a conserved amino acid in an a-helix at the B3 Homoz Y92H Y256H structural yes 2,16 external site of the protein

Homoz B4 Protein destroyed no E336X

Substitution of a non-conserved amino acid in a non- B5 T81A N245A structural yes 1,19 structured segment at the internal part of the protein Substitution of a conserved amino acid at the center of the Y140C Y305C functional no 2,87 active site

Substitution of a non-conserved amino acid in a b-sheet at the B6 V77G I241G structural yes 4,51 external side of the protein Substitution of a non-conserved amino acid in a non- H414R M570R structural yes 0,84 structured part at the internal part of the protein

Homoz Substitution of a non-conserved amino acid in a non- B7 A655G structural yes 1,93 V501G structured segment at the external part of the protein

Table 25: Structural and energetic analysis of all used Sanfilippo B cell lines (continued from previous page):

Energetic Patient No. Mutation Structural analysis analysis

in in Type of  G in Details Rescue possible? H. sapiens C. perfringens mutation kcal/mol

Homoz Substitution of a non-conserved amino acid in a non- B8 A655G structural yes 1,93 V501G structured segment at the external part of the protein

Substitution of a conserved amino acid at the center of the B9 Y140C Y305C functional no 2,87 active site Substitution of conserved amino acid in an a-helix at the W156C W321C structural yes 2,95 internal part of the protein

Homoz Deletion of an a-helix facing the active site, no rescue B10 no R626X possible

B11 L682R L849R structural Substitution of a conserved amino acid in an a-helix yes 1,53

Substitution of a conserved amino acid at the center of the Y140C Y305C functional no 2,87 active site

Homoz Substitution of a non-conserved amino acid in an a-helix B12 F318K structural yes 3,04 E153K adjacent to the active site

B13 R297X Protein destroyed no

Substitution of a conserved amino acid in an a-helix R643H R805H structural yes 0,98 adjacent to the active site

Homoz Substitution of a conserved amino acid in a non-structured B14 P521L structural yes 1,39 P358L segment at the external side of the protein

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4.8. Trehalose metabolism in mice

The results of this thesis suggest that trehalose might serve as a potential therapeutic compound for the therapy of LSDs and other diseases. The other dietary disaccharides, sucrose, maltose and lactose, are absorbed poorly and hydrolyzed by their specific disaccharidases sucrase, maltase and lactase into monosaccharides which are absorbed easily. However, as described above, there are numerous reports of effects of trehalose treatment in vivo which cannot be explained by an excess of glucose (Tanaka et al., 2004, Davies et al., 2006, Rodriguez-Navarro et al., 2010).

While the metabolism of other dietary disaccharides has been studied extensively, to date little is known about absorption, turnover and metabolism of trehalose in mammals. However, knowledge about trehalose metabolism is crucial for a potential trehalose-based therapy.

The disaccharidases sucrase and maltase are known to be regulated by dietary sugars. Diets containing sucrose, maltose or other carbohydrates can increase the activity of sucrase and maltase, respectively (Deren et al., 1967, Reddy et al., 1968, Rosensweig and Herman, 1969). Based on these reports it was hypothesized that a treatment with trehalose might have an effect on intestinal trehalase activity and subsequently on the absorption of trehalose.

To test this hypothesis, C57BL/6 wild-type mice were treated with 2% or 5% trehalose in drinking water (ad libitum) over seven weeks. Every week three mice out of each of the two groups were sacrificed. Subsequently, trehalase activity was assessed along with trehalose concentrations in liver and brain. In addition, the expression levels of Treh, the gene encoding for trehalase, were assessed on intestinal total RNA extracts with qPCR. Twelve untreated mice served as control group.

4.8.1. Intestinal trehalase activity Intestinal trehalase activity was assessed in extracts from mice jejuna as described in chapter 3.12.4.

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In the trehalose-treated groups, the intestinal trehalase activity increased over time from 1.71 to 4.94 µmol/mg protein/h in the 2% group and from 1.64 to 4.61 µmol/mg protein/h in the 5% group, respectively. The trehalase activity in the control mice was 1.89 µmol/mg protein/h (Fig. 33), which is much lower than in trehalose-treated mice.

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Intestinal Trehalase Activity Trehalase Intestinal 1,00

0,00 1 w 2w 3 w 4 w 5 w 6 w 7 w Time of treatment Figure 33: Intestinal trehalase activity over time in trehalose-treated mice C57BL/6 mice were treated with either 2% or 5% trehalose in the drinking water. Each data point of the 2% and 5% group represents the mean ±SEM of three mice. The control group consisted of twelve mice.

4.8.2. Expression of Treh The expression levels of Treh, the gene encoding for trehalase, were assessed in the 5% group. For this purpose, total mRNA was extracted from mice jejuna and retrotranscribed into cDNA. Then, quantitative real-time PCR was performed. All used methods are described in chapters 3.8 and 3.7.5, respectively. Results are expressed as fold changes of expression compared to the untreated control mice.

The expression of Treh remained on wild-type levels for the first three weeks and started then to increase. The expression level peaks after six weeks of treatment and decreases again in the seventh week (Fig. 34).

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2,0 Fold expression compared to compared expression Fold

0,0 1w 2w 3w 4w 5w 6w 7w Time of treatment

Figure 34: Expression of Treh over time in trehalose-treated mice Expression of treh was assessed in C57BL/6 mice treated with 5% trehalose in the drinking water. Each data point represents the mean ±SEM of three mice compared to the mean of the twelve control mice.

4.8.3. Trehalose concentrations in liver and brain 4.8.3.1. Trehalose concentrations in livers and brains over time The concentrations of trehalose in extracts from liver and brain were assessed as described in chapter 3.12.3.

Trehalose could be found in extracts from livers in both treated and untreated mice. There was no obvious correlation between trehalose concentration in the liver and intestinal trehalase activity. Comparing trehalose treated mice with mice of the control group, concentrations in livers of trehalose-treated mice were significantly higher than in livers of untreated mice (p-value < 0.001 and = 0.001 comparing the 2% and 5% with the control group, respectively). The average concentrations were 167 (SEM: ±4.7) nmol trehalose/mg protein in the 2% group and 147 (SEM: ±4.4) nmol trehalose/mg protein in the 5% group. Average concentrations in the control group were 121 (SEM: ±5.2) nmol trehalose/mg protein (Fig.s 35 and 36). Remarkably, trehalose levels were significantly higher in mice treated with 2% trehalose compared to mice treated with 5% trehalose in drinking water (p=0.002).

In extracts of brain no trehalose could be detected, neither in treated nor in untreated mice.

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trehalose concetration concetration trehalose 50,00 ( in nmol trehalose/mg protein) trehalose/mgnmol in (

0,00 1w 2w 3w 4w 5w 6w 7w time of treatment

Figure 35: Trehalose concentrations in mouse livers over time Trehalose concentrations in liver extracts from mice after one to seven weeks of treatment with trehalose. Each data point of the 2% and 5% group represents the mean ±SEM of three mice. The control group consisted of twelve mice.

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Figure 36: Trehalose concentrations in mouse livers Trehalose concentrations in liver extracts from mice treated with trehalose compared to untreated mice. Data is given as mean ±SEM, n=21 for the treatment groups and n=12 for the control group.

4.8.3.2. Trehalose concentrations in liver under additional treatment with the trehalase inhibitor Validamycin A. We then determined whether the absorption of trehalose could be increased by inhibiting intestinal trehalase. Validamycin A is a trehalase inhibitor which is used as

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Results a pesticide in husbandry against organisms that use trehalose as main sugar. It is highly specific for trehalase and has been shown to have low toxicity in mice (LD50: 2000 mg/kg) (Salleh and Honek, 1990, NIOSH, 1993, Kyosseva et al., 1995).

In the previous experiment trehalose concentrations in livers were clearly higher in treated mice after two weeks of treatment. In general, trehalose levels were higher in mice treated with 2% trehalose in drinking water. For this experiment, trehalose levels were determined in three groups of mice with three mice per group. One group was treated with 2% trehalose in drinking water, the second group was treated with 2% trehalose and Validamycin A, the third group was untreated. The mice were sacrificed after two weeks of treatment.

The average trehalose concentrations were 103 (SEM: ±10.5) nmol trehalose/mg protein in the trehalose only group, 121 (SEM: ±7.8) nmol trehalose/mg protein in the trehalose + Validamycin A group and 97 (SEM: ±1.7) nmol trehalose/mg protein in the untreated control group. The concentrations were highest in the trehalose + Validamycin A group (Fig. 37). However, the analyzed mice showed a large individual variation. Given the small number of animals per group, the differences are statistically not significant.

n.s.

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trehalsoe concentration concentration trehalsoe 70,00 (in nmol trehalose/mg protein) trehalose/mg nmol (in 60,00 Trehalose 2% only Trehalose 2% + Untreated Validamycin A

Figure 37: Trehalose concentrations in mouse livers under Validamycin A treatment Trehalose concentrations in liver extracts from mice after two weeks of treatment with trehalose ±Validamycin A. Each group consisted of three mice. Values are mean ±SEM.

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4.8.3.3. Trehalose concentrations in mouse chow Surprisingly, in the previous experiments trehalose was found not only in the livers of treated but also in the livers of untreated control mice. For this reason, the standard mouse food was checked for trehalose. Indeed, trehalose was found in the mouse food used in our facility. According to the manufacturer (Harlan Laboratories Inc., Indianapolis, IN), the used diet (2920 Teklad) contains Brewer’s yeast which is a known source of trehalose.

Since a trehalose-free diet would be of interest for future experiments, four samples of different rodent diets were tested for their trehalose content. Trehalose was found in the diets TD.97184 and TD.98090, while no trehalose was detectable in the rodent diets 2014 Teklad and TD.96208 (Fig. 38). The diet TD.96208 was selected for the use in future experiments, since its nutritional composition is more similar to the original standard rodent diet than the diet 2014 Teklad.

25,00

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15,00

10,00 Trehalose content content Trehalose

5,00 (in mg trehalose/g diet) trehalose/g mg (in

0,00 2014 Teklad TD.96208 TD.97184 TD.98090 Diet

Figure 38: Trehalose content of various rodent diets

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4.9. Summary of results

 Trehalose is able to permeate mammalian cell membranes

 Trehalose treatment can reduce GAG levels in MPS III patient-derived fibroblasts

 GAG levels are reduced in MPS IIIB fibroblasts expressing Tps1

 Trehalose induces nuclear translocation of TFEB

 Trehalose upregulates the expression of genes involved in lysosomal biogenesis and function and in lysosomal exocytosis

 An assay for N-acetylglucosaminidase activity has been developed and validated for high-throughput screening experiments

 Trehalose does not rescue N-acetylglucosaminidase activity in MPS IIIB fibroblasts carrying a wide array of missense mutations

 An obvious correlation between type and location of mutations and decrease of GAG levels upon trehalose treatment could not be observed

 Trehalose treatment increases intestinal trehalose activity over time in mice

 Hepatic trehalose concentrations are higher in trehalose-treated mice and may be increased further with the specific trehalase inhibitor Validamycin A

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Summary

5 DISCUSSION 5.1. Trehalose can permeate human cell membranes In order to determine whether trehalose is able to permeate mammalian cell membranes, intracellular trehalose levels were measured. The used method has been previously described (Parrou and Francois, 1997, Chen et al., 2003) and was adjusted to the specific conditions. HeLa cells were used instead of fibroblasts due to their fast growth rate since a large amount of cells was needed for the assay. Differences in the trehalose uptake behavior between HeLa cells and fibroblasts are possible.

After 72 h trehalose treatment, the intracellular trehalose level in HeLa cells was 29.94 nmol trehalose/mg protein while no trehalose was detectable in the control treated cells (chapter 4.1). Hence, trehalose is able to permeate human cell membranes.

To date, the mechanism of transportation of trehalose into mammalians cell remains unclear. A trehalose-specific transporter protein was described in the insect, Polypedilum vanderplanki (Kikawada et al., 2007). The trehalose transporter 1 (TRET1) is highly specific for trehalose and was found to function also when introduced into mammalian cells. The gene sequence of TRET1 is conserved in insects, but the trehalose affinity of the encoded transporter protein differs from species to species. Several conserved amino acid motifs among the TRET1 orthologues of different insects were found to correspond approximately to those of the human GLUT/SLC2A family, a well-characterized sugar transporter family (Kanamori et al., 2010). A homolog of TRET 1, H-TRET1, was shown to function as an pH-dependent trehalose transporter in the reabsorption of trehalose in Malpighian tubules of Xenopus oocytes (Kikuta et al., 2012). Although no specific transporter has been described in mammals, the results of this work suggest the existence of a trehalose transportation mechanism in mammalian cells. The trehalose uptake may be regulated by a specific trehalose transporter or also by general endocytosis processes. Trehalose has been found before in mammals in brain and liver extracts from mice treated with trehalose in drinking water, although all trehalose should have

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Summary been converted into glucose by intestinal trehalase (Tanaka et al., 2004). Also in the in vivo experiments of this work trehalose has been found in mice liver as described in chapter 4.8.3 and discussed in chapter 5.8.

5.2. Trehalose reduces HS levels in MPS III patient- derived fibroblasts Sanfilippo syndrome is caused by the accumulation of the glycosaminoglycan heparan sulfate in cells due to genetic defects in GAG degradation pathways. Potential therapeutic approaches aim at the reduction of intracellular GAGs. Therefore, the potential of trehalose to reduce stored GAGs was evaluated by measuring GAG levels in MPS III patient-derived fibroblasts under trehalose treatment.

Intracellular HS deposits in MPS III fibroblasts were found to be significantly reduced in most cell lines after 8 days of treatment with trehalose.

Several previous studies have shown the involvement of trehalose in the clearance of storage macromolecules. The suggested mechanisms are induction of autophagy and stabilization of proteins due to the chaperon-like properties of trehalose. In summary, cell-based studies have shown that trehalose can act as an mTOR- independent activator of autophagy, which in turn enhances the clearance of substrates like expanded huntingtin, mutated alpha-synuclein, total and phosphorylated tau and polyubiquinated proteins, all proteins associated with diseases with pathologic storage (HD, AD, PD) (Sarkar et al., 2007, Gomes et al., 2010, Casarejos et al., 2011, Krüger et al., 2011, Lan et al., 2012). Trehalose prevents insulin amyloid formation (Arora et al., 2004) and aggregation of beta- amyloid in vitro (Liu et al., 2005) and can induce clearance of mutant androgen receptor in cell-based models of spinal and bulbur muscular atrophy (Rusmini et al., 2013). Trehalose also counteracts cellular prion infection through autophagy activation (Aguib et al., 2009) and is able to extend the life span of the nematode Caenorhabditis elegans by decreasing the accumulation of age-related lipofuscin (Honda et al., 2010). In vivo analyses have shown that trehalose alleviates polyalanin- and polyglutamine-mediated pathology in mouse models of

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Summary oculopharyngeal muscular dystrophy and Huntington’s disease, respectively (Tanaka et al., 2004, Davies et al., 2006). Moreover, trehalose has been shown to ameliorate dopaminergic and tau pathology in mice with a deletion in the PD-associated gene Parkin and overexpressing tau as well as a reduction of tau aggregations in a human mutant P301S tauopathy mouse model (Rodriguez-Navarro et al., 2010, Schaeffer et al., 2012). Recent studies showed an increased life span and delay of disease onset in a mouse model of amyotrophic lateral sclerosis, prevention of neural tube defects in type 1 diabetic mice and antidepressant-like effects related to the autophagy enhancing properties of trehalose (Castillo et al., 2013, Kara et al., 2013, Xu et al., 2013) .

The described effects were seen after treatment with trehalose over a period of 8 days, which is fairly long considering that TFEB migrated into the nucleus of HeLa cells within 24 h after start of treatment. Although TFEB is induced rather fast the clearance of stored molecules takes considerably more time. This is in agreement with the observation that the expression of most lysosomal genes peaks after one week of treatment. Interestingly, HS levels decreased considerably within 48 h when the Drosophila trehalose synthesizing gene Tps1 was inserted into the genome of MPS IIIB fibroblasts. The discrepancy between cells treated with exogenous trehalose and cells producing trehalose suggests that trehalose-producing cells reach higher trehalose levels in shorter time and thus show faster effects. As mentioned before, no specific trehalose transporter has been described in mammalian cells to date. The uptake of trehalose into fibroblasts might be too slow to have a prompt effect on gene expression. There might also be differences between the uptake behavior of fibroblasts and HeLa cells, which may explain the discrepancy between rapid nuclear migration of TFEB in HeLa cells and slow effects on clearance pathways in primary fibroblasts. In addition, the used HeLa cells were overexpressing TFEB-3xFLAG and thus they might be more sensitive to lysosomal stimuli. The use of an antibody against endogenous TFEB in MPSIII fibroblasts would help understanding this observed discrepancy. However, at the time of the analysis, no antibody against endogenous TFEB was available which could work in immunofluorescence analysis.

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It may also be hypothesized that TFEB present in the cytosol is migrating into the nucleus, activates its target genes and also stimulates its own expression. When more TFEB protein is present, the activation of target genes is much stronger and robust enough to stimulate efficiently cellular clearance. This is again in agreement with the observation that the expression of most lysosomal genes peaks after one week of treatment, i.e. at the same time when the expression of TFEB peaks.

In three out of 14 tested MPS IIIB patient derived cell lines no reduction of intracellular HS levels could be observed. The underlying mechanism of this non- response remains unclear. Non-responding cell lines may have a general impairment of degradation pathways. Interestingly, the mutations of cell lines # B2 and # B4 were predicted to be impossible to be rescued by a chaperon. However, a correlation between type of mutation and response to trehalose treatment is not obvious. In cell line # B4 also technical problems of the experimental procedure may be the underlying cause since that cell line showed a very different growth behavior than other cell lines which may have influenced the normalization procedure or the experiment itself. In those non-responding cell lines the experiment should be repeated in order to exclude technical problems of the experimental procedures and to confirm that these cell lines are truly non-responding. If this is really the case, these cell lines could be analyzed for other mutations that may affect clearance pathways. A first step of further investigations on the mechanism of this non- response could then be the expression analysis of genes involved in lysosomal exocytosis and autophagy.

5.3. HS levels are reduced in MPS IIIB fibroblasts expressing Tps1 Fibroblasts were transduced with the gene Tps1 which encodes for a trehalose synthase from the insect Drosophila melanogaster. HS levels of MPS IIIB fibroblasts transduced with Tps1 decreased considerably within 48 h. In contrast, 8 days of incubation were necessary to see a significant decrease in HS levels when trehalose was administered externally. Drosophila Tps1 catalyzes the synthesis of trehalose-6- phosphate from UDP-glucose and glucose-6-phosphate and the subsequent dephosphorylation of trehalose-6-phosphate into free trehalose (Chen et al., 2002).

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Summary

HEK 293 cells artificially expressing Tps1 have been shown before to be able to produce trehalose and thus being more tolerant to hypoxia (Chen et al., 2003). In contrast to D. melanogaster, E. coli has two genes (OtsA and OtsB) for trehalose synthesis, as has S. cerevisiae (Tps1 and Tps2). Overexpression of OtsA and OtsB yielded trehalose in N2a cells, a model of HD, and significantly reduced polyglutamine-induced aggregation (Tanaka et al., 2004). OtsA and OtsB introduced in human fibroblasts increased their desiccation tolerance (Guo et al., 2000).

The results of this work suggest that trehalose-synthesizing cells reach higher trehalose levels in shorter time and thus show faster effects. The uptake of exogenous trehalose into fibroblasts appears to be rather slow and, given the lack of any specific trehalose transporter, is likely mediated by general endocytosis processes.

5.4. Trehalose induces nuclear translocation of TFEB

To assess the intracellular localization of TFEB under trehalose treatment, HeLa cells overexpressing TFEB-3xFLAG were treated with trehalose for 24 h. The subcellular localization of TFEB was subsequently visualized by confocal microscopy.

Under treatment with trehalose, within 24 h TFEB gradually migrated from a diffuse localization in the cytoplasm, where it predominantly resides in untreated cells, into the nucleus. TFEB nuclear translocation has been associated with its activation and the subsequent activation of its target genes (Sardiello et al., 2009, Palmieri et al., 2011).

A similar phenomenon was recently observed by Dehay and colleagues. After 24 h of treatment with trehalose in a concentration of 1 mM TFEB had migrated partially into the nucleus in BE-M17 neuroblastoma cells (Dehay et al., 2010). While TFEB migrated almost completely from the cytoplasm into the nucleus in HeLa cells, it only migrated partially into the nucleus in BE-M17 cells. This may be due to the 100 times lower concentration of trehalose causing a slower uptake of trehalose. However, also differences in the uptake behavior of the different cell types have to be considered.

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Summary

5.5. Induction of lysosomal genes under trehalose treatment

As described in chapter 4.2 and discussed in chapter 5.2, treatment with trehalose results in a significant reduction of intracellular deposits of HS. The presence of trehalose in cells and subsequent migration of cytosolic TFEB into the nucleus has been demonstrated (chapters 4.1 and 4.4, respectively).

Since nuclear translocation of TFEB has been associated with activation of its target genes (Sardiello et al., 2009), the underlying mechanism of the observed reduction of stored GAGs in MPS III fibroblasts under trehalose treatment may be an induction of lysosomal genes and thus lysosomal enhancement. TFEB has been shown to enhance the clearance of pathogenic storage material by promoting lysosomal pathways and thus counteract disease progression in several models of neurodegenerative diseases including LSDs, Huntington disease, Alzheimer disease and Parkinson disease (Sardiello et al., 2009, Dehay et al., 2010, Medina et al., 2011, Parr et al., 2012, Tsunemi et al., 2012, Decressac et al., 2013, Pastore et al., 2013a, Pastore et al., 2013b, Song et al., 2013, Spampanato et al., 2013). To confirm a correlation between trehalose and the induction of lysosomal genes, the expression of a set of lysosomal genes was analyzed by quantitative real-time PCR in trehalose- treated MPS IIIB fibroblasts at several time points and compared to the expression profile of untreated cells.

The expression profile of the genes TFEB, MCOLN1, ATP6V0D1, NAGLU, TPP1, HEXA, LAMP1, STX4, GRN and SGSH was assessed showing a significant increase of lysosomal gene expression under trehalose treatment. These results support the hypothesis that trehalose can enhance lysosomal function via activation of TFEB.

TFEB itself showed in most cell lines an undulating expression pattern over time with moderate fold-inductions. This might be due to its function as a transcription factor that works by migrating into the nucleus and binding to its promoter. An increase in protein transcript of a transcription factor is not essential to promote the induction of target genes. The highest fold inductions of all genes including TFEB were seen after one week of treatment. This may be due to the undulating behavior of TFEB: upon trehalose treatment, TFEB migrates into the nucleus and stimulates its target genes.

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Summary

It is conceivable that TFEB present in the cytosol migrates into the nucleus, activates target genes and also stimulates its own expression, thus further reinforcing the activation of downstream pathways.

In all cell lines substantial fold-inductions of the genes MCOLN1 (~ 4 – 16 fold) and STX4 (~ 2.35 – 10 fold) were seen after one week of treatment. Interestingly, both MCOLN1 and STX4 have been reported to be involved in cellular clearance pathways. Impaired lysosomal Ca2+ homeostasis can play a role in pathogenesis of LSDs (Lloyd-Evans and Platt, 2011). MCOLN1 (Mucolipin 1) codes for an unspecific cation channel that has recently been described to play a key role in lysosomal exocytosis. Triggered by an elevation of intracellular Ca2+ concentration, lysosomes fuse with the plasma membrane and subsequently discharge their content outside the cell (Medina et al., 2011, Lima et al., 2012).

STX4 (Syntaxin 4) encodes for a plasma membrane SNARE (soluble NSF attachment protein receptor) enriched in microdomains where exocytosis occurs. SNAREs are membrane components that are important for the fusion of transport vesicles with the plasma membrane (resulting in exocytosis) or with target compartments such as lysosomes and are involved in vesicular transport, endocytosis and phagocytosis. STX4 interacts with synaptotagmin VII in the regulation of lysosomal exocytosis (Rao et al., 2004) and also positively influences granulocyte exocytosis (Logan et al., 2006).

The results suggest that the mechanism underlying the clearance of HS in MPS III fibroblasts is the stimulation of cellular clearance pathways, especially lysosomal exocytosis. Stored GAGs are transferred out of the cell by exocytosis. Genes involved in lysosomal exocytosis were upregulated under treatment with trehalose. Interestingly, exocytosis pathways have recently been shown to be impaired in MPS IIIA cells (Keating et al., 2012). The stimulation of other cellular clearance pathways like autophagy is probable since autophagy is also under the control of TFEB (Settembre et al., 2011). The question whether autophagic pathways are induced upon trehalose treatment could be addressed in future studies, e.g. by assessing the expression profile of autophagy genes by qPCR. Candidates could be ATG9, MAPLC3B, WIPI, SQSTM1, UVRAG, VPS11 and VPS18 which are all genes known to be involved in different steps of autophagy (Settembre and Ballabio, 2011).

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Summary

However, autophagy is not likely the underlying mechanism of GAG clearance in MPS III since the required enzymes for GAG degradation should be deficient even after autophagy induction.

The genes SGSH, NAGLU and HEXA code for lysosomal hydrolases involved in the degradation of heparan sulfate and the ganglioside GM2, respectively. A deficiency in one of these genes results in the LSDs MPS IIIA, MPS IIIB or GM2 gangliosidoses. NAGLU is the gene mutated in MPS IIIB patients. Its expression is induced under trehalose treatment. Apparently more mutant protein is translated, which does not result in an increased N-acetylglucosaminidase activity.

ATP6V0D1, TPP1, LAMP1 and GRN code for other lysosomal proteins and were also induced upon trehalose treatment. This underlines the correlation between trehalose and TFEB induction since they are all TFEB targets (Palmieri et al., 2011).

In general, the expression of most monitored TFEB targets peaks after one week of trehalose treatment. This corresponds to the decrease of intracellular HS levels. A reduction of GAGs could be seen best after 8 days of trehalose treatment.

It is unknown how trehalose triggers the nuclear translocation of TFEB. Since fibroblasts do not express trehalase, they are unable to degrade trehalose. The incorporated trehalose might accumulate in lysosomes and provoke that way a response of the CLEAR network. For better insight into the underlying pathways, a microarray experiment should be carried out in the future.

5.6. N-acetylglucosaminidase activity The N-acetylglucosaminidase, encoded by the NAGLU gene, is the mutated protein in Sanfilippo syndrome type B. Among others, chaperon-like properties have been attributed to trehalose (Singer and Lindquist, 1998, Jain and Roy, 2009). Currently, the use of chemical and pharmaceutical chaperons is tested as a novel approach for the treatment of LSDs (Arakawa et al., 2006, Parenti, 2009, Valenzano et al., 2011). For MPS IIIB, PCT has been proposed (Ficko-Blean et al., 2008), but to date no studies have been published. Residual activity of lysosomal hydrolases can be enhanced by using chemical chaperons in a variety of LSDs including Gaucher,

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Summary

Fabry, Pompe, Tay-Sachs, Sandhoff and GM1 gangliosidosis (Fan et al., 1999, Tropak et al., 2004, Fan and Ishii, 2007, Yu et al., 2007, Mu et al., 2008, Suzuki, 2008).

To test whether in addition to induction of lysosomal genes also chaperon-like properties of trehalose may play a role in the reduction of HS deposits in MPS IIIB fibroblasts, N-acetylglucosaminidase activity was assessed. Since no suitable experimental procedure was available for this purpose, in a first step an improved N- acetylglucosaminidase assay suitable for high-throughput screening experiments was developed.

5.6.1. Development and validation of a N-acetylglucosaminidase assay To date, several methods are available to measure the activity of N- acetylglucosaminidase in fibroblasts. The principle of all assays is the processing of an artificial substrate effecting a change in color that can be measured either spectrophotometrically or fluorometrically.

O’Brien incubated homogenates of fibroblast pellets 14 days after subculture with the colorimetric substrate p-nitrophenyl--D-N-acetylglucosaminide in a citrate- phosphate buffer at pH 4.5. After incubation the reaction was stopped with trichloroacetic acid. At pH 10, the released nitrophenyl changes color, which can be measured spectrophotometrically at 420 nm. Normalized on protein content, the released color correlates with the amount of metabolized substrate (O'Brien, 1972).

A similar, improved assay uses the fluorogenic substrate 4-Methylumbelliferyl--N- acetyl-D-glucosaminide that has advantages in sensitivity and ease of use over the colorimetric substrate. This method also requires large amounts of cells, cell disruption by sonication or freeze-thawing and normalization on protein content (Chow and Weissmann, 1981, Marsh and Fensom, 1985).

Alternative and more laborious assays are based on radiolabelled oligosaccharide substrates (Hopwood and Elliott, 1982) or tandem mass spectrometry (Gerber et al., 2001, Wolfe et al., 2012).

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Summary

All described assays require several steps of sample preparation before the actual incubation with the substrate. Fibroblasts are collected from their culture dishes and pelleted, then fibroblast pellets are homogenized by either sonication of freeze- thawing. Finally, after incubation with the substrate, the measured activity has to be normalized on protein content. Moreover, a large amount of cells is needed. For example, the method of O’Brien requires two T 75 tissue culture flasks two weeks post confluence for each condition in order to get reliable results.

All the mentioned steps complicate the assay and introduce potential sources of error. Especially cell disruption by sonication or freeze-thawing is difficult to standardize. The rate of cell disruption varies with cell density, which again depends on the individual growth rate of each cell line. Moreover, sonication and freeze- thawing can affect protein integrity and alter enzyme activity. Therefore, variation only due to sample preparation has to be expected. In addition, the normalization on protein content may introduce another source of error. The assay described by Hopwood and Elliot using radiolabeled oligosaccharides requires a special laboratory setting and protection measures.

Aim of this study was to determine the impact of trehalose on proper folding of N- acetylglucosaminidase mutants. Therefore, several cell lines had to be incubated repeatedly with various amounts of trehalose. All currently available methods are not suitable for high-throughput screenings since a large amount of cells is needed for each tested condition and experimental procedures are complicated. For all these reasons, a protocol for a rapid and easy but nevertheless sensitive assay was developed which can be carried out completely in a 96-well plate.

Briefly, fibroblasts were plated at confluency in a 96-well plate. Cells were kept overnight in culture to achieve attachment and treatment was started the next day. After treatment, cells were incubated with the specific fluorescent substrate, 4MU- alpha-N-acetyl-D-glucosaminide, in an acetate buffer, pH 4.5 for 17 h at 37°C. Finally, the released fluorescence was measured on a plate reader.

To determine the best assay conditions, different cell numbers per well were incubated with various substrate concentrations. The results showed that the best performance was obtained when incubating 104 fibroblasts per well with substrate in

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Summary a concentration of 2 mM. Since the activity of lysosomal enzymes in cultured fibroblasts correlates with time after subculture and hence with confluency at time of assay (Okada et al., 1971), fibroblasts were plated already confluent. In that way fibroblasts were from the beginning in a metabolic state in which highly differentiated cellular functions, including lysosomal hydrolase enzyme synthesis, become more important than cell division (Okada et al., 1971). The increase of fluorescence was shown to be linear over a time period of 5 to 24 h, indicating that the assay signal is far from reaching a plateau at the selected incubation time of 17 h and also suggesting that shorter incubation times could be used if time is critical in the set up of the experiment.

Comparing the enzyme activity of MPS IIIB patient derived fibroblasts with wild-type fibroblasts, the average readout was ~ 18.8 times lower in MPS IIIB fibroblasts (p < 0.002). Thus, the developed assay shows high sensitivity to detect NAG deficiency, and is therefore suitable for the screening of Sanfilippo patients. Differences among MPS IIIB lines or wild-type lines were not statistically significant, demonstrating that the assay is highly reliable. Most used MPS IIIB fibroblast lines carried NAGLU alleles with no residual NAG activity (L35F, V77G, Y92H, Y140C, E153K, W156C, E336X, P358L, H414R, V501G, R626X, W649C, L682R), as previously demonstrated upon transfection of plasmids with the mutated NAGLU cDNAs in COS-7 or CHO cells (Schmidtchen et al., 1998, Tessitore et al., 2000, Lee-Chen et al., 2002, Beesley et al., 2004, Beesley et al., 2005). Another allele carried an early truncating mutation (R297X) that is also expected to result in a complete loss of NAG activity (Beesley et al., 1998). Finally, there is no published evidence reporting measurements of residual activity of three of the alleles tested (T81A, G292R, R643H). This suggests that the low signal resulting from the analysis of these cells (corresponding to 4–7% of the signal obtained from wild-type cells) represents the background noise of the assay, which is comparable to, or lower than, the level of noise from all previously reported assays (O'Brien, 1972, Butterworth, 1978, Chow and Weissmann, 1981, Hopwood and Elliott, 1982, Marsh and Fensom, 1985, Beesley et al., 1998, Schmidtchen et al., 1998, Tessitore et al., 2000, Lee-Chen et al., 2002, Beesley et al., 2004, Beesley et al., 2005, Wolfe et al., 2012).

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Summary

Lysosomal stimulation of fibroblasts with sucrose via activation of TFEB showed an increase of NAGLU expression on mRNA and protein level in a dose-dependent manner. Hence the system is able to show discrete changes in enzyme activity sensitively. Also increased levels of activity can be measured reliably in the context of variation of signaling pathways when TFEB is introduced artificially into cells. Both approaches showed a correlation between increased mRNA concentrations of lysosomal genes and N-acetylglucosaminidase activity.

An assessment of reproducibility showed Z′-factors for this assay of 0.73 and 0.63 in the comparison of H vs. L signals. A Z’-factor greater than 0.5 indicates high reproducibility and is considered excellent for a high-throughput screen (Zhang et al., 1999). The average fold change associated with the H vs. U comparison was 1.7±0.04. The comparison of L vs. U signals generated Z’-factors equal to 0.37 and 0.38 in the two experiments, respectively, with an average fold change of 1.4±0.05. Based on the definition of Z’-factor (see chapter 3.13 for details) and taking into account the observed standard deviation of ~5%, a 1.6-fold increase in NAG activity would be sufficient to reliably define a hit compound that enhances NAG activity in the context of a high-throughput screen. Notably, TFEB overexpression resulted in a 3-fold increase in NAG activity–thus much above the threshold required to define a reliable hit, which would make TFEB a strong candidate in a genetic screen.

In summary, the developed assay complements existing methods and presents desirable characteristics that make it particularly attractive for primary screening in high-throughput applications. The NAG assay herein described (i) involves a reduced number of steps, resulting in a shorter protocol; (ii) requires a reduced number of cells, enabling the use of the 96-well plate format, which, in turn, allows testing multiple mutations, culturing and treatment conditions simultaneously and with a higher number of replicates; (iii) is performed in a single plate from start to finish, with no requirement for transfer of samples or material across plates; (iv) benefits from reduced protein inactivation due to denaturation or degradation, thus resulting in more reproducible results; (v) has desirable characteristics of sensitivity and reproducibility. Since the measured fluorescence values are not normalized to protein content, the assay does not provide an absolute value of N-acetylglucosaminidase activity. Enzyme activity can be expressed either as a relative value comparing

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Summary treated and untreated cells or as a percentage of wild-type activity. However, the focus of screening experiments is to find molecules that enhance enzyme activity compared to untreated cells. In this regard, the percent increase of activity is of much higher interest than the absolute value of activity.

LSDs are perfect targets for pharmacological chaperon therapy and SCRT because rescue of up to as little as 5-10% of the corresponding wild-type activity may ameliorate clinical symptoms (Brooks et al., 2006, Parenti, 2009, Desnick and Schuchman, 2012). In general, the modulation of the proteostasis network is a promising pharmacological strategy to promote folding of unstable, degradation- prone enzymes containing missense mutations (Mu et al., 2008, Lu et al., 2011, Wang et al., 2011a, Wang et al., 2011b, Wang et al., 2011c, Shen et al., 2012, Song et al., 2013). Recently, candidates for pharmacological chaperon therapy have been identified for several LSDs by performing high-throughput screening of chemical libraries (Maegawa et al., 2007, Tropak et al., 2007, Zheng et al., 2007, Tropak et al., 2008, Urban et al., 2008, Maegawa et al., 2009). The developed assay meets the requirements for an HTS and may be used in future studies to find candidates for PCT or SCRT in MPS III.

5.6.2. Effects of trehalose on N-acetylglucosaminidase activity in WT and MPS IIIB fibroblasts One aim of this work was to assess the chaperon-like properties of trehalose and therefore determine its impact on the proper folding of mutant N- acetylglucosaminidase. For this purpose, a rapid and sensitive assay for high- throughput screening has been developed and validated as described and discussed in chapters 4.6.1 and 5.6.1, respectively.

Wild-type fibroblasts treated with trehalose showed a significant increase of N- acetylglucosaminidase activity after 4 days. However, no difference in N- acetylglucosaminidase activity could be detected between treated and untreated MPS IIIB patient-derived fibroblasts.

As described in chapter 4.5, trehalose stimulates the TFEB-mediated induction of lysosomal genes, including the expression of the NAGLU gene. Apparently, the

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Summary induction of NAGLU is the underlying mechanism of increased N- acetylglucosaminidase activity in WT fibroblasts. Also, in Sanfilippo B patient-derived fibroblasts the expression of NAGLU is induced under trehalose treatment. However, this appears to result only in an increase of dysfunctional mutant protein. Based on the presented data there is no evidence for chaperon-mediated enhancement of N- acetylglucosaminidase activity. The results do not support a chaperon-like function of trehalose in MPS IIIB fibroblasts. Either the residual activity in MPS IIIB fibroblasts cannot be enhanced by chemical chaperons in general, or trehalose is not able to act as a chaperon in Sanfilippo cells.

5.7. Mutation mapping and energetic analysis

For this work, the mutations of the 14 used MPS IIIB cell lines were analyzed regarding (i) location and quality of the mutation, (ii) conservation of the mutated amino acid through species and (iii) change in folding free energy upon mutation.

The influence of a certain mutation on the function of a protein depends on several criteria. Mutations affecting an amino acid in the active site of the protein may more likely alter the enzymatic function than in the case of mutations in structural parts of the protein. Mutations in structural parts of the enzyme may influence stability and folding of the protein. Most missense mutations do not directly impair the enzymatic function but destabilize the protein’s native structure (Durand et al., 2000). As a result, misfolded proteins are recognized by the ER-related quality control system and subsequently degraded faster than wild-type protein although some residual activity might remain (Ellgaard and Helenius, 2003). Structural mutations are also more likely rescuable by chemical chaperons than mutations affecting the active site of the protein, while truncating mutations affecting a consistent part of the protein are not rescuable at all. A number of mutated enzymes in other diseases have been shown to retain catalytic activity if forced to fold into their native structure (Sawkar et al., 2002, Mu et al., 2008). The presented mutation mapping results are in agreement with the results of a previous study (Ficko-Blean et al., 2008).

The importance of a certain amino acid can be estimated by investigating the conservation of this amino acid through different species. Highly conserved amino

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Summary acids more likely play an important role in the function of a protein than non- conserved amino acids. The NAG of C. perfringens has ~30% overall amino acid sequence identity to the human NAG. The amino acid sequence of the active site is almost entirely conserved (Ficko-Blean et al., 2008).

The extent of degradation and the rescuability by a chemical chaperon of enzyme variants containing misfolding, non-inactivating mutations depends on the destabilizing effect of the specific substitution, which in turn determines the residual enzymatic activity in the lysosome (Parenti, 2009, Wang et al., 2011c, Wang and Segatori, 2013). These changes of protein stability upon mutation were predicted with the online tool PoPMuSiC v2.1 (Dehouck et al., 2009, Dehouck et al., 2011), which analyzed as base the same three-dimensional model of the NAG enzyme of C. perfringens that was used for the mutation mapping. Higher energy values indicate a higher destabilization of the mutant protein, which in turn will be degraded more rapidly.

It is not easy to use type and location of a mutation to correlate genotype with phenotype. However, a correlation between mutation and NAG activity has been reported: a compound heterozygous MPS IIIB patient (mutations: R297X/F48L) showed an attenuated phenotype due to residual activity of the F48L mutation. (Yogalingam et al., 2000). Also other mutations which are only structural and do not involve the active site of the enzyme, have been associated with an attenuated phenotype (Yogalingam and Hopwood, 2001, Selmer et al., 2012). This might be due to a residual NAG activity of one of the alleles. But also patients with a more severe phenotype may benefit from a chaperon-mediated therapy, since many LSDs would have a much more attenuated phenotype if the enzyme activity would exceed 5 -10% of the normal activity (von Figura, 1991, Hasilik et al., 2009, Desnick and Schuchman, 2012).

Under normal conditions, mutant enzymes are recognized as misfolded and degraded rapidly. The presented analysis tried to predict whether a certain mutation can be retrieved by a chemical chaperon. Since trehalose did not have any chaperon-like effects under the chosen conditions, the results are yet to be confirmed experimentally. The known NAG-inhibitors 2AcDNJ and 6AcCAS were suggested to

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Summary be used as an initial lead for the development of chemical and pharmaceutical chaperons specific for NAG (Ficko-Blean et al., 2008).

A correlation between potential rescuability by a chaperon and observed reduction of stored GAGs upon trehalose treatment was not obvious.

5.8. Trehalose metabolism in mice

To date, little is known about absorption, turnover and metabolism of trehalose in mammals. However, knowledge about trehalose metabolism is crucial for a potential trehalose-based therapy. To get insight into trehalose metabolism, C57BL/6 wild-type mice were treated with 2% or 5% trehalose in the drinking water over seven weeks. Every week the intestinal trehalase activity was assessed along with the expression of the trehalase-encoding gene Treh and levels of trehalose in brain and liver extracts.

Over time, the activity of the intestinal trehalase increased in both treated groups, beginning from the second week. Hence, trehalose treatment has a regulatory effect on the activity of the intestinal trehalase. Other disaccharidases have been reported before to be regulated by dietary sugars (Rosensweig et al., 1971). The activity of sucrase and maltase increased in rats when fed with diets containing sucrose or maltose, respectively. Lactase activity was upregulated by lactose in one study, but found unaffected in another (Deren et al., 1967, Reddy et al., 1968). In addition, disaccharidase activity may also be regulated by non-specific sugars. So was trehalase found to be upregulated in rats on a maltose diet (Reddy et al., 1968). In humans, sucrose supplementation increased both sucrase and maltase activity. Dietary maltose also upregulated maltase activity. However, lactase activity remained unaffected by lactose in humans (Rosensweig and Herman, 1968, Rosensweig and Herman, 1969). Hence, the presented induction of trehalase in mice might not be the same in humans and further investigation is required.

The expression of Treh, the gene encoding for trehalase, increased under treatment with trehalose. However, the expression remained on wild-type level for the first three weeks and then started to increase. The expression level peaked after six weeks of treatment and decreased again in the seventh week. There is no direct correlation to

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Summary trehalase activity. It is unclear, however, how robust these data are, since mRNA- extraction from intestinal tissue was a challenge and thus RNA quality might have been poor, which is reflected by the generous standard deviations resulting from the average of the various samples.

In addition to trehalase activity and Treh expression, trehalose concentrations were determined in extracts from brain and liver. Although dietary disaccharides are absorbed easily only after hydrolysis by their specific disaccharidases, there are several reports of effects of trehalose treatment in vivo, which cannot be explained by an excess of glucose. (Tanaka et al., 2004, Davies et al., 2006, Rodriguez-Navarro et al., 2010, Schaeffer et al., 2012, Kara et al., 2013, Xu et al., 2013).

In this work, trehalose was found in livers of both treated and untreated mice. In a previous study, the metabolism of trehalose was studied in Treh knock-out mice (Treh -/-). In the blood of those mice no trehalose could be detected by HPLC (Kamiya et al., 2004). Other tissues were not examined. Trehalose has been found before in homogenates of brain (110 nmol trehalose/mg protein) and liver (11.6 µmol trehalose/mg protein) from mice treated 12 weeks with 2% trehalose in the drinking water (Tanaka et al., 2004). Given that the molecular weight of trehalose is 342 g/mol, we calculated that these values result in the unexpectedly high amount of ~4 grams of trehalose for 1 gram of protein in liver tissues (and about 100 times less in brain). In our study, the measured trehalose levels in liver tissues (167 nmol trehalose/mg protein) were ~70 times lower than this value, and no trehalose was detectable in brain tissues. A third, independent study would help clarify the issue and contribute to establishing what the actual amount of trehalose stored in these organs following oral ingestion is.

The level of trehalose in liver homogenates was significantly higher in mice treated with 2% trehalose than in mice treated with 5% trehalose in the drinking water. It was previously reported that R6/2 Huntington disease mice had a greater benefit from a treatment with 2% trehalose in drinking water than with 5% (Tanaka et al., 2004). Treatment with 5% trehalose may increase the trehalase activity faster than a treatment with 2% trehalose, and thus decrease the amount of absorbed trehalose. Based on the data presented in this work, there might be a faster induction of trehalase in mice treated with 5% trehalose. However, the activity differences

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Summary between the 2% and the 5% groups were statistically not significant, thus not supporting a differential induction of trehalase activity.

Looking at the trehalose concentrations at each week, there is no obvious correlation between trehalase activity and trehalose concentration. Although trehalase activity is increasing over time, there is no clear trend of the trehalose concentration. From the data presented, a conclusion is not possible whether an upregulated trehalase results in lower intracorporal trehalose levels.

Surprisingly, trehalose was also detected in liver homogenates of untreated control mice. Correspondence with the manufacturer revealed that the used food contains brewer’s yeast, which is a known source of trehalose. Ingested and absorbed trehalose might accumulate in the liver up to a certain level. It is conceivable that the trehalose present in the normal diet of the test mice has influenced the outcomes of experiments, masking the effects of trehalase induction on trehalose uptake and storage in the liver. For future experiments, a trehalose-free diet should be considered in order to have a clean background.

In a small experiment it was determined whether the absorption of trehalose could be increased by inhibiting the intestinal trehalase. Validamycin A is a trehalase inhibitor widely used as a pesticide in husbandry against organisms that use trehalose as main sugar. It has been shown to be highly specific for trehalase and to have low toxicity in mice (LD50: 2000 mg/kg) (Salleh and Honek, 1990, NIOSH, 1993, Kyosseva et al., 1995). Trehalose levels in liver homogenates were on average higher in mice treated with 2% trehalose and Validamycin A compared to mice treated with trehalose 2% only. Since only three animals were used per group, the observed differences were statistically not significant. These results have to be considered preliminary and would require further investigation with larger groups of mice.

In the present work, no trehalose was detectable in brain homogenates. For the treatment of Sanfilippo disease or other neurodegenerative diseases, trehalose has to reach the brain. The mentioned previous studies, however, reported amelioration of CNS pathology in mouse models of Huntington and Parkinson diseases. When thinking about a potential therapy with trehalose, the presented results have to be

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Summary considered. An oral trehalose therapy might increase the intestinal trehalase activity resulting in reduced trehalose uptake and thus a failure of therapy. In order to prevent that, an interval therapy should be considered: trehalose could be given for a certain time period, followed by a treatment-free period in order to get back to lower trehalase activity levels.

For higher trehalose levels in the target tissues, other ways of administration have to be considered. An option is to administer trehalose intravenously. However, a trehalase is also expressed in the kidney (Elbein et al., 2003). It hydrolyzes plasmatic trehalose, resulting in an increase of blood glucose. In order to circumvent intestinal and renal trehalase and to reach high trehalose concentrations in the brain, trehalose could be injected directly into the CSF. The different options, i.e. oral interval therapy, IV therapy and intrathecal therapy, should be evaluated in future studies.

5.9. Conclusion

The results of this thesis shed new light on the relationship between trehalose and the lysosomal system and on the mammalian metabolism of trehalose. The experiments here described demonstrate that trehalose activates TFEB, which in turn induces the expression of lysosomal genes including genes involved in cellular clearance pathways. This enhances the cell’s capability for the degradation of HS and other pathologically stored molecules. Since trehalose stimulates the lysosomal system in general and does not specifically aim at a certain degradation pathway, this concept may also be adjustable to other LSDs and diseases with related pathogenesis. The high solubility and low toxicity of trehalose make it a promising molecule for the treatment of LSDs and other more common neurodegenerative diseases. However, the results of this work show that, toward a possible therapeutic application of trehalose, a deep investigation of its metabolism is necessary.

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5.10. Future directions

In this work, trehalose-mediated effects on the expression of lysosomal genes were shown, which resulted in a decrease of stored glycosaminoglycans.

These seminal results suggesting use of trehalose as a potential treatment in LSDs raise several questions that would require further investigations:

1. The genes MCOLN1 and STX4, involved in lysosomal exocytosis, have been shown to be upregulated upon treatment with trehalose. The actual ongoing exocytosis could be assessed by measuring the release of lysosomal enzymes into the cell culture medium (Medina et al., 2011). When exocytosis is ongoing, a higher fraction of enzyme is secreted into the cell culture medium.

2. An even better understanding of the underlying mechanisms of trehalose action could be achieved with a microarray experiment. Based on the data of this work, a concentration of 100 mM trehalose and a treatment time of 1 week seem to be the best conditions for such an experiment. This would provide insight into a broad variety of signaling pathways and could not only help understand the mechanism of TFEB action but also the pathway on which trehalose stimulates TFEB. Also shorter incubation times have to be considered in order to understand the gap of action between TFEB nuclear translocation and the actual decrease of macromolecules.

3. An expression profile of lysosomal genes could also be assessed in Tps1 transduced fibroblasts. Apparently, similar pathways are involved but the degradation of macromolecules is much faster. Further investigation will provide insight into the underlying mechanisms.

4. Out of 14 MPS IIIB cell lines, three did not show a decrease of intracellular GAGs under trehalose treatment. As suggested above, these may have a general impairment of lysosomal pathways. A first step towards understanding the differences could be the assessment of an expression profile of lysosomal genes.

5. The presented in vitro results strongly suggest a TFEB mediated reduction of glycosaminoglycans under trehalose treatment. The high solubility and low toxicity of trehalose make it a promising molecule for the use in patients. Thus,

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Summary

the effects of trehalose should be evaluated in vivo in a mouse model of MPS III. Trehalose can be administered orally by dissolving trehalose in the drinking water. Since mice express an intestinal trehalase, other ways of trehalose administration have to be considered, e.g. intravenous injection of a trehalose solution into the tail vein or intrathecal administration in order to circumvent the blood-brain barrier. These in vivo experiments are being currently performed in Dr. Sardiello’s lab.

6. This work and several other reports show that trehalose is taken up from the culture medium into the mammalian cell. However, to date little is known about the underlying mechanism. Further studies are required.

7. Although trehalose is a promising candidate for a TFEB-mediated treatment of neurodegenerative diseases, additional candidates should be identified and evaluated in respect of their ability to induce TFEB and its depending pathways. A molecule would preferably be non-toxic in mammals, pass the intestinal brush border without degradation and cross the BBB in order to have an effect in the brain. A screen of already FDA-approved drugs might be a suitable approach, since this would shortcut years of safety studies.

8. Chaperon-like properties have been attributed to trehalose. However, in the experiments of this work no chaperon-like mediated effects could be observed. Since there is now a suitable assay for a screening experiment available, other known chemical chaperons or compound libraries could be screened and evaluated for their potential use in patients.

9. In a proof-of-principle experiment, it was tried to determine whether inhibiting the intestinal trehalase with a specific trehalase inhibitor could increase the absorption of trehalose. This experiment could be repeated with larger numbers of mice in order to get more robust data. Another option would be the use of a knock-out mouse, deficient of the Treh gene and thus unable to hydrolyze trehalose.

10. Further evaluation of trehalose metabolism and intracorporal distribution upon different ways of administration could be carried out. Considered should be oral interval therapy, IV therapy and intrathecal administration.

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Summary

6 Summary Sanfilippo disease (or MPS III) is a devastating autosomal recessive LSD caused by deficiency of enzymes involved in the degradation of the glycosaminoglycan heparan sulfate leading to its progressive lysosomal accumulation. Patients typically show severe signs of neurodegeneration including behavioral symptoms and mental deterioration, which finally leads to severe dementia and early death. To date there is no established therapy.

Trehalose is a disaccharide of glucose naturally found in lower eukaryotes, which is able to act as a protectant against a variety of stress conditions and has been shown to be beneficial in models of storage diseases. Based on these, a role of trehalose in stabilization of protein folding and activation of autophagy pathways has been proposed. In this work, the therapeutic potential of trehalose for the treatment of LSDs has been tested in a cellular model of MPS III.

In a first step, we showed that the cellular accumulation of heparan sulfate can be decreased in MPS III patient-derived fibroblasts under treatment with trehalose.

In a second step the underlying mechanisms of action were investigated, both the activation of cellular clearance pathways and the chaperon effects. Since lysosomal processes including autophagy and lysosomal exocytosis have been shown to be under coordinated transcriptional control of the master gene TFEB, and trehalose is known to stimulate clearance of pathologically stored deposits, it was hypothesized that trehalose might enhance lysosomal function through TFEB activation.

Immunofluorescence analysis of subcellular TFEB localization under trehalose treatment revealed its gradual migration from the cytosol to the nucleus over time, which was associated with the activation of its target genes. Moreover, the expression of several lysosomal genes including genes involved in cellular clearance pathways was found to be upregulated upon trehalose treatment.

The analysis of chaperon properties of trehalose was performed by assaying enzymatic activity of N-acetylglucosaminidase, which is the deficient enzyme in Sanfilippo B disease, in fibroblasts from Sanfilippo B patients. Since no suitable assays existed, an experimental protocol was developed and validated. Bioinformatic

128

Summary data were integrated in order to determine the influence of a certain mutation on folding and stability of a protein and to predict the likelihood of a mutation to be retrievable by a chemical chaperon. However, trehalose did not rescue N- acetylglucosaminidase activity in MPS IIIB fibroblasts carrying a wide array of missense mutations. The bioinformatic predictions of rescuability by chaperons have still to be confirmed experimentally.

The in vivo studies of this work were focused on the trehalose metabolism in C57BL/6 mice. Administration of trehalose in drinking water increased the intestinal trehalase activity significantly compared to untreated mice. Moreover, hepatic trehalose levels were increased in trehalose-treated mice.

The findings presented in this thesis provide new insights into the mechanisms underlying trehalose-mediated effects. High solubility and lack of toxicity make trehalose a promising molecule for the treatment of neurodegenerative diseases caused by protein misfolding and/or defects in autophagic pathways. Lysosomal enhancement with trehalose or other small molecules might be a novel therapeutic approach for the treatment of LSDs including Sanfilippo syndrome and other, more common neurodegenerative diseases like Alzheimer disease or Parkinson desease. However, further research on the molecular mechanisms as well as trehalose metabolism in mammals has to be conducted to fully investigate the potential of this molecule in the treatment of neurodegenerative storage disorders.

129

Zusammenfasung

7 Zusammenfassung Das Sanfilippo Syndrom (Mukopolysaccharidose III) ist eine autosomal rezessiv vererbte lysosomale Speichererkrankung. Ein Mangel an Enzymen, die für den Abbau des Glykosaminoglykans Heparansulfat notwendig sind, führt zu dessen fortschreitender lysosomaler Akkumulation. Die Patienten zeigen typischerweise schwere Zeichen der neurodegenerativen Veränderungen mit Verhaltensauffälligkeiten und Abnahme ihrer geistigen Fähigkeiten, die schließlich zu schwerer Demenz und frühem Tod führen. Derzeit gibt es keine etablierte Behandlung für MPS III.

Trehalose ist ein natürliches, in niederen Eukaryoten vorkommendes Glukose- Dissachharid, das einer Vielzahl zellulärer Stressfaktoren entgegenwirken kann, sowie positive Effekte in verschiedenen Tier- und Zellmodellen von Speichererkrankungen haben kann. Aufgrund dieser Daten wurden Trehalose- vermittelte Effekte auf die Proteinfaltung sowie auf die Aktivierung von Autophagozytose-Signalwege postuliert. In dieser Arbeit wurde in einem MPS III- Zellmodell das therapeutische Potential von Trehalose für die Behandlung lysosmaler Speicherkrankheiten getestet.

Zunächst wurde gezeigt, dass die Behandlung mit Trehalose zelluläre Heparansulfat- Ablagerungen in Fibroblasten von MPS III-Patienten vermindert.

Die möglichen zugrunde liegenden Mechanismen wurden anschließend weiter untersucht, sowohl die Aktivierung zellulärer Abbau-Signalkaskaden als auch ein Chaperon-Effekt. Da lysosomale Prozesse (inkl. Autophagozytose und lysosomale Exozytose) unter koordinierter transkriptioneller Kontrolle des Tanskriptionsfaktors EB (TFEB) stehen und Trehalose bekannterweise zelluläre Stoffwechselwege stimuliert, wurde die Hypothese aufgestellt, dass Trehalose die lysosomale Funktion durch TFEB-Aktivierung verbessert.

Eine Untersuchung der subzellulären TFEB-Lokalisation mit Immunofluoreszenztechniken zeigte unter Trehalose-Behandlung die schrittweise Migration von TFEB in den Zellkern, was mit der Aktivierung von TFEB-Zielgenen assoziiert ist. Darüber hinaus waren mehrere lysosomale Gene, die in zelluläre

130

Zusammenfasung

Abbauwege involviert sind, unter Behandlung mit Trehalose transkriptionell hochreguliert.

Die Analyse der Chaperon-Eigenschaften von Trehalose wurde durch Messung der N-Acetylglukosaminidase-Aktivität, die in MPS IIIB Patienten vermindert ist, in Fibroblasten von Sanfilippo B Patienten durchgeführt. Da hierfür kein passendes Protokoll zur Verfügung stand, wurde ein solches entwickelt und validiert. Bioinformatische Daten wurden in die Analyse integriert, um den Einfluss einer bestimmten Mutation auf Proteinfaltung und –stabilität zu bestimmen, sowie um die Wahrscheinlichkeit einer Verbesserung der NAG-Aktivität durch ein Chaperon vorherzusagen. Allerdings konnte die NAG-Aktivität verschiedener Mutationen mit Trehalose nicht gesteigert werden; die bioinformatischen Vorhersagen müssen noch experimentell validiert werden.

Die in vivo Experimente dieser Arbeit untersuchen den Metabolismus von Trehalose in C57BL/6 Mäusen. Die Gabe von Trehalose im Trinkwasser der Mäuse erhöhte deren intestinale Trehalaseaktivität signifikant im Vergleich zu unbehandelten Mäusen. Darüber hinaus war die hepatische Trehalosekonzentration in Trehalose- behandelten Mäusen erhöht.

Die Ergebnisse dieser Arbeit geben neue Einsichten in die Wirkungsweise von Trehalose-vermittelten Effekten. Hohe Löslichkeit und gute Verträglichkeit machen Trehalose zu einem vielversprechenden Molekül für die Behandlung neurodegenerativer Erkrankungen aufgrund von Proteinfaltungsstörungen und/oder Defekten in zellulären Stoffwechselwegen. Die Stimulierung des lysosomalen Systems durch Trehalose oder andere kleine Moleküle könnte ein neuer Therapieansatz für lysosomale Speichererkrankungen und andere, häufigere neurodegenerative Erkrankungen sein.

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References

8 References

1. Aguib Y., Heiseke A., Gilch S., Riemer C., Baier M., Schatzl H. M. & Ertmer A. 2009. Autophagy induction by trehalose counteracts cellular prion infection. Autophagy, 5, 361-9.

2. Applegarth D. A., Toone J. R. & Lowry R. B. 2000. Incidence of inborn errors of metabolism in British Columbia, 1969-1996. Pediatrics, 105, e10.

3. Arakawa T., Ejima D., Kita Y. & Tsumoto K. 2006. Small molecule pharmacological chaperones: From thermodynamic stabilization to pharmaceutical drugs. Biochim Biophys Acta, 1764, 1677-87.

4. Arfi A., Richard M., Gandolphe C. & Scherman D. 2010. Storage correction in cells of patients suffering from mucopolysaccharidoses types IIIA and VII after treatment with genistein and other isoflavones. J Inherit Metab Dis, 33, 61-7.

5. Arora A., Ha C. & Park C. B. 2004. Inhibition of insulin amyloid formation by small stress molecules. FEBS Lett, 564, 121-5.

6. Baehner F., Schmiedeskamp C., Krummenauer F., Miebach E., Bajbouj M., Whybra C., Kohlschutter A., Kampmann C. & Beck M. 2005. Cumulative incidence rates of the mucopolysaccharidoses in Germany. J Inherit Metab Dis, 28, 1011-7.

7. Bahr B. A. & Bendiske J. 2002. The neuropathogenic contributions of lysosomal dysfunction. J Neurochem, 83, 481-9.

8. Ballabio A. & Gieselmann V. 2009. Lysosomal disorders: from storage to cellular damage. Biochim Biophys Acta, 1793, 684-96.

9. Bax M. C. & Colville G. A. 1995. Behaviour in mucopolysaccharide disorders. Arch Dis Child, 73, 77-81.

10. Beattie G. M., Crowe J. H., Lopez A. D., Cirulli V., Ricordi C. & Hayek A. 1997. Trehalose: a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage. Diabetes, 46, 519-23.

11. Beesley C., Moraitou M., Winchester B., Schulpis K., Dimitriou E. & Michelakakis H. 2004. Sanfilippo B syndrome: molecular defects in Greek patients. Clin Genet, 65, 143-9.

12. Beesley C. E., Jackson M., Young E. P., Vellodi A. & Winchester B. G. 2005. Molecular defects in Sanfilippo syndrome type B (mucopolysaccharidosis IIIB). J Inherit Metab Dis, 28, 759-67.

13. Beesley C. E., Young E. P., Vellodi A. & Winchester B. G. 1998. Identification of 12 novel mutations in the alpha-N-acetylglucosaminidase gene in 14

132

References

patients with Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB). J Med Genet, 35, 910-4.

14. Bellettato C. M. & Scarpa M. 2010. Pathophysiology of neuropathic lysosomal storage disorders. J Inherit Metab Dis, 33, 347-62.

15. Benaroudj N., Lee D. H. & Goldberg A. L. 2001. Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals. J Biol Chem, 276, 24261-7.

16. Bidou L., Allamand V., Rousset J. P. & Namy O. 2012. Sense from nonsense: therapies for premature stop codon diseases. Trends Mol Med, 18, 679-88.

17. Boelens J. J., Prasad V. K., Tolar J., Wynn R. F. & Peters C. 2010. Current international perspectives on hematopoietic stem cell transplantation for inherited metabolic disorders. Pediatr Clin North Am, 57, 123-45.

18. Bondar R. J. & Mead D. C. 1974. Evaluation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides in the hexokinase method for determining glucose in serum. Clin Chem, 20, 586-90.

19. Brooks D. A., Muller V. J. & Hopwood J. J. 2006. Stop-codon read-through for patients affected by a lysosomal storage disorder. Trends Mol Med, 12, 367- 73.

20. Bull D. A., Connors R. C., Reid B. B., Albanil A., Stringham J. C. & Karwande S. V. 2000. Improved biochemical preservation of lung slices during cold storage. J Surg Res, 90, 144-8.

21. Butterworth J. 1978. Diagnosis of the mucopolysaccharidoses using cultured skin fibroblasts and amniotic fluid cells. J Inherit Metab Dis, 1, 25-8.

22. Casarejos M. J., Solano R. M., Gomez A., Perucho J., De Yebenes J. G. & Mena M. A. 2011. The accumulation of neurotoxic proteins, induced by proteasome inhibition, is reverted by trehalose, an enhancer of autophagy, in human neuroblastoma cells. Neurochem Int, 58, 512-20.

23. Castillo K., Nassif M., Valenzuela V., Rojas F., Matus S., Mercado G., Court F. A., Van Zundert B. & Hetz C. 2013. Trehalose delays the progression of amyotrophic lateral sclerosis by enhancing autophagy in motoneurons. Autophagy, 9.

24. Cejkova J., Ardan T., Cejka C. & Luyckx J. 2011. Favorable effects of trehalose on the development of UVB-mediated antioxidant/pro-oxidant imbalance in the corneal epithelium, proinflammatory cytokine and matrix metalloproteinase induction, and heat shock protein 70 expression. Graefes Arch Clin Exp Ophthalmol, 249, 1185-94.

25. Chen Q., Behar K. L., Xu T., Fan C. & Haddad G. G. 2003. Expression of Drosophila trehalose-phosphate synthase in HEK-293 cells increases hypoxia tolerance. J Biol Chem, 278, 49113-8.

133

References

26. Chen Q. & Haddad G. G. 2004. Role of trehalose phosphate synthase and trehalose during hypoxia: from flies to mammals. J Exp Biol, 207, 3125-9.

27. Chen Q., Ma E., Behar K. L., Xu T. & Haddad G. G. 2002. Role of trehalose phosphate synthase in anoxia tolerance and development in Drosophila melanogaster. J Biol Chem, 277, 3274-9.

28. Chow P. & Weissmann B. 1981. 4-Methylumbelliferyl 2-acetamido-2-deoxy- alpha-D-glucopyranoside, a fluorogenic substrate for N-acetyl-alpha-D- glucosaminidase. Carbohydr Res, 96, 87-93.

29. Cleary M. A. & Wraith J. E. 1993. Management of mucopolysaccharidosis type III. Arch Dis Child, 69, 403-6.

30. Coutinho M. F., Lacerda L. & Alves S. 2012a. Glycosaminoglycan storage disorders: a review. Biochem Res Int, 2012, 471325.

31. Coutinho M. F., Prata M. J. & Alves S. 2012b. Mannose-6-phosphate pathway: a review on its role in lysosomal function and dysfunction. Mol Genet Metab, 105, 542-50.

32. Coutinho M. F., Prata M. J. & Alves S. 2012c. A shortcut to the lysosome: the mannose-6-phosphate-independent pathway. Mol Genet Metab, 107, 257-66.

33. Crawley A. C., Marshall N., Beard H., Hassiotis S., Walsh V., King B., Hucker N., Fuller M., Jolly R. D., Hopwood J. J. & Hemsley K. M. 2011. Enzyme replacement reduces neuropathology in MPS IIIA dogs. Neurobiol Dis, 43, 422-34.

34. Cressant A., Desmaris N., Verot L., Brejot T., Froissart R., Vanier M. T., Maire I. & Heard J. M. 2004. Improved behavior and neuropathology in the mouse model of Sanfilippo type IIIB disease after adeno-associated virus-mediated gene transfer in the striatum. J Neurosci, 24, 10229-39.

35. Crowe J. H., Tablin F., Wolkers W. F., Gousset K., Tsvetkova N. M. & Ricker J. 2003. Stabilization of membranes in human platelets freeze-dried with trehalose. Chem Phys Lipids, 122, 41-52.

36. Davies J. E., Sarkar S. & Rubinsztein D. C. 2006. Trehalose reduces aggregate formation and delays pathology in a transgenic mouse model of oculopharyngeal muscular dystrophy. Hum Mol Genet, 15, 23-31.

37. De Duve C., Pressman B. C., Gianetto R., Wattiaux R. & Appelmans F. 1955. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J, 60, 604-17.

38. De Jong J. G., Wevers R. A. & Liebrand-Van Sambeek R. 1992. Measuring urinary glycosaminoglycans in the presence of protein: an improved screening procedure for mucopolysaccharidoses based on dimethylmethylene blue. Clin Chem, 38, 803-7.

134

References

39. De Ruijter J., De Ru M. H., Wagemans T., Ijlst L., Lund A. M., Orchard P. J., Schaefer G. B., Wijburg F. A. & Van Vlies N. 2012a. Heparan sulfate and dermatan sulfate derived disaccharides are sensitive markers for newborn screening for mucopolysaccharidoses types I, II and III. Mol Genet Metab, 107, 705-10.

40. De Ruijter J., Maas M., Janssen A. & Wijburg F. A. 2013. High prevalence of femoral head necrosis in Mucopolysaccharidosis type III (Sanfilippo disease): a national, observational, cross-sectional study. Mol Genet Metab, 109, 49-53.

41. De Ruijter J., Valstar M. J., Narajczyk M., Wegrzyn G., Kulik W., Ijlst L., Wagemans T., Van Der Wal W. M. & Wijburg F. A. 2012b. Genistein in Sanfilippo disease: a randomized controlled crossover trial. Ann Neurol, 71, 110-20.

42. De Virgilio C., Hottiger T., Dominguez J., Boller T. & Wiemken A. 1994. The role of trehalose synthesis for the acquisition of thermotolerance in yeast. I. Genetic evidence that trehalose is a thermoprotectant. Eur J Biochem, 219, 179-86.

43. De Virgilio C., Simmen U., Hottiger T., Boller T. & Wiemken A. 1990. Heat shock induces enzymes of trehalose metabolism, trehalose accumulation, and thermotolerance in Schizosaccharomyces pombe, even in the presence of cycloheximide. FEBS Lett, 273, 107-10.

44. Decressac M. & Björklund A. 2013. TFEB: Pathogenic role and therapeutic target in Parkinson disease. Autophagy, 9, 1181-1180.

45. Decressac M., Mattsson B., Weikop P., Lundblad M., Jakobsson J. & Bjorklund A. 2013. TFEB-mediated autophagy rescues midbrain dopamine neurons from alpha-synuclein toxicity. Proc Natl Acad Sci U S A, 110, E1817- 26.

46. Dehay B., Bove J., Rodriguez-Muela N., Perier C., Recasens A., Boya P. & Vila M. 2010. Pathogenic lysosomal depletion in Parkinson's disease. J Neurosci, 30, 12535-44.

47. Dehouck Y., Grosfils A., Folch B., Gilis D., Bogaerts P. & Rooman M. 2009. Fast and accurate predictions of protein stability changes upon mutations using statistical potentials and neural networks: PoPMuSiC-2.0. Bioinformatics, 25, 2537-43.

48. Dehouck Y., Kwasigroch J. M., Gilis D. & Rooman M. 2011. PoPMuSiC 2.1: a web server for the estimation of protein stability changes upon mutation and sequence optimality. BMC Bioinformatics, 12, 151.

49. Delgadillo V., O'callaghan Mdel M., Artuch R., Montero R. & Pineda M. 2011. Genistein supplementation in patients affected by Sanfilippo disease. J Inherit Metab Dis, 34, 1039-44.

135

References

50. Deren J. J., Broitman S. A. & Zamcheck N. 1967. Effect of diet upon intestinal disaccharidases and disaccharide absorption. J Clin Invest, 46, 186-95.

51. Desnick R. J. & Schuchman E. H. 2012. Enzyme replacement therapy for lysosomal diseases: lessons from 20 years of experience and remaining challenges. Annu Rev Genomics Hum Genet, 13, 307-35.

52. Di Malta C., Fryer J. D., Settembre C. & Ballabio A. 2012. Astrocyte dysfunction triggers neurodegeneration in a lysosomal storage disorder. Proc Natl Acad Sci U S A, 109, E2334-42.

53. Di Natale P., Di Domenico C., Gargiulo N., Castaldo S., Gonzalez Y. R. E., Mithbaokar P., De Felice M., Follenzi A., Naldini L. & Villani G. R. 2005. Treatment of the mouse model of mucopolysaccharidosis type IIIB with lentiviral-NAGLU vector. Biochem J, 388, 639-46.

54. Dionisi-Vici C., Rizzo C., Burlina A. B., Caruso U., Sabetta G., Uziel G. & Abeni D. 2002. Inborn errors of metabolism in the Italian pediatric population: a national retrospective survey. J Pediatr, 140, 321-7.

55. Durand P., Fabrega S., Henrissat B., Mornon J. P. & Lehn P. 2000. Structural features of normal and mutant human lysosomal glycoside hydrolases deduced from bioinformatics analysis. Hum Mol Genet, 9, 967-77.

56. Elbein A. D. 1974. The metabolism of alpha,alpha-trehalose. Adv Carbohydr Chem Biochem, 30, 227-56.

57. Elbein A. D., Pan Y. T., Pastuszak I. & Carroll D. 2003. New insights on trehalose: a multifunctional molecule. Glycobiology, 13, 17R-27R.

58. Ellgaard L. & Helenius A. 2003. Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol, 4, 181-91.

59. Ellinwood N. M., Ausseil J., Desmaris N., Bigou S., Liu S., Jens J. K., Snella E. M., Mohammed E. E., Thomson C. B., Raoul S., Joussemet B., Roux F., Cherel Y., Lajat Y., Piraud M., Benchaouir R., Hermening S., Petry H., Froissart R., Tardieu M., Ciron C., Moullier P., Parkes J., Kline K. L., Maire I., Vanier M. T., Heard J. M. & Colle M. A. 2011. Safe, efficient, and reproducible gene therapy of the brain in the dog models of Sanfilippo and Hurler syndromes. Mol Ther, 19, 251-9.

60. Elrick M. J., Yu T., Chung C. & Lieberman A. P. 2012. Impaired proteolysis underlies autophagic dysfunction in Niemann-Pick type C disease. Hum Mol Genet, 21, 4876-87.

61. Fan J. Q. & Ishii S. 2007. Active-site-specific chaperone therapy for Fabry disease. Yin and Yang of enzyme inhibitors. FEBS J, 274, 4962-71.

62. Fan J. Q., Ishii S., Asano N. & Suzuki Y. 1999. Accelerated transport and maturation of lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor. Nat Med, 5, 112-5.

136

References

63. Fan X., Tkachyova I., Sinha A., Rigat B. & Mahuran D. 2011. Characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis IIIC. PLoS ONE, 6, e24951.

64. Fan X., Zhang H., Zhang S., Bagshaw R. D., Tropak M. B., Callahan J. W. & Mahuran D. J. 2006. Identification of the gene encoding the enzyme deficient in mucopolysaccharidosis IIIC (Sanfilippo disease type C). Am J Hum Genet, 79, 738-44.

65. Feldhammer M., Durand S. & Pshezhetsky A. V. 2009. Protein misfolding as an underlying molecular defect in mucopolysaccharidosis III type C. PLoS ONE, 4, e7434.

66. Ficko-Blean E., Stubbs K. A., Nemirovsky O., Vocadlo D. J. & Boraston A. B. 2008. Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB. Proc Natl Acad Sci U S A, 105, 6560-5.

67. Fraldi A., Hemsley K., Crawley A., Lombardi A., Lau A., Sutherland L., Auricchio A., Ballabio A. & Hopwood J. J. 2007. Functional correction of CNS lesions in an MPS-IIIA mouse model by intracerebral AAV-mediated delivery of sulfamidase and SUMF1 genes. Hum Mol Genet, 16, 2693-702.

68. Fu H., Dirosario J., Kang L., Muenzer J. & Mccarty D. M. 2010. Restoration of central nervous system alpha-N-acetylglucosaminidase activity and therapeutic benefits in mucopolysaccharidosis IIIB mice by a single intracisternal recombinant adeno-associated viral type 2 vector delivery. J Gene Med, 12, 624-33.

69. Fu H., Dirosario J., Killedar S., Zaraspe K. & Mccarty D. M. 2011. Correction of neurological disease of mucopolysaccharidosis IIIB in adult mice by rAAV9 trans-blood-brain barrier gene delivery. Mol Ther, 19, 1025-33.

70. Fu H., Kang L., Jennings J. S., Moy S. S., Perez A., Dirosario J., Mccarty D. M. & Muenzer J. 2007. Significantly increased lifespan and improved behavioral performances by rAAV gene delivery in adult mucopolysaccharidosis IIIB mice. Gene Ther, 14, 1065-77.

71. Fuller M., Tucker J. N., Lang D. L., Dean C. J., Fietz M. J., Meikle P. J. & Hopwood J. J. 2011. Screening patients referred to a metabolic clinic for lysosomal storage disorders. J Med Genet, 48, 422-5.

72. Gerber S. A., Turecek F. & Gelb M. H. 2001. Design and synthesis of substrate and internal standard conjugates for profiling enzyme activity in the Sanfilippo syndrome by affinity chromatography/electrospray ionization mass spectrometry. Bioconjug Chem, 12, 603-15.

73. Gomes C., Escrevente C. & Costa J. 2010. Mutant superoxide dismutase 1 overexpression in NSC-34 cells: effect of trehalose on aggregation, TDP-43 localization and levels of co-expressed glycoproteins. Neurosci Lett, 475, 145- 9.

137

References

74. Greiner-Tollersrud O. K. & Berg T. 2005. Lysosomal storage disorders. In: Saftig P. (ed.) Lysosomes. Georgetown, Tex.; New York, N.Y.: Landes Bioscience/Eurekah.com; Springer Science+Business Media.

75. Guffon N., Bin-Dorel S., Decullier E., Paillet C., Guitton J. & Fouilhoux A. 2011. Evaluation of miglustat treatment in patients with type III mucopolysaccharidosis: a randomized, double-blind, placebo-controlled study. J Pediatr, 159, 838-844 e1.

76. Guo N., Puhlev I., Brown D. R., Mansbridge J. & Levine F. 2000. Trehalose expression confers desiccation tolerance on human cells. Nat Biotechnol, 18, 168-71.

77. Hasilik A., Wrocklage C. & Schroder B. 2009. Intracellular trafficking of lysosomal proteins and lysosomes. Int J Clin Pharmacol Ther, 47 Suppl 1, S18-33.

78. Hawkins-Salsbury J. A., Reddy A. S. & Sands M. S. 2011. Combination therapies for lysosomal storage disease: is the whole greater than the sum of its parts? Human Molecular Genetics, 20, R54-R60.

79. Heldermon C. D., Qin E. Y., Ohlemiller K. K., Herzog E. D., Brown J. R., Vogler C., Hou W., Orrock J. L., Crawford B. E. & Sands M. S. 2013. Disease correction by combined neonatal intracranial AAV and systemic lentiviral gene therapy in Sanfilippo Syndrome type B mice. Gene Ther.

80. Hemsley K. M. & Hopwood J. J. 2011. Emerging therapies for neurodegenerative lysosomal storage disorders - from concept to reality. J Inherit Metab Dis, 34, 1003-12.

81. Hemsley K. M., King B. & Hopwood J. J. 2007. Injection of recombinant human sulfamidase into the CSF via the cerebellomedullary cistern in MPS IIIA mice. Mol Genet Metab, 90, 313-28.

82. Honda Y., Tanaka M. & Honda S. 2010. Trehalose extends longevity in the nematode Caenorhabditis elegans. Aging Cell, 9, 558-69.

83. Hopwood J. J. & Elliott H. 1982. Detection of the Sanfilippo type B syndrome using radiolabelled oligosaccharides as substrates for the estimation of alpha- N-acetylglucosaminidase. Clin Chim Acta, 120, 77-86.

84. Hottiger T., De Virgilio C., Hall M. N., Boller T. & Wiemken A. 1994. The role of trehalose synthesis for the acquisition of thermotolerance in yeast. II. Physiological concentrations of trehalose increase the thermal stability of proteins in vitro. Eur J Biochem, 219, 187-93.

85. Hrebicek M., Mrazova L., Seyrantepe V., Durand S., Roslin N. M., Noskova L., Hartmannova H., Ivanek R., Cizkova A., Poupetova H., Sikora J., Urinovska J., Stranecky V., Zeman J., Lepage P., Roquis D., Verner A., Ausseil J., Beesley C. E., Maire I., Poorthuis B. J., Van De Kamp J., Van Diggelen O. P., Wevers R. A., Hudson T. J., Fujiwara T. M., Majewski J., Morgan K., Kmoch S. &

138

References

Pshezhetsky A. V. 2006. Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome). Am J Hum Genet, 79, 807-19.

86. Jagdale G. B., Grewal P. S. & Salminen S. O. 2005. Both heat-shock and cold-shock influence trehalose metabolism in an entomopathogenic nematode. J Parasitol, 91, 988-94.

87. Jain N. K. & Roy I. 2009. Effect of trehalose on protein structure. Protein Sci, 18, 24-36.

88. Jakobkiewicz-Banecka J., Piotrowska E., Narajczyk M., Baranska S. & Wegrzyn G. 2009. Genistein-mediated inhibition of glycosaminoglycan synthesis, which corrects storage in cells of patients suffering from mucopolysaccharidoses, acts by influencing an epidermal growth factor- dependent pathway. J Biomed Sci, 16, 26.

89. Jakobkiewicz-Banecka J., Wegrzyn A. & Wegrzyn G. 2007. Substrate deprivation therapy: a new hope for patients suffering from neuronopathic forms of inherited lysosomal storage diseases. J Appl Genet, 48, 383-8.

90. Jeyakumar M., Dwek R. A., Butters T. D. & Platt F. M. 2005. Storage solutions: treating lysosomal disorders of the brain. Nat Rev Neurosci, 6, 713- 25.

91. Kamiya T., Hirata K., Matsumoto S., Arai C., Yoshizane C., Kyono F., Ariyasu T., Hanaya T., Arai S., Okura T., Yamamoto K., Ikeda M. & Kurimoto M. 2004. Targeted disruption of the trehalase gene: determination of the digestion and absorption of trehalose in trehalase-deficient mice. Nutrition Research, 24, 185-196.

92. Kanamori Y., Saito A., Hagiwara-Komoda Y., Tanaka D., Mitsumasu K., Kikuta S., Watanabe M., Cornette R., Kikawada T. & Okuda T. 2010. The trehalose transporter 1 gene sequence is conserved in insects and encodes proteins with different kinetic properties involved in trehalose import into peripheral tissues. Insect Biochem Mol Biol, 40, 30-7.

93. Kandror O., Bretschneider N., Kreydin E., Cavalieri D. & Goldberg A. L. 2004. Yeast adapt to near-freezing temperatures by STRE/Msn2,4-dependent induction of trehalose synthesis and certain molecular chaperones. Mol Cell, 13, 771-81.

94. Kandror O., Deleon A. & Goldberg A. L. 2002. Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures. Proc Natl Acad Sci U S A, 99, 9727-32.

95. Kara N. Z., Toker L., Agam G., Anderson G. W., Belmaker R. H. & Einat H. 2013. Trehalose induced antidepressant-like effects and autophagy enhancement in mice. Psychopharmacology (Berl).

139

References

96. Keating D. J., Winter M. A., Hemsley K. M., Mackenzie K. D., Teo E. H., Hopwood J. J., Brooks D. A. & Parkinson-Lawrence E. J. 2012. Exocytosis is impaired in mucopolysaccharidosis IIIA mouse chromaffin cells. Neuroscience, 227, 110-8.

97. Kegg-Map00531. 2011. Kyoto Encyclopedia of Genes and Genomes: Glycosaminoglycan degradation (map 00531) [Online]. Kyoto: Kanehisa Laboratories. Available: http://www.genome.jp/dbget- bin/www_bget?pathway+map00531 [Accessed August 12th 2013].

98. Kikawada T., Saito A., Kanamori Y., Nakahara Y., Iwata K., Tanaka D., Watanabe M. & Okuda T. 2007. Trehalose transporter 1, a facilitated and high- capacity trehalose transporter, allows exogenous trehalose uptake into cells. Proc Natl Acad Sci U S A, 104, 11585-90.

99. Kikuta S., Hagiwara-Komoda Y., Noda H. & Kikawada T. 2012. A novel member of the trehalose transporter family functions as an h(+)-dependent trehalose transporter in the reabsorption of trehalose in malpighian tubules. Front Physiol, 3, 290.

100. Kikuta S., Kikawada T., Hagiwara-Komoda Y., Nakashima N. & Noda H. 2010. Sugar transporter genes of the brown planthopper, Nilaparvata lugens: A facilitated glucose/fructose transporter. Insect Biochem Mol Biol, 40, 805-13.

101. Krivit W., Sung J. H., Shapiro E. G. & Lockman L. A. 1995. Microglia: the effector cell for reconstitution of the central nervous system following bone marrow transplantation for lysosomal and peroxisomal storage diseases. Cell Transplant, 4, 385-92.

102. Krüger U., Wang Y., Kumar S. & Mandelkow E. M. 2011. Autophagic degradation of tau in primary neurons and its enhancement by trehalose. Neurobiol Aging.

103. Kyosseva S. V., Kyossev Z. N. & Elbein A. D. 1995. Inhibitors of pig kidney trehalase. Arch Biochem Biophys, 316, 821-6.

104. Lan D. M., Liu F. T., Zhao J., Chen Y., Wu J. J., Ding Z. T., Yue Z. Y., Ren H. M., Jiang Y. P. & Wang J. 2012. Effect of trehalose on PC12 cells overexpressing wild-type or A53T mutant alpha-synuclein. Neurochem Res, 37, 2025-32.

105. Lee-Chen G. J., Lin S. P., Lin S. Z., Chuang C. K., Hsiao K. T., Huang C. F. & Lien W. C. 2002. Identification and characterisation of mutations underlying Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB). J Med Genet, 39, E3.

106. Lee D. H. & Goldberg A. L. 1998. Proteasome inhibitors cause induction of heat shock proteins and trehalose, which together confer thermotolerance in Saccharomyces cerevisiae. Molecular and Cellular Biology, 18, 30-38.

140

References

107. Lefrancois S., Zeng J. B., Hassan A. J., Canuel M. & Morales C. R. 2003. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin. Embo Journal, 22, 6430-6437.

108. Lehman T. J., Miller N., Norquist B., Underhill L. & Keutzer J. 2011. Diagnosis of the mucopolysaccharidoses. Rheumatology (Oxford), 50 Suppl 5, v41-8.

109. Lieberman A. P., Puertollano R., Raben N., Slaugenhaupt S., Walkley S. U. & Ballabio A. 2012. Autophagy in lysosomal storage disorders. Autophagy, 8, 719-30.

110. Lima W. C., Leuba F., Soldati T. & Cosson P. 2012. Mucolipin controls lysosome exocytosis in Dictyostelium. J Cell Sci, 125, 2315-22.

111. Linde L. & Kerem B. 2008. Introducing sense into nonsense in treatments of human genetic diseases. Trends Genet, 24, 552-63.

112. Liu R., Barkhordarian H., Emadi S., Park C. B. & Sierks M. R. 2005. Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid 40 and 42. Neurobiol Dis, 20, 74-81.

113. Lloyd-Evans E. & Platt F. M. 2011. Lysosomal Ca(2+) homeostasis: role in pathogenesis of lysosomal storage diseases. Cell Calcium, 50, 200-5.

114. Logan M. R., Lacy P., Odemuyiwa S. O., Steward M., Davoine F., Kita H. & Moqbel R. 2006. A critical role for vesicle-associated membrane protein-7 in exocytosis from human eosinophils and neutrophils. Allergy, 61, 777-84.

115. Lu J., Yang C., Chen M., Ye D. Y., Lonser R. R., Brady R. O. & Zhuang Z. 2011. Histone deacetylase inhibitors prevent the degradation and restore the activity of glucocerebrosidase in Gaucher disease. Proc Natl Acad Sci U S A, 108, 21200-5.

116. Lübke T., Lobel P. & Sleat D. E. 2009. Proteomics of the lysosome. Biochim Biophys Acta, 1793, 625-35.

117. Lüllmann-Rauch R. 2005. History and Morphology of the Lysosome. In: Saftig P. (ed.) Lysosomes. Georgetown, Tex.; New York, N.Y.: Landes Bioscience/Eurekah.com; Springer Science+Business Media.

118. Luyckx J. & Baudouin C. 2011. Trehalose: an intriguing disaccharide with potential for medical application in ophthalmology. Clin Ophthalmol, 5, 577-81.

119. Luzio J. P., Pryor P. R. & Bright N. A. 2007. Lysosomes: fusion and function. Nature Reviews Molecular Cell Biology, 8, 622-632.

120. Maegawa G. H., Tropak M., Buttner J., Stockley T., Kok F., Clarke J. T. & Mahuran D. J. 2007. Pyrimethamine as a potential pharmacological chaperone for late-onset forms of GM2 gangliosidosis. J Biol Chem, 282, 9150-61.

141

References

121. Maegawa G. H., Tropak M. B., Buttner J. D., Rigat B. A., Fuller M., Pandit D., Tang L., Kornhaber G. J., Hamuro Y., Clarke J. T. & Mahuran D. J. 2009. Identification and characterization of ambroxol as an enzyme enhancement agent for Gaucher disease. J Biol Chem, 284, 23502-16.

122. Malcolm C., Hain R., Gibson F., Adams S., Anderson G. & Forbat L. 2012. Challenging symptoms in children with rare life-limiting conditions: findings from a prospective diary and interview study with families. Acta Paediatr, 101, 985-92.

123. Malinowska M., Wilkinson F. L., Bennett W., Langford-Smith K. J., O'leary H. A., Jakobkiewicz-Banecka J., Wynn R., Wraith J. E., Wegrzyn G. & Bigger B. W. 2009. Genistein reduces lysosomal storage in peripheral tissues of mucopolysaccharide IIIB mice. Mol Genet Metab, 98, 235-42.

124. Malinowska M., Wilkinson F. L., Langford-Smith K. J., Langford-Smith A., Brown J. R., Crawford B. E., Vanier M. T., Grynkiewicz G., Wynn R. F., Wraith J. E., Wegrzyn G. & Bigger B. W. 2010. Genistein improves neuropathology and corrects behaviour in a mouse model of neurodegenerative metabolic disease. PLoS ONE, 5, e14192.

125. Marsh J. & Fensom A. H. 1985. 4-Methylumbelliferyl alpha-N- acetylglucosaminidase activity for diagnosis of Sanfilippo B disease. Clin Genet, 27, 258-62.

126. Martina J. A., Chen Y., Gucek M. & Puertollano R. 2012. MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB. Autophagy, 8, 903-14.

127. Medina D. L., Fraldi A., Bouche V., Annunziata F., Mansueto G., Spampanato C., Puri C., Pignata A., Martina J. A., Sardiello M., Palmieri M., Polishchuk R., Puertollano R. & Ballabio A. 2011. Transcriptional activation of lysosomal exocytosis promotes cellular clearance. Dev Cell, 21, 421-30.

128. Meikle P. J., Hopwood J. J., Clague A. E. & Carey W. F. 1999. Prevalence of lysosomal storage disorders. JAMA, 281, 249-54.

129. Meyer A., Kossow K., Gal A., Muhlhausen C., Ullrich K., Braulke T. & Muschol N. 2007. Scoring evaluation of the natural course of mucopolysaccharidosis type IIIA (Sanfilippo syndrome type A). Pediatrics, 120, e1255-61.

130. Mu T. W., Ong D. S., Wang Y. J., Balch W. E., Yates J. R., 3rd, Segatori L. & Kelly J. W. 2008. Chemical and biological approaches synergize to ameliorate protein-folding diseases. Cell, 134, 769-81.

131. Nelson J. 1997. Incidence of the mucopolysaccharidoses in Northern Ireland. Hum Genet, 101, 355-8.

132. Nelson J., Crowhurst J., Carey B. & Greed L. 2003. Incidence of the mucopolysaccharidoses in Western Australia. Am J Med Genet A, 123A, 310- 3.

142

References

133. Niosh 1993. Registry of toxic effects of chemical substances. In: National Institute for Occupational Safety and Health (ed.) 19 -1978: DHEW publication no (NIOSH). Cincinnati, Ohio; Washington, D.C.: U.S. Dept. of Health and Human Services, Public Health Service, Center for Disease Control.

134. O'brien J. S. 1972. Sanfilippo syndrome: profound deficiency of alpha- acetylglucosaminidase activity in organs and skin fibroblasts from type-B patients. Proc Natl Acad Sci U S A, 69, 1720-2.

135. Okada S., Veath M. L., Leroy J. & O'brien J. S. 1971. Ganglioside GM2 storage diseases: hexosaminidase deficiencies in cultured fibroblasts. Am J Hum Genet, 23, 55-61.

136. Okumiya T., Kroos M. A., Vliet L. V., Takeuchi H., Van Der Ploeg A. T. & Reuser A. J. 2007. Chemical chaperones improve transport and enhance stability of mutant alpha-glucosidases in glycogen storage disease type II. Mol Genet Metab, 90, 49-57.

137. Osellame L. D., Rahim A. A., Hargreaves I. P., Gegg M. E., Richard-Londt A., Brandner S., Waddington S. N., Schapira A. H. & Duchen M. R. 2013. Mitochondria and quality control defects in a mouse model of Gaucher disease-links to Parkinson's disease. Cell Metab, 17, 941-53.

138. Palmieri M., Impey S., Kang H., Di Ronza A., Pelz C., Sardiello M. & Ballabio A. 2011. Characterization of the CLEAR network reveals an integrated control of cellular clearance pathways. Human Molecular Genetics, 20, 3852-3866.

139. Parenti G. 2009. Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics. EMBO Mol Med, 1, 268-79.

140. Parenti G., Zuppaldi A., Gabriela Pittis M., Rosaria Tuzzi M., Annunziata I., Meroni G., Porto C., Donaudy F., Rossi B., Rossi M., Filocamo M., Donati A., Bembi B., Ballabio A. & Andria G. 2007. Pharmacological enhancement of mutated alpha-glucosidase activity in fibroblasts from patients with Pompe disease. Mol Ther, 15, 508-14.

141. Parr C., Carzaniga R., Gentleman S. M., Van Leuven F., Walter J. & Sastre M. 2012. Glycogen synthase kinase 3 inhibition promotes lysosomal biogenesis and autophagic degradation of the amyloid-beta precursor protein. Molecular and Cellular Biology, 32, 4410-8.

142. Parrou J. L. & Francois J. 1997. A simplified procedure for a rapid and reliable assay of both glycogen and trehalose in whole yeast cells. Anal Biochem, 248, 186-8.

143. Pastore N., Ballabio A. & Brunetti-Pierri N. 2013a. Autophagy master regulator TFEB induces clearance of toxic SERPINA1/alpha-1-antitrypsin polymers. Autophagy, 9, 1094-1096.

144. Pastore N., Blomenkamp K., Annunziata F., Piccolo P., Mithbaokar P., Maria Sepe R., Vetrini F., Palmer D., Ng P., Polishchuk E., Iacobacci S., Polishchuk

143

References

R., Teckman J., Ballabio A. & Brunetti-Pierri N. 2013b. Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency. EMBO Mol Med, 5, 397-412.

145. Pena-Llopis S., Vega-Rubin-De-Celis S., Schwartz J. C., Wolff N. C., Tran T. A., Zou L., Xie X. J., Corey D. R. & Brugarolas J. 2011. Regulation of TFEB and V-ATPases by mTORC1. EMBO J, 30, 3242-58.

146. Pinto R., Caseiro C., Lemos M., Lopes L., Fontes A., Ribeiro H., Pinto E., Silva E., Rocha S., Marcao A., Ribeiro I., Lacerda L., Ribeiro G., Amaral O. & Sa Miranda M. C. 2004. Prevalence of lysosomal storage diseases in Portugal. Eur J Hum Genet, 12, 87-92.

147. Piotrowska E., Jakobkiewicz-Banecka J., Baranska S., Tylki-Szymanska A., Czartoryska B., Wegrzyn A. & Wegrzyn G. 2006. Genistein-mediated inhibition of glycosaminoglycan synthesis as a basis for gene expression-targeted isoflavone therapy for mucopolysaccharidoses. Eur J Hum Genet, 14, 846-52.

148. Piotrowska E., Jakobkiewicz-Banecka J., Maryniak A., Tylki-Szymanska A., Puk E., Liberek A., Wegrzyn A., Czartoryska B., Slominska-Wojewodzka M. & Wegrzyn G. 2011. Two-year follow-up of Sanfilippo Disease patients treated with a genistein-rich isoflavone extract: assessment of effects on cognitive functions and general status of patients. Med Sci Monit, 17, CR196-202.

149. Piotrowska E., Jakóbkiewicz-Banecka J., Tylki-Szymanska A., Liberek A., Maryniak A., Malinowska M., Czartoryska B., Puk E., Kloska A., Liberek T., Baranska S., Wegrzyn A. & Wegrzyn G. 2008. Genistin-rich soy isoflavone extract in substrate reduction therapy for Sanfilippo syndrome: An open-label, pilot study in 10 pediatric patients. Current Therapeutic Research, 69, 166- 179.

150. Platt F. M. & Walkley S. U. 2004. Lysosomal defects and storage. In: Platt F. M. & Walkley S. U. (eds.) Lysosomal disorders of brain : recent advances in molecular and cellular pathogenesis and treatment. Oxford ; New York: Oxford University Press.

151. Poorthuis B. J. H. M., Wevers R. A., Kleijer W. J., Groener J. E. M., De Jong J. G. N., Van Weely S., Niezen-Koning K. E. & Van Diggelen O. P. 1999. The frequency of lysosomal storage diseases in The Netherlands. Human Genetics, 105, 151-156.

152. Rao S. K., Huynh C., Proux-Gillardeaux V., Galli T. & Andrews N. W. 2004. Identification of SNAREs involved in synaptotagmin VII-regulated lysosomal exocytosis. J Biol Chem, 279, 20471-9.

153. Reczek D., Schwake M., Schroder J., Hughes H., Blanz J., Jin X., Brondyk W., Van Patten S., Edmunds T. & Saftig P. 2007. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta- glucocerebrosidase. Cell, 131, 770-83.

144

References

154. Reddy B. S., Pleasants J. R. & Wostmann B. S. 1968. Effect of dietary carbohydrates on intestinal disaccharidases in germfree and conventional rats. J Nutr, 95, 413-9.

155. Reina-Bueno M., Argandona M., Salvador M., Rodriguez-Moya J., Iglesias- Guerra F., Csonka L. N., Nieto J. J. & Vargas C. 2012. Role of trehalose in salinity and temperature tolerance in the model halophilic bacterium Chromohalobacter salexigens. PLoS ONE, 7, e33587.

156. Reuser A. J., Verheijen F. W., Bali D., Van Diggelen O. P., Germain D. P., Hwu W. L., Lukacs Z., Muhl A., Olivova P., Piraud M., Wuyts B., Zhang K. & Keutzer J. 2011. The use of dried blood spot samples in the diagnosis of lysosomal storage disorders--current status and perspectives. Mol Genet Metab, 104, 144-8.

157. Richards A. B., Krakowka S., Dexter L. B., Schmid H., Wolterbeek A. P., Waalkens-Berendsen D. H., Shigoyuki A. & Kurimoto M. 2002. Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies. Food Chem Toxicol, 40, 871-98.

158. Robertson D. A., Callen D. F., Baker E. G., Morris C. P. & Hopwood J. J. 1988. Chromosomal localization of the gene for human glucosamine-6- sulphatase to 12q14. Hum Genet, 79, 175-8.

159. Roczniak-Ferguson A., Petit C. S., Froehlich F., Qian S., Ky J., Angarola B., Walther T. C. & Ferguson S. M. 2012. The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis. Sci Signal, 5, ra42.

160. Rodriguez-Navarro J. A., Rodriguez L., Casarejos M. J., Solano R. M., Gomez A., Perucho J., Cuervo A. M., Garcia De Yebenes J. & Mena M. A. 2010. Trehalose ameliorates dopaminergic and tau pathology in parkin deleted/tau overexpressing mice through autophagy activation. Neurobiol Dis, 39, 423-38.

161. Rohrbach M. & Clarke J. T. 2007. Treatment of lysosomal storage disorders : progress with enzyme replacement therapy. Drugs, 67, 2697-716.

162. Rosensweig N. S. & Herman R. H. 1968. Control of jejunal sucrase and maltase activity by dietary sucrose or fructose in man. A model for the study of enzyme regulation in man. J Clin Invest, 47, 2253-62.

163. Rosensweig N. S. & Herman R. H. 1969. Diet and disaccharidases. Am J Clin Nutr, 22, 99-102.

164. Rosensweig N. S., Herman R. H. & Stifel R. B. 1971. Dietary regulation of small intestinal enzyme activity in man. Am J Clin Nutr, 24, 65-9.

165. Rozaklis T., Beard H., Hassiotis S., Garcia A. R., Tonini M., Luck A., Pan J., Lamsa J. C., Hopwood J. J. & Hemsley K. M. 2011. Impact of high-dose, chemically modified sulfamidase on pathology in a murine model of MPS IIIA. Exp Neurol, 230, 123-30.

145

References

166. Ruijter G. J., Valstar M. J., Van De Kamp J. M., Van Der Helm R. M., Durand S., Van Diggelen O. P., Wevers R. A., Poorthuis B. J., Pshezhetsky A. V. & Wijburg F. A. 2008. Clinical and genetic spectrum of Sanfilippo type C (MPS IIIC) disease in The Netherlands. Mol Genet Metab, 93, 104-11.

167. Rusmini P., Crippa V., Giorgetti E., Boncoraglio A., Cristofani R., Carra S. & Poletti A. 2013. Clearance of the mutant androgen receptor in motoneuronal models of spinal and bulbar muscular atrophy. Neurobiol Aging.

168. Saftig P. & Klumperman J. 2009. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol, 10, 623- 35.

169. Salleh H. M. & Honek J. F. 1990. Time-dependent inhibition of porcine kidney trehalase by aminosugars. FEBS Lett, 262, 359-62.

170. Sanfilippo S. J., Podosin R., Langer L. & Good R. A. 1963. Mental retardation associated with acid mucopolysacchariduria (heparitin sulfate type). J Pediatr, 63, 837-838.

171. Sardiello M. & Ballabio A. 2009. Lysosomal enhancement: a CLEAR answer to cellular degradative needs. Cell Cycle, 8, 4021-2.

172. Sardiello M., Palmieri M., Di Ronza A., Medina D. L., Valenza M., Gennarino V. A., Di Malta C., Donaudy F., Embrione V., Polishchuk R. S., Banfi S., Parenti G., Cattaneo E. & Ballabio A. 2009. A gene network regulating lysosomal biogenesis and function. Science, 325, 473-7.

173. Sarkar S., Davies J. E., Huang Z., Tunnacliffe A. & Rubinsztein D. C. 2007. Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein. J Biol Chem, 282, 5641- 52.

174. Sawkar A. R., Cheng W. C., Beutler E., Wong C. H., Balch W. E. & Kelly J. W. 2002. Chemical chaperones increase the cellular activity of N370S beta - glucosidase: a therapeutic strategy for Gaucher disease. Proc Natl Acad Sci U S A, 99, 15428-33.

175. Schaeffer V., Lavenir I., Ozcelik S., Tolnay M., Winkler D. T. & Goedert M. 2012. Stimulation of autophagy reduces neurodegeneration in a mouse model of human tauopathy. Brain, 135, 2169-77.

176. Schmidtchen A., Greenberg D., Zhao H. G., Li H. H., Huang Y., Tieu P., Zhao H. Z., Cheng S., Zhao Z., Whitley C. B., Di Natale P. & Neufeld E. F. 1998. NAGLU mutations underlying Sanfilippo syndrome type B. Am J Hum Genet, 62, 64-9.

177. Schröder B. A., Wrocklage C., Hasilik A. & Saftig P. 2010. The proteome of lysosomes. Proteomics, 10, 4053-76.

146

References

178. Schultz M. L., Tecedor L., Chang M. & Davidson B. L. 2011. Clarifying lysosomal storage diseases. Trends Neurosci, 34, 401-10.

179. Scott H. S., Blanch L., Guo X. H., Freeman C., Orsborn A., Baker E., Sutherland G. R., Morris C. P. & Hopwood J. J. 1995. Cloning of the sulphamidase gene and identification of mutations in Sanfilippo A syndrome. Nat Genet, 11, 465-7.

180. Selmer K. K., Gilfillan G. D., Stromme P., Lyle R., Hughes T., Hjorthaug H. S., Brandal K., Nakken S., Misceo D., Egeland T., Munthe L. A., Braekken S. K. & Undlien D. E. 2012. A mild form of Mucopolysaccharidosis IIIB diagnosed with targeted next-generation sequencing of linked genomic regions. Eur J Hum Genet, 20, 58-63.

181. Settembre C. & Ballabio A. 2011. TFEB regulates autophagy: An integrated coordination of cellular degradation and recycling processes. Autophagy, 7, 1379-81.

182. Settembre C., Di Malta C., Polito V. A., Arencibia M. G., Vetrini F., Erdin S., Erdin S. U., Huynh T., Medina D., Colella P., Sardiello M., Rubinsztein D. C. & Ballabio A. 2011. TFEB Links Autophagy to Lysosomal Biogenesis. Science, 332, 1429-1433.

183. Settembre C., Fraldi A., Jahreiss L., Spampanato C., Venturi C., Medina D., De Pablo R., Tacchetti C., Rubinsztein D. C. & Ballabio A. 2008. A block of autophagy in lysosomal storage disorders. Hum Mol Genet, 17, 119-29.

184. Settembre C., Fraldi A., Medina D. L. & Ballabio A. 2013. Signals from the lysosome: a control centre for cellular clearance and energy metabolism. Nat Rev Mol Cell Biol, 14, 283-96.

185. Settembre C., Zoncu R., Medina D. L., Vetrini F., Erdin S., Huynh T., Ferron M., Karsenty G., Vellard M. C., Facchinetti V., Sabatini D. M. & Ballabio A. 2012. A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J, 31, 1095-108.

186. Shen D., Wang X., Li X., Zhang X., Yao Z., Dibble S., Dong X. P., Yu T., Lieberman A. P., Showalter H. D. & Xu H. 2012. Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release. Nat Commun, 3, 731.

187. Singer M. A. & Lindquist S. 1998. Multiple effects of trehalose on protein folding in vitro and in vivo. Mol Cell, 1, 639-48.

188. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J. & Klenk D. C. 1985. Measurement of protein using bicinchoninic acid. Anal Biochem, 150, 76-85.

147

References

189. Song W., Wang F., Savini M., Ake A., Di Ronza A., Sardiello M. & Segatori L. 2013. TFEB regulates lysosomal proteostasis. Hum Mol Genet, 22, 1994- 2009.

190. Sorrentino N. C., D'orsi L., Sambri I., Nusco E., Monaco C., Spampanato C., Polishchuk E., Saccone P., De Leonibus E., Ballabio A. & Fraldi A. 2013. A highly secreted sulphamidase engineered to cross the blood-brain barrier corrects brain lesions of mice with mucopolysaccharidoses type IIIA. EMBO Mol Med, 5, 675-90.

191. Soto T., Fernandez J., Vicente-Soler J., Cansado J. & Gacto M. 1999. Accumulation of trehalose by overexpression of tps1, coding for trehalose-6- phosphate synthase, causes increased resistance to multiple stresses in the fission yeast schizosaccharomyces pombe. Appl Environ Microbiol, 65, 2020- 4.

192. Spampanato C., Feeney E., Li L., Cardone M., Lim J. A., Annunziata F., Zare H., Polishchuk R., Puertollano R., Parenti G., Ballabio A. & Raben N. 2013. Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. EMBO Mol Med, 5, 691-706.

193. Stambuk B. U., Panek A. D., Crowe J. H., Crowe L. M. & De Araujo P. S. 1998. Expression of high-affinity trehalose-H+ symport in Saccharomyces cerevisiae. Biochim Biophys Acta, 1379, 118-28.

194. Suzuki Y. 2008. Chemical chaperone therapy for GM1-gangliosidosis. Cell Mol Life Sci, 65, 351-3.

195. Swan M. & Saunders-Pullman R. 2013. The association between ss- glucocerebrosidase mutations and parkinsonism. Curr Neurol Neurosci Rep, 13, 368.

196. Tanaka M., Machida Y., Niu S., Ikeda T., Jana N. R., Doi H., Kurosawa M., Nekooki M. & Nukina N. 2004. Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease. Nat Med, 10, 148-54.

197. Tessitore A., Villani G. R., Di Domenico C., Filocamo M., Gatti R. & Di Natale P. 2000. Molecular defects in the alpha-N-acetylglucosaminidase gene in Italian Sanfilippo type B patients. Hum Genet, 107, 568-76.

198. Tomanin R., Zanetti A., Zaccariotto E., D'avanzo F., Bellettato C. M. & Scarpa M. 2012. Gene therapy approaches for lysosomal storage disorders, a good model for the treatment of mendelian diseases. Acta Paediatr, 101, 692-701.

199. Tropak M. B., Blanchard J. E., Withers S. G., Brown E. D. & Mahuran D. 2007. High-throughput screening for human lysosomal beta-N-Acetyl hexosaminidase inhibitors acting as pharmacological chaperones. Chem Biol, 14, 153-64.

200. Tropak M. B., Kornhaber G. J., Rigat B. A., Maegawa G. H., Buttner J. D., Blanchard J. E., Murphy C., Tuske S. J., Coales S. J., Hamuro Y., Brown E. D.

148

References

& Mahuran D. J. 2008. Identification of pharmacological chaperones for Gaucher disease and characterization of their effects on beta- glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry. Chembiochem, 9, 2650-62.

201. Tropak M. B., Reid S. P., Guiral M., Withers S. G. & Mahuran D. 2004. Pharmacological enhancement of beta-hexosaminidase activity in fibroblasts from adult Tay-Sachs and Sandhoff Patients. J Biol Chem, 279, 13478-87.

202. Tsunemi T., Ashe T. D., Morrison B. E., Soriano K. R., Au J., Roque R. A., Lazarowski E. R., Damian V. A., Masliah E. & La Spada A. R. 2012. PGC- 1alpha rescues Huntington's disease proteotoxicity by preventing oxidative stress and promoting TFEB function. Sci Transl Med, 4, 142ra97.

203. Urban D. J., Zheng W., Goker-Alpan O., Jadhav A., Lamarca M. E., Inglese J., Sidransky E. & Austin C. P. 2008. Optimization and validation of two miniaturized glucocerebrosidase enzyme assays for high throughput screening. Comb Chem High Throughput Screen, 11, 817-24.

204. Urbanelli L., Magini A., Polchi A., Polidoro M. & Emiliani C. 2011. Recent developments in therapeutic approaches for lysosomal storage diseases. Recent Pat CNS Drug Discov, 6, 1-19.

205. Valenzano K. J., Khanna R., Powe A. C., Boyd R., Lee G., Flanagan J. J. & Benjamin E. R. 2011. Identification and characterization of pharmacological chaperones to correct enzyme deficiencies in lysosomal storage disorders. Assay Drug Dev Technol, 9, 213-35.

206. Valstar M. J., Marchal J. P., Grootenhuis M., Colland V. & Wijburg F. A. 2011. Cognitive development in patients with Mucopolysaccharidosis type III (Sanfilippo syndrome). Orphanet J Rare Dis, 6, 43.

207. Valstar M. J., Neijs S., Bruggenwirth H. T., Olmer R., Ruijter G. J., Wevers R. A., Van Diggelen O. P., Poorthuis B. J., Halley D. J. & Wijburg F. A. 2010. Mucopolysaccharidosis type IIIA: clinical spectrum and genotype-phenotype correlations. Ann Neurol, 68, 876-87.

208. Valstar M. J., Ruijter G. J. G., Diggelen O. P., Poorthuis B. J. & Wijburg F. A. 2008. Sanfilippo syndrome: A mini-review. Journal of Inherited Metabolic Disease, 31, 240-252.

209. Vitner E. B., Platt F. M. & Futerman A. H. 2010. Common and uncommon pathogenic cascades in lysosomal storage diseases. J Biol Chem, 285, 20423-7.

210. Von Figura K. 1991. Molecular recognition and targeting of lysosomal proteins. Curr Opin Cell Biol, 3, 642-6.

211. Wang F., Agnello G., Sotolongo N. & Segatori L. 2011a. Ca2+ homeostasis modulation enhances the amenability of L444P glucosylcerebrosidase to

149

References

proteostasis regulation in patient-derived fibroblasts. ACS Chem Biol, 6, 158- 68.

212. Wang F., Chou A. & Segatori L. 2011b. Lacidipine remodels protein folding and Ca 2+ homeostasis in Gaucher's disease fibroblasts: a mechanism to rescue mutant glucocerebrosidase. Chem Biol, 18, 766-76.

213. Wang F. & Segatori L. 2013. Remodeling the proteostasis network to rescue glucocerebrosidase variants by inhibiting ER-associated degradation and enhancing ER folding. PLoS ONE, 8, e61418.

214. Wang F., Song W., Brancati G. & Segatori L. 2011c. Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases. J Biol Chem, 286, 43454-64.

215. Watanabe M., Kikawada T., Minagawa N., Yukuhiro F. & Okuda T. 2002. Mechanism allowing an insect to survive complete dehydration and extreme temperatures. Journal of Experimental Biology, 205, 2799-2802.

216. Weber B., Guo X. H., Kleijer W. J., Van De Kamp J. J., Poorthuis B. J. & Hopwood J. J. 1999. Sanfilippo type B syndrome (mucopolysaccharidosis III B): allelic heterogeneity corresponds to the wide spectrum of clinical phenotypes. Eur J Hum Genet, 7, 34-44.

217. Welsh J. D., Poley J. R., Bhatia M. & Stevenson D. E. 1978. Intestinal disaccharidase activities in relation to age, race, and mucosal damage. Gastroenterology, 75, 847-55.

218. White K. K., Karol L. A., White D. R. & Hale S. 2011. Musculoskeletal manifestations of Sanfilippo Syndrome (mucopolysaccharidosis type III). J Pediatr Orthop, 31, 594-8.

219. Wijburg F. A., Wegrzyn G., Burton B. K. & Tylki-Szymanska A. 2013. Mucopolysaccharidosis type III (Sanfilippo syndrome) and misdiagnosis of idiopathic developmental delay, attention deficit/hyperactivity disorder or autism spectrum disorder. Acta Paediatr, 102, 462-70.

220. Winder-Rhodes S. E., Garcia-Reitbock P., Ban M., Evans J. R., Jacques T. S., Kemppinen A., Foltynie T., Williams-Gray C. H., Chinnery P. F., Hudson G., Burn D. J., Allcock L. M., Sawcer S. J., Barker R. A. & Spillantini M. G. 2012. Genetic and pathological links between Parkinson's disease and the lysosomal disorder Sanfilippo syndrome. Mov Disord, 27, 312-5.

221. Wolfe B. J., Ghomashchi F., Kim T., Abam C. A., Sadilek M., Jack R., Thompson J. N., Scott C. R., Gelb M. H. & Turecek F. 2012. New substrates and enzyme assays for the detection of mucopolysaccharidosis III (Sanfilippo Syndrome) types A, B, C, and D by tandem mass spectrometry. Bioconjug Chem, 23, 557-64.

222. Wolkers W. F., Walker N. J., Tablin F. & Crowe J. H. 2001. Human platelets loaded with trehalose survive freeze-drying. Cryobiology, 42, 79-87.

150

References

223. Wraith J. E. 2004. Clinical aspects and diagnosis. In: Platt F. M. & Walkley S. U. (eds.) Lysosomal disorders of brain: recent advances in molecular and cellular pathogenesis and treatment. Oxford; New York: Oxford University Press.

224. Wyatt G. R. & Kalf G. F. 1957. The chemistry of insect hemolymph. II. Trehalose and other carbohydrates. J Gen Physiol, 40, 833-47.

225. Xu C., Li X., Wang F., Weng H. & Yang P. 2013. Trehalose prevents neural tube defects by correcting maternal diabetes-suppressed autophagy and neurogenesis. Am J Physiol Endocrinol Metab.

226. Yogalingam G. & Hopwood J. J. 2001. Molecular genetics of mucopolysaccharidosis type IIIA and IIIB: Diagnostic, clinical, and biological implications. Hum Mutat, 18, 264-81.

227. Yogalingam G., Weber B., Meehan J., Rogers J. & Hopwood J. J. 2000. Mucopolysaccharidosis type IIIB: characterisation and expression of wild-type and mutant recombinant alpha-N-acetylglucosaminidase and relationship with sanfilippo phenotype in an attenuated patient. Biochim Biophys Acta, 1502, 415-25.

228. Yu W. H., Zhao K. W., Ryazantsev S., Rozengurt N. & Neufeld E. F. 2000. Short-term enzyme replacement in the murine model of Sanfilippo syndrome type B. Mol Genet Metab, 71, 573-80.

229. Yu Z., Sawkar A. R. & Kelly J. W. 2007. Pharmacologic chaperoning as a strategy to treat Gaucher disease. FEBS J, 274, 4944-50.

230. Zhang J. H., Chung T. D. & Oldenburg K. R. 1999. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen, 4, 67-73.

231. Zhang Q. & Yan T. 2012. Correlation of intracellular trehalose concentration with desiccation resistance of soil Escherichia coli populations. Appl Environ Microbiol, 78, 7407-13.

232. Zhao H. G., Li H. H., Bach G., Schmidtchen A. & Neufeld E. F. 1996. The molecular basis of Sanfilippo syndrome type B. Proc Natl Acad Sci U S A, 93, 6101-5.

233. Zheng W., Padia J., Urban D. J., Jadhav A., Goker-Alpan O., Simeonov A., Goldin E., Auld D., Lamarca M. E., Inglese J., Austin C. P. & Sidransky E. 2007. Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease. Proc Natl Acad Sci U S A, 104, 13192-7.

234. Zhou J., Tan S. H., Nicolas V., Bauvy C., Yang N. D., Zhang J., Xue Y., Codogno P. & Shen H. M. 2013. Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion. Cell Res, 23, 508-23.

151

Preliminary Publications

9 Preliminary Publications

Mauri V., Lotfi P., Segatori L. & Sardiello M. 2013. A rapid and sensitive method for measuring N-acetylglucosaminidase activity in cultured cells. PLoS ONE, 8, e68060.

Appendix

10 Appendix

Figure I: Sequence of Clone C1’ and C2’ from Tps1-cloning and alignment to reference sequence of Tps1 Reference sequence is taken from the UCSC Genome Browser. Missmatches are marked in red. All shown missmatches are polymorphisms.

Tps1 Ref 1>ATGCCCGACACGGAAATCATCGTTACCAATGCCGGGGAGCCCTCCACCAAGGCGAGTCTCATCGTGGTATCCAATCGGTTGCCGTTTGTGCTTATCCGAG C1’ 1>ATGCCCGACACGGAAATCATCGTTACCAATGCCGGGGAGCCCTCCACCAAGGCGAGTCTCATCGTGGTATCCAATCGGTTGCCGTTTGTGCTTATCCGAG C2’ 1>ATGCCCGACACGGAAATCATCGTTACCAATGCCGGGGAGCCCTCCACCAAGGCGAGTCTCATCGTGGTATCCAATCGGTTGCCGTTTGTGCTTATCCGAG

Tps1 Ref 101>ATCCGAAGACCGATGAGTTGGAACGCAGGGCCAGTGCCGGTGGCCTGGTGACTGCAGTGTGCCCGGTGGTGATCAAGGGCAGTGGTCTCTGGGTGGGCTG C1’ 101>ATCCGAAGACCGATGAGTTGGAACGCAGGGCCAGTGCCGGTGGCCTGGTGACTGCAGTGTGCCCGGTGGTGATCAAGGGCAGTGGTCTCTGGGTGGGCTG C2’ 101>ATCCGAAGACCGATGAGTTGGAACGCAGGGCCAGTGCCGGTGGCCTGGTGACTGCAGTGTGCCCGGTGGTGATCAAGGGCAGTGGTCTCTGGGTGGGCTG

Tps1 Ref 201>GTCGGGTATCCACTTGAAGGATCCTAACGAGGCCATTCCCGAGTCCAATCCCAACGATCAGACTCCGACTGCTGGCCTCAAGTCCGAGCAGGTGGTGTCC C1’ 201>GTCGGGTATCCACTTGAAGGATCCTAACGAGGCCATTCCCGAGTCCAATCCCAACGATCAGACTCCGACTGCTGGCCTCAAGTCCGAGCAGGTGGTGTCC C2’ 201>GTCGGGTATCCACTTGAAGGATCCTAACGAGGCCATTCCCGAGTCCAATCCCAACGATCAGACTCCGACTGCTGGCCTCAAGTCCGAGCAGGTGGTGTCC

Tps1 Ref 301>GTCAACATCGATTCGAAGATCTTCGATAGCTACTACAACGGATGCTGCAACAAGATCTTCTGGCCACTGTTCCACTCGATGCCGGGAAGAGCCAACTTCG C1’ 301>GTGAACATCGATTCGAAGATCTTCGATAGCTACTACAACGGATGCTGCAACAAGATCTTCTGGCCACTGTTCCACTCGATGCCGGGAAGAGCCAACTTCG C2’ 301>GTGAACATCGATTCGAAGATCTTCGATAGCTACTACAACGGATGCTGCAACAAGATCTTCTGGCCACTGTTCCACTCGATGCCGGGAAGAGCCAACTTCG

Tps1 Ref 401>GAGGCGAGCACTGGCACGACTATGTCACTGTCAACAAGCACTTCGCCGTGCGGACCATCGAGGCTCTGGAGAAGTGCCTGGCCAAAAACCAGGGCAGCGA C1’ 401>GAGGCGAGCACTGGCACGACTATGTCACTGTCAACAAGCACTTCGCCGTGCGGACCATTGAGGCTCTGGAGAAGTGCCTGGCCAAAAACCAGGGCAGCGA C2’ 401>GAGGCGAGCACTGGCACGACTATGTCACTGTCAACAAGCACTTCGCCGTGCGGACCATTGAGGCTCTGGAGAAGTGCCTGGCCAAAAACCAGGGCAGCGA

Tps1 Ref 501>GAAGAGTCCACCGATTGTCTGGATCCACGACTACCATCTCATGCTGGCCGCCAATTGGGTGCGCGAGCACGCCGAGGAGAAGAACCTGCCCTGCCGACTG C1’ 501>GAAGAGTCCACCGATTGTCTGGATCCACGACTACCATCTCATGCTGGCCGCCAATTGGGTGCGCGAGCACGCCGAGGAGAAGAACCTGCCCTGCCGACTG C2’ 501>GAAGAGTCCACCGATTGTCTGGATCCACGACTACCATCTCATGCTGGCCGCCAATTGGGTGCGCGAGCACGCCGAGGAGAAGAACCTGCCCTGCCGACTG

Tps1 Ref 601>GCTTTCTTCCTGCACATCCCATTCCCGCCATGGGACATCTTCCGCCTGCTGCCCTGGTCCGATGAGATCCTACAGGGTATGTTGGGTTGTGACCTGGTAG C1’ 601>GCTTTCTTCCTGCACATTCCATTCCCGCCATGGGACATCTTCCGCCTGCTGCCCTGGTCCGATGAGATCCTACAGGGTATGTTGGGTTGTGACCTGGTAG C2’ 601>GCTTTCTTCCTGCACATTCCATTCCCGCCATGGGACATCTTCCGCCTGCTGCCCTGGTCCGATGAGATCCTACAGGGTATGTTGGGTTGTGACCTGGTAG

Tps1 Ref 701>GCTTCCATATTCAGGACTATTGCCTTAACTTTGTGGACTGCTGCCAGCGCAATCTCGGCTGCCGTGTGGACAGAAACAATCTGCTGGTGGAACATGGCGG C1’ 701>GCTTCCATATTCAGGACTATTGCCTGAACTTTGTGGACTGCTGCCAGCGCAATCTCGGCTGCCGTGTGGACAGAAACAATCTGCTGGTGGAACATGGCGG C2’ 701>GCTTCCATATTCAGGACTATTGCCTGAACTTTGTGGACTGCTGCCAGCGCAATCTCGGCTGCCGTGTGGACAGAAACAATCTGCTGGTGGAACATGGCGG

Tps1 Ref 801>GCGCACCGTTCGCGTCCGCCCGCTTCCCATTGGTATTCCCTACGAACGCTTCGTTAATTTGGCCACGACCGCGCCCAAGGTGCTAAAGACATCCAAGATG C1’ 801>ACGCACCGTTCGCGTCCGTCCGCTTCCCATTGGTATTCCCTACGAACGCTTCGTTAATTTGGCCACGACCGCGCCCAAGGTGCTAAAGACATCCAAGATG C2’ 801>ACGCACCGTTCGCGTCCGTCCGCTTCCCATTGGTATTCCCTACGAACGCTTCGTTAATTTGGCCACGACCGCGCCCAAGGTGCTAAAGACATCCAAGATG

Tps1 Ref 901>CAAATCATCCTGGGAGTGGACCGACTGGACTACACCAAGGGCCTCGTCCATCGCCTTATGGCTTTTGAGGCGCTGCTGCTGAAGTATCCGCAGCACAAGG C1’ 901>CAAATCATCCTGGGAGTGGACCGACTGGACTACACCAAGGGCCTCGTCCATCGCCTTATGGCTTTTGAGGCGCTGCTGCTGAAGTATCCGCAGCACAAGG C2’ 901>CAAATCATCCTGGGAGTGGACCGACTGGACTACACCAAGGGCCTCGTCCATCGCCTTATGGCTTTTGAGGCGCTGCTGCTGAAGTATCCGCAGCACAAGG

Tps1 Ref 1001>AGAAGGTGAGCCTGCTGCAGATCTCGGTGCCGTCGCGAACCGATGTAAAAGAGTACCGGGAGCTGAAGGAGGAGGTGGACCAGCTGGTGGGCCGCATCAA C1’ 1001>AGAAGGTGAGCCTGCTGCAGATCTCGGTGCCGTCGCGAACCGATGTAAAAGAGTACCGGGAGCTGAAGGAGGAGGTGGACCAGCTGGTGGGCCGCATCAA C2’ 1001>AGAAGGTGAGCCTGCTGCAGATCTCGGTGCCGTCGCGAACCGATGTAAAAGAGTACCGGGAGCTGAAGGAGGAGGTGGACCAGCTGGTGGGCCGCATCAA

Tps1 Ref 1101>CGGACGCTTTACCACAGCCAACTGGGCGCCCATACGCTACATCTACGACTATGTCAGCCAAGACGAGTTGGCTGCTTTGTACAGGGATGCGGCTGTTTGC C1’ 1101>CGGACGCTTTACCACAGCCAACTGGGCGCCCATACGCTACATCTACGACTATGTCAGCCAAGACGAGTTGGCTGCTTTGTACAGGGATGCGGCTGTTTGC C2’ 1101>CGGACGCTTTACCACAGCCAACTGGGCGCCCATACGCTACATCTACGACTATGTCAGCCAAGACGAGTTGGCTGCTTTGTACAGGGATGCGGCTGTTTGC

Tps1 Ref 1201>CTGGTAACTCCACTTCGCGATGGCATGAACCTGGTGGCCAAGGAGTTCGTGGCCTGCCAGATCAACGAGGTGCCCGGCGTGCTGGTCATCTCACCGTTCG C1’ 1201>CTGGTAACTCCACTTCGCGATGGCATGAACCTGGTGGCCAAGGAGTTCGTGGCCTGCCAGATCAACGAGGTGCCCGGCGTGCTGGTCATCTCACCGTTCG C2’ 1201>CTGGTAACTCCACTTCGCGATGGCATGAACCTGGTGGCCAAGGAGTTCGTGGCCTGCCAGATCAACGAGGTGCCCGGCGTGCTGGTCATCTCACCGTTCG

Tps1 Ref 1301>CAGGAGCCGGCGAGATGATGCACGAGGCGCTGCTCTGCAATCCGTACGAGGTGAACGAGGCCGCCGAGGTGATCCACCGAGCACTGACCATGCCCGAGGA C1’ 1301>CAGGAGCCGGCGAGATGATGCACGAGGCGCTGCTCTGCAATCCGTACGAGGTGAACGAGGCCGCCGAGGTGATCCACCGAGCACTGACCATGCCCGAGGA C2’ 1301>CAGGAGCCGGCGAGATGATGCACGAGGCGCTGCTCTGCAATCCGTACGAGGTGAACGAGGCCGCCGAGGTGATCCACCGAGCACTGACCATGCCCGAGGA

Tps1 Ref 1401>TGAGCGCGTCCTACGTATGGCTCGGCTGCGCCGTCGTGAAGCCGAGTGCGATGTGAGCCACTGGATGCGTTGTTTCCTAAAGGCGGTGGGCGCTCTGGAG C1’ 1401>TGAGCGCGTCCTACGTATGGCTCGGCTGCGCCGTCGTGAAGCCGAGTGCGATGTGAGCCACTGGATGCGTTGTTTCCTAAAGGCGGTGGGCGCTCTGGAG C2’ 1401>TGAGCGCGTCCTACGTATGGCTCGGCTGCGCCGTCGTGAAGCCGAGTGCGATGTGAGCCACTGGATGCGTTGTTTCCTAAAGGCGGTGGGCGCTCTGGAG

Tps1 Ref 1501>ATGGATGATGTGGGCACCACCATTATGCAGCCAGTGTCCGTGGACGATTTCGATGACTACTTACTGAAATACATCGGCTATAACCACAAGTTGGCTCTGC C1’ 1501>ATGGATGATGTGGGCACCACCATTATGCAGCCAGTGTCCGTGGACGATTTCGATGACTACTTACTGAAATACATCGGCTATAACCACAAGTTGGCTCTGC C2’ 1501>ATGGATGATGTGGGCACCNCCATTATGCAGCCAGTGTCCGTGGACGATTTCGATGACTACTTACTGAAATACATCGGCTATAACCACAAGTTGGCTCTGC

Appendix

Tps1 Ref 1601>TGCTGGACTACGACGGAACCTTGGCGCCCATTGCTCCTCATCCTGATCTGGCCACGCTCTCGCCCGAAATTAAGAATGTACTATATAAGCTGTCCAATCA C1’ 1601>TGCTGGACTACGACGGAACCTTGGCGCCCATTGCTCCTCATCCTGATCTGGCCACGCTCTCGCCCGAAATTAAGAATGTACTATATAAGCTGTCCAATCA C2’ 1601>TGCTGGACTACGACGGAACCTTGGCGCCCATTGCTCCTCATCCTGATCTGGCCACGCTCTCGCCCGAAATTAAGAATGTACTATATAAGCTGTCCAATCA

Tps1 Ref 1701>CTCGGACGTCTACGTGGCCGTCATCTCGGGTCGCAACGTGGACAATGTCAAGAAGATGGTTGGCATCGAGGGCATAACCTATGCGGGCAATCACGGTCTA C1’ 1701>CTCGGACGTCTACGTGGCCGTCATCTCGGGTCGCAACGTGGACAATGTCAAGAAGATGGTTGGCATCGAGGGCATAACCTATGCGGGCAATCACGGTCTA C2’ 1701>CTCGGACGTCTACGTGGCCGTCATCTCGGGTCGCAACGTGGACAATGTCAAGAAGATGGTTGGCATCGAGGGCATAACCTATGCGGGCAATCACGGTCTA

Tps1 Ref 1801>GAGATCCTTCATCCGGACGGCAGCAAGTTCGTACACCCCATGCCCATGGAGTACGAAAAGAAGGTCAGTGACCTGCTGAAGGCTCTGCAGGATTCCGTTT C1’ 1801>GAGATCCTTCATCCGGACGGCAGCAAGTTCGTACACCCCATGCCCATGGAGTACGAAAAGAAGGTCAGTGACCTGCTGAAGGCTCTGCAGGATTCCGTTT C2’ 1801>GAGATCCTTCATCCGGACGGCAGCAAGTTCGTACACCCCATGCCCATGGAGTACGAAAAGAAGGTCAGTGACCTGCTGAAGGCTCTGCAGGATTCCGTTT

Tps1 Ref 1901>GCCGCGACGGCGCTTGGGTGGAGAATAAGGGGGCGTTGCTAACGTTCCACTACCGAGAGACGCCCAATCATCTGAGGGGAGCTATGGTTGACAAGGCGCG C1’ 1901>GCCGCGACGGCGCTTGGGTGGAGAATAAGGGGGCGTTGCTAACGTTCCACTACCGAGAGACGCCCAATCATCTGAGGGGAGCTATGGTTGACAAGGCGCG C2’ 1901>GCCGCGACGGCGCTTGGGTGGAGAATAAGGGGGCGTTGCTAACGTTCCACTACCGAGAGACGCCCAATCATCTGAGGGGAGCTATGGTTGACAAGGCGCG

Tps1 Ref 2001>CTCCTTGATCGAGAAGTACGGCTTCAAGGCCACGGAGGCGCATTGCGCTCTGGAAGCTCGTCCGCCGGTGCAGTGGAATAAGGGTCGTGCCTCGATCTAC C1’ 2001>CTCCTTGATCGAGAAGTACGGCTTCAAGGCCACGGAGGCGCATTGCGCTCTGGAAGCTCGTCCGCCGGTGCAGTGGAATAAGGGTCGTGCCTCGATCTAC C2’ 2001>CTCCTTGATCGAGAAGTACGGCTTCAAGGCCACGGAGGCGCATTGCGCTCTGGAAGCTCGTCCGCCGGTGCAGTGGAATAAGGGTCGTGCCTCGATCTAC

Tps1 Ref 2101>ATCCTGCGCACATCCTTCGGCGTGGACTGGAACGAACGCATCAAGATTATCTATGTGGGTGACGATCTAACAGACGAGGACGCCATGGTGGCTCTGAAGG C1’ 2101>ATCCTGCGCACATCCTTCGGCGTGGACTGGAACGAACGCATCAAGATTATCTATGTGGGTGACGATCTAACAGACGAGGACGCCATGGTGGCTCTGAAGG C2’ 2101>ATCCTGCGCACATCCTTCGGCGTGGACTGGAACGAACGCATCAAGATTATCTATGTGGGTGACGATCTAACAGACGAGGACGCCATGGTGGCTCTGAAGG

Tps1 Ref 2201>GTATGGCGCGAACTTTCCGTGTGACATCGTCGGATATTGTGAAGACCGCTGCGGATCATCGACTGCCCTCCACAGACTCCGTGTACACCCTGCTGAAATG C1’ 2201>GTATGGCGCGAACTTTCCGTGTGACATCGTCGGATATTGTGAAGACCGCTGCGGATCATCGACTGCCCTCCACAGACTCCGTGTACACCCTGCTGAAATG C2’ 2201>GTATGGCGCGAACTTTCCGTGTGACATCGTCGGATATTGTGAAGACCGCTGCGGATCATCGACTGCCCTCCACAGACTCCGTGTACACCCTGCTGAAATG

Tps1 Ref 2301>GGTGGAACGACACTTCATGGGACGCAAGGCGCGCGCCAATTCGCTGACCTACAGGCCCACCAAGGGCGACGGTGTCCAGATGCAGATGTCCCTGGAGGTG C1’ 2301>GGTGGAACGA~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ C2’ 2301>GGTGGAACGACACTTCATGGGACGCAAGGCGCGCGCCAATTCGCTGACCTACAGGCCCACCAAGGGCGACGGTGTCCAGATGCAGATGTCCCTGGAGGTG

Tps1 Ref 2401>GCCGCTTCAGCGAACAATCTGGAGGTGTGA>2430 C1’ 2310>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~>2310 C2’ 2401>GCCGCTTCAGCGAACAATCTGGAGGTGTGA>2430

Appendix

Figure II: Alignment of NAG protein sequence through different species Human, mouse, Zebrafish, Xenopus, Drosophila melanogaster and Clostridium perfringens. The numbers above each row give the position in the human NAG, the numbers below each row give the position in the NAG of C. perfringens. Different colors indicate different levels of conservation through the species (green, blue or red: amino acid conserved through three, four or five or more species, respectively).

Human MEAVAVAAAVGVLLLAGAGGAAGDEAREAAAVRALVARLLG Mouse MEAAGLAVILGFLLLAG GSVGDEAREAKAVRELVVRLLG Zebrafish MIHCIRCALIALLFMTLLHSVCGD FQTLAHLRAKASDKS QSRAVLDLLRRLLG Xenopus MKAALLVLLLLSCSETVVPTRVFPTLSHLKPQKGDDV QTAAVRELLARLLG Drosophila ...ALLAVLCLQLAQGSELNWDAASGPEMGLQMAAHLAPSTPKDVQETAAM AVISRVIG Bacterial ...VFGKNTGESLPEVKKIATSNFSETPWATEYEKFNSDSAYANEKTLNEIKNLVGRVIG 159

42 Human PGPAADFSVSVERALAAKPGLDTYSLGGGGAARVRVRGSTGVAAAAGLHRYLRDFCGCHV Mouse PGPAANFLVSVERALADESGLDTYSLSGGGGVPVLVRGSTGVAAAAGLHRYLRDFCGCQV Zebrafish NRAREFIVSVNRTLS ADGLDVCELRSAKNNKVVAVGSTGVAVATGIYNYLKYFCNCHV Xenopus GRASDFSVTVNSSLA EGGRDTYRLSSGAGGTVLVVGSSGVAAASGCYTYLKAFCGAHL Drosophila ERSSQLFKVQVNKNM DLRSFQISMLDDGRILLMGWDGVSVCKALHHYLKYVLNKDV Bacterial REFKDKFIFEIRDQLNGN DVFEVSDSGDGKVLIKGNNGVSLASGFNYYLKNYCNVSY 209

102 Human A WSGSQLRLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDWARWEREIDWMALN Mouse A WSSAQLHLPWPLPAVPDGLTETTPNRYRYYQNVCTHSYSFVWWDWARWEQEIDWMALN Zebrafish S WAGDQLNLPRPLPALTGVLRISTPHRFRYYQNVCTASYSSVWWDWPRWQREIDWMALN Xenopus S WSGAQLQLPPALPPVPAPLTRSAPHRFRYYQNVCTSSYSFVWWDWARWEKEIDWMALS Drosophila D WFKMRIELPTNLQLPNVTIESKSASPIIYHQNVCTWSYSFAWWGIEQWRRHLDWMALM Bacterial NPIMGSNLKMPETMPSVGERVVIDTPYEHRYALNFCTYSYTMSFWDWDQYEEFLDWCAMN 266

161 Human GINLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLAWGRMGNLHTWDGPLPPSWHI Mouse GINLALAWNGQEAIWQRVYLALGLTQSEIDTYFTGPAFLAWGRMGNLHTWDGPLPRSWHL Zebrafish GINLPLAFTGQEVLWQEVYLSLGLNQTELDRFFSGPAFLAWNRMGNLFQWGGPLPQSWHV Xenopus GINMPLAFTGQEAIWYKVYLSLGLNESEIFDFFTGPAFLAWGRMGNIHTWGGPLSISWME Drosophila GISLTIA PVQEAIWVKVYTDMGLRMEEIDEHLAGPAFQAWQRMGNIRGWAGPLTPAWRR Bacterial GVNLVLDIIGQEEVLRRTLNEFGYSDEEVKEFISGPAYFAWFYMQNMTGFGGPLPNDWFE 326

221 Human KQLYLQHRVLDQMRSFGMTPVLPAFAGHVPEAVTRVFPQVNVTKMGSWGHFNCSYSC SF Mouse SQVYLQHRILDRMRSFGMIPVLPAFAGHVPKAITRVFPQVNVIKLGSWGHFNCSYSC SF Zebrafish KQLYLQFKILDRMRSFGMIPVLPAFSGIVPEGITRLFPKANVTKLSPWSHFNCTYSC AY Xenopus KRLSLQLQITERMRSLGMITVLPAFAGHIPEGILRVFPKVTVSRLGGWSNFNCTYSC SY Drosophila YQLLLQQEIITAQRNLGMSVALPAFAGHVPRALKRLNPESTFMEVQRWNQFPDRYCC GL Bacterial QRAELGRKMHDRMQSFGINPVLQGYSGMVPRDFKEKNQEAQTISQGGWCGFDRPDMLKTY 386

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280 Human LLAPEDPIFPIIGSLFLRELIKEFGT DHIYGADTFNEMQPPSSEPSYLAAATTAVYEAM Mouse LLAPGDPMFPLIGNLFLRELTKEFGT DHIYGADTFNEMQPPFSDPSYLAATTAAVYEAM Zebrafish VLDPRDPLFHRIGALFLTQVIEEFGT DHIYNTDTFNEMPPASSDPTYLASISRAIFNTM Xenopus LLDPEDPLFQWIGELFLSQMVQSFGT DHIYSADTFNEMSPTSSDPGYLSAVSGAIFKSM Drosophila FVEPTENLFKEIASRFLHNIITKYGS NHIFFCDPFNELEPPVAKPEYMRSTAAAIYESM Bacterial VNEGEADYFQKVADVFYEKQKEVFGDVTNFYGVDPFHEGGNTGDLDN GKIYEIIQNKM 446

339 Human TAVDTEAVWLLQGWLFQHQPQFWGPAQIRAVLGAVPRGRLLVLDLFAESQPVYTRTASFQ Mouse VTVDPDAVWLLQGWLFQHQPQFWGPSQIRAVLEAVPRGRLLVLDLFAESHPVYMHTASFH Zebrafish TSVDPQAIWLMQGWLFISDPSFWKADQVKALLHGVPLGRMIVLDLFAESMPVYSSTNSFY Xenopus AKVDPDAIWLMQGWLFINNPSFWRPAQTKALLHGAPIGRIIVLDLFAETVPVYLTTESFY Drosophila RGIDPQAIWLLQGWMFVKNP FWTTDMAEAFLTAAPRGRILVLDLQSEQFPQYELTRSYF Bacterial IEHDNDAVWVIQNW QGNPS NNKLEGLTKKDQAMVLDLFSEVSPDWNRLEE R 504

399 Human GQPFIWCMLHNFGGNHGLFGALEAVNGGPEAARLFPNSTMVGTGMAPEGISQNEVVYSLM Mouse GQPFIWCMLHNFGGNHGLFGALEDVNRGPQAARLFPNSTMVGTGIAPEGIGQNEVVYALM Zebrafish GQPFIWCMLHNFGGNSGLFGTVDSINSGPFNAVRFPNSTLVGLGMTPEGIEQNPVIYELM Xenopus GQPFIWCMLNNFGGNHGLFGNIEGVNRGPFDAAKFPNSTMVGTGLTPEGIEQNDMIYEFM Drosophila GQPFIWCMLHNFGGTLGMFGSAKLINSGIEEARRLPNSSLVGTGITPEGIGQNYVMYSFT Bacterial DLPWIWNMLHNFGGRMGMDAAPEKLATEIPKALA NSEHMVGIGITPEAINTNPLAYELL 555

459 Human AELGWRKDPVPDLAAWVTSFAARRYGVSHPDAGAAWRLLLRSVYNCSGEACRGHNRSPLV Mouse AELGWRKDPVPDLMAWVSSFAIRRYGVSQPDAVAAWKLLLRSVYNCSGEACSGHNRSPLV Zebrafish SELAWRKDPV NLYKWVSLYALRRYGSMDENLALAWQLLFRSVYNCTLPKYKNHNRSPLV Xenopus NEIGWSSQPI NLTKWISNYSDRRYGQSNTDARMAWQILLRSVYNCTQILH NHNHSPLV Drosophila LERGWSNTSL DLDSWFTNFSHSRYGVKDERLEQAWLLLKNSVYSFRG LQKMRGQYVVT Bacterial FDMAWTRDQI NFRTWTEDYIERRYGKTNKEILEAWNIILDTAYKKRNDYYQGAAESIIN 614

519 Human RRPSLQMNT SIWYNRSDVFEA WRLLLTSAPSL ATSPAFRYDLLDLTRQAVQEL Mouse KRPSLQMST AVWYNRSDVFEA WRLLLTAAPNL TTSPAFRYDLLDVTRQAVQEL Zebrafish HRPSLHMQT DIWYDPADFYRA WKLLFEAAPGL VTLETFRYDLVDVTRQALQLL Xenopus RRPSLNMNT DICYNKADIYEA WRFMHNASFAL GKSATFLYDLVDITREAVQQL Drosophila RRPSFNQEP FTWYNASAVLDA WHLLLTFRAIIPLEDNRYEIYEHDLVDITRQFLQIS Bacterial ARPGFGIKSASTWGHSKIVYDKSEFEKAIEIFAKNYDEFKDSDAFLYDFADILKQLLANS 673

572 Human VSLYYEEARSAYLSKELASLLRAGGVLAYELLPALDEVLASDSRFLLGSWLEQARAAAVS Mouse VSLCYEEARTAYLKQELDLLLRAGGLLVYKLLPTLDELLASSSHFLLGTWLDQARKAAVS Zebrafish TTEFYKDIKSAFQTQKLSDLLTAGGVLVYDLLPELDRILSSNEHFLLGAWLQQAQSQGVD Xenopus VSEYYLEIKEAYGKKSLQQLMTAGGVLVYDLLPELDSLLSSQPGFLLGSWLKAAKSMAST Drosophila ADQLYINLRSAYRKRQVSRFEFLS VKLLKLFDDMELILASSRNFLLGNWLQQAKQAAPN Bacterial AQEYYEVMCNAYNNGNGEKFKFVSG KFLELIKLQERVLSTRPEFLIGNWIEDARTMLKD 733

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632 Human EAE ADFYEQNSRYQLTLWGPE GNILDYANKQLAGLVANYYTPRWRLFLEALVD Mouse EAE AQFYEQNSRYQITLWGPE GNILDYANKQLAGLVADYYQPRWCLFLGTLAH Zebrafish EHE AHLYDINARNQITLWGPD GEILDYASKEWAGLVEDYYLQRWGLFVNTLVE Xenopus PAE AALYDMNARNQITLWGPT GNILDYANKQYGGLVQDYYTERWGLFVWFLVQ Drosophila TGQ QRNFEFNARNQITAWGPD GQILDYACKQWSGLVSDYYRPRWRLFLEDVTV Bacterial SDDWTKDLFEFNARALVTTWGSRNNADGGGLKDYSNRQWSGLTEDYYYARWEKWINGLQA 792

685 Human SVAQGIPFQQHQFDKNV FQLEQAFVLSKQRYPSQPRGDTVDLAKKIFLKYYPRWVAGSW Mouse SLARGVPFQQHEFEKNV FPLEQAFVYNKKRYPSQPRGDTVDLSKKIFLKYHPQPDSL Zebrafish CLDRGRPFKQDVFNQAV FQVEKGFVFNQRKYPTKPLGDTYDIARRIFLKYYPYALKKTE Xenopus SLNKGEHFNQDKFNKAV FVLEEDFVYNGKEYMASPTGDTLEIANKIYLKYQPYARKRFL Drosophila ALHAGRPFNGTAFKLKVSHEIELPFSNKDDVYPVTPVGNTWLISQDIFETWKGYSKDTLF Bacterial ELDGGAKAPNIDW FKMEYDWVNKKSDTDKLYPTEASNENLGELAKIAMESYSVTNMDKI 752

Human Mouse Zebrafish TKKTS Xenopus RRNKGLELNL Drosophila LHNNRLSVPQKAGPK Bacterial L

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