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Biochimica et Biophysica Acta 1762 (2006) 934–944 www.elsevier.com/locate/bbadis

Review From genes to systems: New global strategies for the characterization of NCL biology ⁎ Anu Jalanko a, , Jaana Tyynelä b, Leena Peltonen a,c,d,e

a National Public Health Institute, Department of Molecular Medicine, Biomedicum Helsinki, P.O. Box 104, 00251 Helsinki, Finland b University of Helsinki, Institute of Biomedicine/Biochemistry, Finland c University of Helsinki, Department of Medical Genetics, Finland d Research Programme of Molecular Medicine and Research Programme of Neurosciences, Biomedicum Helsinki, Finland e The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA Received 22 April 2006; received in revised form 1 September 2006; accepted 5 September 2006 Available online 12 September 2006

Abstract

Neuronal ceroid lipofuscinoses (NCL) are rare neurological disorders with a uniform phenotype, caused by mutations in seven known genes. NCL provide a unique model to characterize molecular pathways critical for normal neuronal development and pathological neuronal degeneration. Systems biology based approach utilizes the rapidly developing tools of genomics, proteomics, lipidomics and metabolomics and aims at thorough understanding of the functions of cells, tissues and whole organisms by molecular analysis and biocomputing-assisted modeling. The systems level understanding of NCL is now possible by utilizing different model organisms. Initial work has revealed disturbed metabolic pathways in several NCL disorders and most analyses have utilized the infantile (INCL/CLN1) and juvenile (JNCL/CLN3) disease modeling and utilized mainly human and mouse samples. To date, the data obtained from transcript and lipidomic profiling has pinpointed the role of and synaptic function in the infantile NCL. Changes in glutamate utilization and amino acid metabolism have been a common theme emerging from the transcript and metabolite profiling of the juvenile NCL. Further experimental models are being developed and systematic sample collection as well as data integration projects are needed. The combined analyses of the global information should provide means to expose all the NCL-associated molecular pathways. © 2006 Elsevier B.V. All rights reserved.

Keywords: Neuronal ceroid lopifuscinosis; Global strategies for NCL; NCL systems biology

1. Introduction of lysosomal enzymes cathepsin D [5–8], palmitoyl protein thioesterase 1 (PPT1) [9–11] and tripeptidyl peptidase 1 (TPP1) The Neuronal ceroid-lipofuscinoses (NCLs) are collectively [12,13]. Three NCL types, CLN3, CLN6 and CLN8, are caused the most common inherited progressive encephalopathy of by mutations in genes encoding novel transmembrane proteins childhood, with a frequency of 7–8 per 100,000 live births of which CLN3 is lysosomal and both CLN6 and CLN8 [1,2]. All forms of NCLs are characterized by progressive residents of the endoplasmic reticulum (ER) [14–22]. CLN5 is neuronal death in the central nervous system (CNS), leading to encoding a polypeptide that is found as a soluble but possibly manifestation of epilepsy and progressive mental and physical also a membrane-bound lysosomal protein [23–25]. decline [3,4]. These rare diseases, caused by mutations in seven All known NCL-proteins are expressed ubiquitously, but known genes, provide a unique model to characterize molecular their defective functions produce dramatic consequences solely pathways critical for normal neuronal development and in neurons. Different explanations are proposed for this neuron- pathological neuronal degeneration. Three early clinical types, specificity: neurons may simply be more sensitive to storage congenital NCL, CLN1 and CLN2, are caused by deficiencies materials. Alternatively, NCL proteins may be linked to the development of the nervous system or involved in so far ⁎ Corresponding author. Tel.: +358 9 4744 8392; fax: +358 9 4744 8480. uncharacterized neuron-specific metabolic pathways. Recent E-mail address: [email protected] (A. Jalanko). investigations have provided new data on the functional aspects

0925-4439/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2006.09.001 A. Jalanko et al. / Biochimica et Biophysica Acta 1762 (2006) 934–944 935 of the NCL proteins. Especially the research of the most 2. Utilization of global gene expression profiling to common form, JNCL/CLN3 has provided novel links between approach the disturbed metabolic pathways in NCL the CLN3 protein and cytoskeleton connected to defects in membrane and ion transport [26–30]. However, it remains The mouse is particularly suitable for the analysis of gene obvious that more system-wide, non-hypothesis-based strate- expression since tissues from all stages of development and gies are needed to expose all the NCL-associated pathways. many different strains and mutants are available. To date, nine Experimental species, including both naturally occurring and mouse models for neuronal ceroid lipofuscinoses (NCL) have specifically targeted mouse models for NCL have provided provided us with highly accurate tissue phenotypes but so far excellent tools to address the molecular pathways behind have not significantly contributed to the pathways involved or various forms of NCL. So far the targeted knock-out and knock- to the more detailed understanding of molecular pathogenesis in in mouse models exist for Cln1 and Cln3 and knock-out models CNS. Only a handful of reports have taken genome-wide for Cln2, Cln5 and cathepsin D [31–37]. Naturally occurring strategies to dissect the disease process in details, most studies mouse mutants interrupting the homologous CLN gene exist for have focused on the establishing the relevance of mouse models Cln6 (nclf) and Cln8 (mnd) [38,39]. Collectively these mouse for human diseases. However, first reports of transcript profiles models represent an invaluable resource for investigating the collected from various brain regions have helped to provide molecular pathogenesis in these devastating neurodegenerative some clues of affected metabolic pathways in three of these disorders and novel information has already emerged from mouse models, providing potential targets for more focused mouse studies of NCL. This is well exemplified by the work analyses [32,36,62–65]. that has established the role of selective involvement of GABAergic interneurons combined with disturbances in the 2.1. Transcript profiling in Cln1 deficient mouse models thalamocortical circuits in the neuronal pathogenesis of NCL [40–47]. Critical for future treatment of these devastating The Cln1-knockout mice homozygous for a truncation of the diseases are the gene therapy trials carried out in two mouse Cln1 gene product, were first analysed for global gene models which have also enabled the first human trials [48–51]. expression changes from whole brains at the age of 10 weeks The potential of the NCL mouse models for genome-wide [62] (Table 1). The analysis resulted in the identification of 20 analyses to define all involved pathways still remains to be genes with altered expression >1.5 fold and provided utilized. Except rodent models, the yeast model has exposed immunoblotting data that supported the observed expression critical pathways in NCL, even helped to identify the primary changes of selected genes. The genes with transcript levels gene defects [26]. deviating from controls are known to be involved in various Systems biology-based approach uses the rapidly develop- cellular pathways, including , trafficking, glial ing tools of genomics, proteomics, lipidomics and metabo- activation and calcium homeostasis, all of which have been lomics and aims at thorough understanding of the functions of connected to the tissue pathogenesis of human INCL [2,66–68]. cells, tissues and whole organisms by “holistic” molecular In another study, targeted to the cortical region and reporting analysis and bio computing-assisted modeling. The systems transcript profiles in 6 month old Ppt1Δdex4 mice [32], level understanding of a biological organism will be enabled significant changes were observed for total of 234 genes by combining data collected at different levels of the among which 195 were upregulated (Table 1). The up-regulated biological system, using molecular, cellular, and physiological genes partitioned in a wide variety of developmental, cellular, methods. The basic technologies for global analyses of mRNA physiological and metabolic processes. This study employed a and protein levels as well as genome-wide strategies to statistical algorithm and rigorous two-step data-filtering process systematically block the transcription of selected sets of genes and could pinpoint immune response processes as predominant are becoming available for investigators [52–56]. Additional- disturbed pathways in the knockout mice. The signs of cortical ly, mass spectrometry and NMR spectroscopy have proven inflammation were also analyzed by immunohistochemistry and feasible to screen the changes in and solute metabolites the data implied that inflammation appeared prior to neurode- in a system-wide manner and form the backbone methods for generation in Ppt1Δdex4 mice. Comparison of the two transcript lipidomics and metabolomics [57,58]. Finally advanced profiling analyses does not pinpoint changes in common genes imaging technologies facilitate detailed analyses of specific but both highlight the changes in inflammation related brain regions both in humans and in experimental animals, molecular pathways. like exemplified by the recently produced first versions of the 3D map of human and C57/BL mouse brain, produced by 2.2. Transcript profiling in Cln3 deficient models magnetic imaging [59–61]. All systems biology approaches create huge amount of data, which needs to be carefully For CLN3 deficiency, the mouse was not the first organism quality controlled and analyzed with advanced tools of to be utilized in global gene expression profiling. The early biocomputing in order to decipher the metabolic events analysis utilizing the S. cerevisiae knockout model for the Cln3 within the organism and to evaluate the significance of homolog Btn1p [26], deserves special attention. These first findings in a given system. This review aims to summarize analyses actually brought up implications that the CLN3 protein the genome wide approaches used in the analyses of NCL and is involved in cytoskeletal dynamics and this has been corresponding mouse models. replicated more recently in mammalian cell models [28,69]. 936 A. Jalanko et al. / Biochimica et Biophysica Acta 1762 (2006) 934–944

Table 1 Summary of Gene expression profiling in mouse models of NCL Mouse Age Brain region Samples Chip Data analysis Differentially expressed Affected Ref. genes pathways Cln1 10 weeks whole 3×3 Affymetrix U74 MAS 5 20 (>1.5) ND [62] Ppt1Δdex4 6 months ctx 2×5 Affymetrix U74Av2 MAS 5, RMA Bioconductor 234 (ss) 14 [32] GeneSpring 6.0+additional Cln3 10 weeks whole 3×3 Affymetrix U74 MAS 5 14 (>1.5) ND [62] Cln3 10 weeks cb 2×3 Affymetrix MAS 5 756 (>2.0) 14 [63] Mu19K Cln3 10 weeks retina 2×3 Affymetrix MAS 5 285 (>2.0) 12 [64] Mu19K Cln3 E16.5 ctx neurons 2×8 Affymetrix 430 A2.0 MAS 5 GeneSpring 6.2.1. 88 (ss) 7 [65] 1 3 months ctx 2×5 Affymetrix MAS 5 016 (>2.0) 3 [36] 4.5 months ctx U74Av2 68 (>2.0)

The Cln3 knock-out mouse model has been utilized in four 2.3. Transcript profiling in Cln5 deficient mouse model different types of gene expression profiling sets (Table 1). Firstly, the whole brains of 10 week old Cln3 knockout mice Gene expression profiling for the Cln5 knockout mouse was and wild-type mice were analyzed using a high-density performed utilizing similar sampling, gene chips and data oligonucleotide array [62]. In this array set 14 genes with analysis as with the Ppt1Δdex4 mouse [32,36] except that the altered expression >1.5 fold was identified and two of these mice were analyzed at two different ages, 3 and 4.5 months. The genes were associated to neurotransmitter metabolism and two expression array data revealed changes of >2 fold for 16 genes with glutamate utilization. The downregulation of membrane- in the cerebrums of the 3 month-old knock-out mice and in the bound and soluble catechol-o-methyltransferase (COMT) was 4.5 month-old mice a total of 68 genes showed altered verified also by immunoblotting. Another report presented the expression levels. The affected molecular pathways were gene expression changes from the cerebellum of 10 week old further analyzed in a similar manner as in the case of the Cln3 knockout mice [63]. This experiment was performed Ppt1Δdex4 mice and the pathway analysis indicated upregulation with a different type of oligonucleotide array set (Table 1)as of pathway components related to inflammation and down- compared to the previous report [62]. The cerebellum array regulation of myelin pathway components, evident in both age revealed 756 genes with reproducible change in expression of 2 groups [36]. fold or more. The genes were assigned to 14 different functional classes including trafficking at the synapse, lipid metabolism, 3. Proteomic tools for the analysis of disease mechanisms in inflammatory response, energy metabolism, cytoskeleton, cell NCL death and amino acid metabolism, many of which have been connected to Cln3 deficiency [30]. Gene expression micro- 3.1. Proteomics and brain diseases arrays have also been utilized to assess the changes expressed in the whole eye, a characteristic target organ in human CLN Equally to transcript profiling, proteomic profiling should phenotype, of the Cln3 knockout mouse [64], revealing 285 provide information on the disease process in NCL. The genes with altered transcript levels. This work used similar proteins defective in NCL reside in the secretory pathway of the chips and data analysis procedures as the cerebellum study and cell and to date, CLN2, CLN3 and CLN5 have shown some the authors identified 18 genes that did not appear as changed in evidence to be interconnected [25]. System-wide analysis of the transcript profiles of cerebellum. Particularly, downregula- proteomic alterations is so far missing and should expose novel tion of genes associated with mitochondrial energy production metabolic connections for the NCL disorders. Importantly, in the mitochondria was considered specific to the eye. Changes proteomics is the only way to study post-translational in Mitochondrial energy production have been previously modifications of proteins, for example phosphorylation and detected in human NCL [66]. A very recent transcript profiling glycosylation, being of central importance at the functional study utilized Cln3 deficient mouse cortical neurons and level. In order to cover the complexity and dynamics of the implicated disturbances in mitochondrial, cytoskeletal and eukaryotic proteome, multiple complementary techniques have synaptic functions. This report provided the first functional been developed, and many of the state-of-the-art methods are data supporting the transcript profiling of the Cln3 deficient only facilitated by the recent advances in mass spectrometry mouse model [65]. Similarly to the Cln1 data, no single (MS) [70–72]. similarly affected genes can be detected in the four different Systems level proteomics, particularly, the brain proteomics microarray reports assessing the Cln3 deficiency. The exami- is at its initial state [56,73,74]. Major international efforts to nation of the affected molecular pathways in the different promote education and knowledge in proteomics and to studies suggests a link between mitochondrial dysfunction and accelerate and develop proteomics research have been initiated: cytoskeleton-mediated presynaptic inhibition behind this mouse the European Proteomic association (EuPA) was established in model of NCL. 2001 and the Human Proteome Organization (HUPO) in 2001 A. Jalanko et al. / Biochimica et Biophysica Acta 1762 (2006) 934–944 937

[75]. Within HUPO, the Human Brain Proteome Project focuses systems level analysis of lipids [57,92,93]. Importantly, many on analyzing the brain proteome in health and disease [76,77]. critical brain functions are based on coordinated molecular Brain proteomics have so far provided invaluable information events at the synapse and accumulating evidence both from in on the protein profiles of normal brain tissue as well as for some vitro cellular and in vivo mouse studies have linked the disease-specific alterations in neurological disorders [78–83]. molecular defects in NCL disorders closely to the disturbed The emerging specific applications of proteomics, including synaptic functions [94–97]. These events occur in regulated techniques to study the brain lysosomal proteome, synaptic lipid microenvironments with highly specific lipid composition proteome, or synaptic phosphoproteome, have great potential in and lipid–protein interactions [98,99]. providing an insight into physiological and pathological events occurring in NCL, many related to lysosomal mechanisms 4.2. Lipidomics and NCL diseases [74,84–86]. The lipid profiles of the NCL brain have previously been 3.2. Proteomic studies on NCL diseases studied in autopsy tissues from humans and animals with NCL, and conventional analytical methods have indicated alterations In contrast to many other NCL forms, for which the in many lipid classes, including polyunsaturated fatty acids, causative gene defects were identified by genome-wide cholesterol, , triacylglycerols, lysophosphatidic mapping and positional cloning, the molecular cause of late acid and dolichols [100–103]. More recently, the early data on infantile NCL (CLN2) was initially revealed by proteomics, by dolichol accumulation and lack of polyunsaturated fatty acids in 2D- gel electrophoresis: (TPPI) spot was NCL tissues have been supported by quantitative studies using absent from the lysosomal proteome of CLN2 patient tissues state-of-the-art methods: FACE technique (fluorophore assisted [12]. Later, 2D-gel electrophoresis of the human brain mannose- electrophoresis) revealed a striking accumulation 6-phosphoproteome revealed novel mannose-6-phosphate con- of lipid-linked oligosaccharides in six mouse models of NCL, taining proteins which could be considered as potential and it was suggested that the lipid-linked oligosaccharides candidates for additional NCL forms, and this study confirmed arouse from dolichol-linked sugars during the lysosomal the soluble lysosomal nature of CLN5 protein [85]. Moreover, degradation of autophagosomes [104]. Finally, quantitative 2D-gel electrophoresis has been used to track down the MS analysis of brain lipids has revealed complex and dramatic metabolic defects in CLN6 patient tissues, where MnSOD alterations in the molecular composition of phospholipids, was found to be increased [87]. neutral sphingolipids and , particularly in the poly- Proteomic approach on the systems level can now increas- unsaturated fatty acid species of these lipids, in patients with ingly be applied in research of NCL diseases thanks to the CLN1 and CLN8, but not in patients with CLN2 or CLN3 availability of good and well-characterized mouse models (see [68,92,105]. These studies indicating clear alterations in the above). The mouse models facilitate standardized collection of lipid composition of NCL brains were performed on human samples, analysis, and comparison of results under such strictly autopsy material, for which the post mortem delay and storage controlled circumstances, which would never be possible with conditions vary. Thus, based on the first promising results and the rarely available patient material. Moreover, longitudinal the possibility to utilize the animal models, the emerging field of studies at different stages of the disease process are enabled by lipidomics is likely to provide an invaluable view into the the mouse models of NCL. Within the EU funded research pathogenetic mechanisms of NCL diseases. project “NCL-models” (www.nclmodels.org), we aim to reveal disease-specific alterations in the brain proteome by comparing 5. Metabolomics: Global analysis of small molecules the brain protein profile of NCL mice and controls at different stages of the disease. Moreover, we will search for common Metabolomics refers to characterization of biological themes in the proteomic changes shared by the different NCL variation in a small molecular weight metabolite level mouse models. By these studies we should be able to obtain [58,106,107] and metabolic disorders often result in the information on the affected metabolic events, the temporal secretion of abnormal metabolites into the serum or urine of sequence of the observed alterations, and, most importantly, on patients. It has been long known that the storage material in all the similarities of neuronal defects between different forms of NCLs is highly insoluble and the early studies of NCL patients NCLs. could only describe the accumulation of abnormal dolichols in the urine (see previous section). More recent analyses have 4. The emerging field of lipidomics utilized autopsy brain samples of CLN1 and CLN3 deficient patients and discovered significantly altered neuronal metabo- 4.1. Lipidomics lite profiles in CLN1 [108]. Utilization of the Cln3 mouse model brought up the observation of GAD65 autoantibodies in The crucial role of lipids within cells is demonstrated by the serum of Cln3 deficient mice and this was also observed in multiple human diseases that involve disruption of lipid JNCL patients [109,110]. No system level screening of serum or metabolic pathways; such as cancer and neurodegenerative urinary metabolites has yet been performed in NCL patients, but diseases [57,88–91]. The recent technical advances in liquid interestingly, the screening of amino acid levels in JNCL patient chromatography and mass spectrometry have facilitated the sera apparently led to the discovery of abnormal arginine 938 A. Jalanko et al. / Biochimica et Biophysica Acta 1762 (2006) 934–944 transport [27,29,111]. Metabolic profiles derived from 1HNMR likely to interact or at least the different NCL disorders are spectroscopic analysis of biofluids and tissue extracts, in expected to show similar metabolic disturbances. This technol- conjunction with multivariate statistics have been demonstrated ogy offers the possibility to silence the NCL genes cross the to be highly discriminatory for neurological diseases [112]. For disease spectrum and should offer a highly feasible tool to NCL, the first metabolomics study utilizing the multivariate assess the combined effects of the silencing of NCL genes. statistical approach was performed for the mnd mouse model, Naturally, cross-breeding of different NCL model organisms harboring a mutation in the Cln8 gene and representing a model will lead to more accurate answers at the disease level but this for the CLN8/Northern epilepsy. A number of altered route is much more tedious, expensive and time consuming and metabolite profiles were identified in the mnd mouse brain not suitable as an initial screening tool, making specific gene and blood plasma, exemplified by increased glutamate and N- silencing with RNAi as a lucrative option. acetyl-aspartate (NAA), and decreased creatine and glutamine [113]. A more recent metabolomics approach, based also on 7. Data analysis and databases high resolution solution 1H NMR spectroscopy in conjunction with statistical pattern recognition revealed profound metabolic The interpretation of genome-wide data remains a major changes in 1–6 month-old Cln3 knock out mouse of cortex, challenge because of the complexity of the underlying cerebellum and remaining regions of the brain [114]. More biological networks and problems in sufficiently accurately specifically, concentrations of glutamate were increased in all targeted sample collection of relevant tissues like brain or eye. areas and time points whereas those of GABA were decreased Transcriptional profiling of CNS samples has proven to be a relative to wild type. These changes can potentially be linked to challenging task due to the complexity of the tissue and the observed upregulation of glutamic acid decarboxylase problems in obtaining sufficiently clean cellular populations, autoantibodies (GAD65 autoantibodies) detected in the tissues the heterogeneity of RNA source, diluting the differences in of the Cln3 knockout mice [111]. Also, this type of insult may transcript levels. Characteristic for all studies described here, be explained by the documented selective loss of GABAergic the changes seen in the transcript profiles of individual genes interneurons in the Cln3 knockout mice, although this is have been marginal and often statistically not significant detected only at the age of 7 months [33]. Further variations in [reviewed in [121–123]. Further, the transcript levels of brain myo-inositol, creatine and NAA were identified, possibly specific genes are often low, requiring usage of specific reflecting an early stage glial activation in these knockout statistical methods. Finally, none of the studies so far has mice [44]. Importantly, the metabolic changes could be shown addressed sufficiently the different developmental stages of to be independent from the normal aging process. CNS, a critical parameter for the disease pathogenesis of NCL. In order to recapitulate the possibly perturbed functional 6. Utilization of RNAi to study the role of different genes in biological networks involved in disease process, a detailed NCL pathway analysis is required [124]. To obtain meaningful expression data, it is crucial to develop standardized approaches The animal models for NCL offer the possibility to screen for study design, annotation of resources, quality control, the metabolic defects in a disease state. The brain is a very statistics, data registration and storage. The success requires delicate organ and in the NCL, the primary functional defect accurate, quality controlled technology and good practices have may be masked by a general neuronal insult. The analyses of been adopted [125,126]. affected metabolic pathways in NCL would greatly benefit if the Another critical issue is the data interpretation. A wide analyses were performed both to diseased tissue and to normal diversity of statistical methods have been applied for classifi- tissue or cells that have been induced an instant shutdown of the cation and recognition of expression profiles. The data disease gene. RNAi is a gene silencing technology that is transformation steps include image acquisition and segmenta- spreading rapidly to nearly every aspect of biomedical research tion and although many current methods and software packages [55,115,116]. RNAi utilizes sequence-specific short interfering offer automatic image processing, no consensus exists for the RNA (siRNA), known to form an RNA-induced silencing best possible logistics and the problems encountered in various complex with other cellular proteins, leading to sequence- steps of data cleaning have generated a large amount of specific degradation of RNA [117]. RNAi technology has been literature [126,127]. Finally, extensive developmental work is utilized to study the role of different genes in the disease ongoing in the functional classification of the initially identified pathogenesis and for the identification of new therapeutic involved biological processes. Currently, the microarray data is targets for metabolic diseases. To date, great success has been most often linked to Gene ontology (GO) terms, constituting accomplished in the utilization of this technology in for standardized nomenclature for biological processes, molecular example analysis of insulin signaling in diabetes [118]. C. functions and cellular components [128,129]. Several statistical elegans has been widely utilized in the RNAi screens, tools have been developed that link microarray experimental exemplified by the C. elegans ORFeome resource, offering data to GO terms in an automated way (www.geneontology.org/ the genome-wide C. elegans RNAi library [119,120]. For the GO.tools.shtml). New biocomputational approaches aim at NCL diseases, different model organisms are being developed relating the gene expression patterns with functional networks and it should be of interest to utilize the RNAi technology for and a wide variety of tools have been introduced to connect the gene silencing in each NCL. Many of the NCL proteins are array data to documented biological pathways [127,130]. A. Jalanko et al. / Biochimica et Biophysica Acta 1762 (2006) 934–944 939

Novel pathways involved with neuronal processes disturbed 8. Summary and future prospects by NCL mutations can be addressed using also other types of genome-wide tools and methods of bioinformatics, reaching The unraveling of the molecular mechanisms that underlie a beyond the comparative analyses of transcript profiles. A complex process, such as neuronal development or neurodegen- comprehensive view of the putative protein interactions is eration, is a formidable challenge. NCL disorders comprise a possible using sequence information from multiple species and genetically heterogenic group of diseases that still result in a bioinformatics plays a key role in the construction of genetic (or relatively uniform phenotype in the CNS, characterized by a protein) networks [131]. Homology searches can identify progressive and selective neuronal loss. All the proteins orthologues and paralogues of genes with known functions underlying NCLs, i.e. PPT1, TPP1, CLN3, CLN5, CLN6, and shared regulatory motifs combined with coordinated CLN8 and cathepsin D, must play critical role in pathways expression levels are good indicators of genetic networks. important for the normal development and survival of neurons. Phylogenetic profiling looks for genes showing the same pattern More and less systematic strategies have been utilized in attempts of presence or absence across multiple genomes [132]. The to dissect the disturbed metabolic pathways in NCL. To date, the Rosetta Stone (domain fusion) method identifies proteins that data obtained from transcript and lipidomic profiling have are separate in one organism but fused in another [133]. Finally, provided information on the role of lipid metabolism, synaptic the gene neighbour method identifies genes that are chromo- membrane trafficking and inflammation mediated neuronal death, somally close across many species. Further validation is especially in the Cln1 deficient mouse models [32,62,104]. possible when identified genes have a known function. In this Changes in glutamate utilization has been a common theme event, one can compare the keyword annotations for the emerging from the transcript and metabolic profiling of Cln3 clustered genes in one or more of the current databases. Data deficient mice and also in human samples [62–64,108]. from expression arrays can be extended by using algorithms Importantly, “metabolomics”-based screening of amino acids in aiming at identification of common promoter and enhancer the sera of JNCL patients led to the discovery of defective arginine regions on co-expressed genes in critical tissues or cells transport [27,29,110]. The transcript profiling of Cln5 deficient revealing cross-talking genes under similar cis-regulation mice further suggested astrocytic activation and defects in [134,135]. The shared transcriptional elements in promoter myelinization [36]. Finally, the profiling of lipids and metabolites and enhancer regions can be identified by utilizing both in the CLN8 have highlighted severe aberrations in the lipid experimentally derived transcription factor binding site libraries profiles of human autopsied brains and changes in glutamate (Transfac and JASPAR) and ab initio motif finding techniques metabolism in the mnd mouse [92,113] (Fig. 1). The data so far (AnnSPEC, AlignACE, CompareACE) in the sets of genes collected with system-based tools have been encouraging but only grouped by co-expression or by GO-based biological pathways. rarely exposed totally new metabolic routes or tissue-specific In order to increase the specificity of the ambiguous consequences of NCL mutations. However, these initial findings transcription factor binding motifs, comparative genomics/ should stimulate further system-wide analyses of the currently phylogenetic footprinting techniques can also be used. It seems existing NCL models in experimental species. obvious that various strategies utilizing advanced tools of Setting technical, statistical and biocomputational challenges biocomputing and accumulating data of publicly accessible aside, any systematic comparisons of the genome-wide data databases will shed new light also on the molecular networks of obtained from the different NCL mouse models and human CNS harmed by NCL mutations. Again, a co-ordinated data samples have been hampered by biology-related and data collection and integrated data analyses across different NCL integration problems. The targeted mouse mutants have been types and animal models would greatly benefit the field. produced using different genetic backgrounds, complicating the

Fig. 1. Current status of global analyses in NCL. 940 A. Jalanko et al. / Biochimica et Biophysica Acta 1762 (2006) 934–944 interpretation of the results (Fig. 1). Further, systematic Information about global analyses is critical for understand- sampling, known to be important for correctly aimed compar- ing the molecular mechanisms underlying a disease. For NCL, ative statistical analyses have been lacking, partly due to the development of different model organisms will be crucial utilization of human autopsy samples. To initialize the for the success of the systematic strategies. To date, yeast, harmonization of sample collection, the European Commission nematode and Drosophila models exist only for CLN1 or CLN3 funded NCL models research consortium (www.nclmodels.org) diseases [26,139–143]. Besides producing novel data, it is has taken the task to breed the NCL mouse into the same genetic important to utilize the existing information available in background (C57/BL) to provide a platform for more feasible different databases, providing an integrated view of genome- comparisons [136]. wide information for the different model organisms [144–150]. Key goals of future NCL research include detailed Additionally, databases of the abnormal histology associated characterization of the perturbed metabolic and transcriptional with spontaneous and induced mutations of mice and other pathways using powerful “omics” technologies. Comparative organisms produced by transgenesis, targeted mutagenesis and transcriptome and proteome analyses in mammals are very chemical mutagenesis exist, and also these can be utilized to limited, but evidence suggests a close correlation between expedite the dissection of different disease phenotypes [151]. mRNA and protein expression for the majority of genes [137]. The mouse brain analytics should be simplified by utilization of To date it is clear that the proteomics technology lies somewhat stereological methods as well as 3D brain mapping to first behind the transcript profiling, but efficient methods for the identify the affected brain areas [60]. Additionally, the yeast, analysis of sample quality as well as for the 2D gel nematode and Drosophila models offer efficient systems to electrophoresis have recently emerged [138]. Technologies for track genetic interactions [152,153]. Further, detailed analyses lipid and metabolite profiling do exist and their efficient of the genome-wide expression data of the model organisms are utilization in NCL research only lacks systematic experimen- needed to identify other genes co-regulated with the NCL genes. tation and data integration. Novel approaches have already been One could foresee the combined analyses of the global introduced for data processing and analysis and data integration information of NCL (Fig. 2) as an avenue to resolve the disease will be a challenging task to combine gene expression profiling, mechanisms behind these devastating disorders. Furthermore, proteomic and metabolite analyses. innovative use of these new analytic possibilities has high

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