Progress towards understanding the neurobiology of Batten disease or neuronal ceroid lipofuscinosis Jonathan D. Cooper

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1 Progress towards understanding the neurobiology of Batten disease or neuronal ceroid lipofuscinosis Jonathan D Cooper Purpose of review The identification of genes mutated in the neuronal ceroid lipofuscinoses has accelerated research into the mechanisms that underlie these fatal autosomal recessive storage disorders, which are often referred to as Batten disease This review summarizes progress in this field since October 2001, describing advances in cell biology, the characterization of new animal models of neuronal ceroid lipofuscinosis, and the impact of novel methodology to reveal insights into its pathogenesis Recent findings Gene products for six of the eight forms of neuronal ceroid lipofuscinosis have now been discovered, and concerted efforts are underway to understand the normal biology of each gene product and how this may be altered by mutation Several lines of evidence point to functions for the CLN genes in the endosomal lysosomal system, and suggest neuron-specific roles for these proteins Indeed, a requirement for appropriate protein trafficking within neurons may explain the profound and selective effects of these disorders upon the central nervous system The development of mouse and large animal models has enabled comparative studies of the progressive effects of disease, including characterization by morphological and biochemical means supplemented by metabonomic and microarray techniques Summary Insights into disease mechanisms are building a detailed profile of the impact of neuronal ceroid lipofuscinosis upon the brain With the eventual aim of developing successful therapeutic strategies, it will be equally important to characterize the clinical progression of the disorder, and to identify quantifiable endpoints that can ultimately be used in clinical trials Keywords lysosomal storage disorders, neurodegeneration, neuronal ceroid lipofuscinosis, pathological mechanisms Curr Opin Neurol 16: # 2003 Lippincott Williams & Wilkins Pediatric Storage Disorders Laboratory, Department of Neuropathology, Institute of Psychiatry, King s College London, London, UK Correspondence to Jonathan D Cooper, Pediatric Storage Disorders Laboratory, Department of Neuropathology, P040, Institute of Psychiatry, King s College London, De Crespigny Park, London SE5 8AF, UK Tel: ; fax ; jcooper@iopkclacuk Current Opinion in Neurology 2003, 16: Abbreviations ANCL adult neuronal ceroid lipofuscinosis CNS central nervous system GABA a-aminobutyric acid GAD65 glutamic acid decarboxylase INCL infantile neuronal ceroid lipofuscinosis JNCL juvenile neuronal ceroid lipofuscinosis LINCL late infantile neuronal ceroid lipofuscinosis NCL neuronal ceroid lipofuscinosis PPT palmitoyl-protein thioesterase TPP-1 tripeptidyl peptidase-1 # 2003 Lippincott Williams & Wilkins Introduction The neuronal ceroid lipofuscinoses (NCLs) are a significant cause of childhood progressive intellectual and neurological deterioration [1 ] Collectively, this group of at least eight genetically distinct disorders is considered the most common pediatric neurodegenerative disease Originally described as a form of amaurotic familial idiocy, these fatal disorders were subsequently renamed because of the characteristic intracellular accumulation of ceroid and lipofuscin The precise relationship between the appearance of these lipopigments and cellular dysfunction remains unclear, but these disorders exert a profound effect upon the central nervous system (CNS) of affected individuals [2,3] The substantial impact of the NCLs upon carers has only recently begun to be evaluated [4 ] As comprehensively reviewed elsewhere, the NCLs are typified by their progressive nature, presenting with visual disturbances leading to blindness, neurocognitive decline, an increased severity of untreatable seizures and ultimately premature death [2,3,5,6] The vast majority of cases manifest during childhood with an infantile (INCL), late infantile (LINCL) or juvenile (JNCL) onset; although rare adult onset forms (ANCL) and an increasing number of variant forms are also recognized This spectrum of NCL subtypes makes accurate diagnosis complicated, relying on combined histological, ultrastructural and enzymatic criteria, together with subtle differences in clinical presentation [5 7] As with all fatal genetically predetermined disorders, counselling is of great importance for newly diagnosed families, and parents organizations play an invaluable role providing support and information (wwwbdsraorg and wwwbdfaukorguk) Genotyping is the definitive means of distinguishing between forms of NCL, although enzymatic analysis is DOI: /01wco b 121

2 122 Developmental disorders informative as a first approach [2,6,8,9] Since 1995 the individual genes mutated in six forms of NCL have been identified [5,8] The products of these CLN genes fall into two distinct categories comprising either soluble lysosomal enzymes (CLN1 and CLN2) [10] or predicted transmembrane proteins of unknown structure and function (CLN3, CLN5, CLN6 and CLN8) A comprehensive catalogue of mutations in each gene has been compiled and is available online (wwwuclacuk/ncl), facilitating the establishment of phenotype genotype correlations [2,9,11] Despite the identification of their molecular basis, very little is known about how mutations in these different genes ultimately result in this profoundly disabling disorder This is particularly true for CLN3, CLN5, CLN6 and CLN8, in which basic knowledge of the normal roles of these proteins, and how their functions are compromised by mutation, is incomplete Indeed, many significant questions about the pathological mechanisms that operate in the NCLs remain unanswered Why is it that mutations in so many different genes result in similar pathological profiles, albeit with different ages of onset and rates of progression? Moreover, why are the effects of these mutations largely confined to the CNS when autofluorescent lipopigments accumulate throughout the body? This review focuses on recent progress towards understanding the neurobiology of the NCLs, highlighting the latest advances in cell biology, and the impact of new animal models and novel methodology to provide a fresh perspective on disease mechanisms As a clearer picture of the pathological events in the NCLs emerges, attention will turn towards therapeutic attempts to halt disease progression To be successful it will require a detailed knowledge of clinical progression together with a series of quantifiable endpoints to judge therapeutic efficacy Progress in identifying mutated genes The most recently identified CLN gene is CLN6, which is mutated in variant LINCL [12,13 ] CLN6 is conserved across vertebrates, and encodes a novel 311 amino acid protein that is predicted to be an integral membrane protein with six or seven transmembrane domains Mutational analysis of affected families revealed at least six different mutations, the majority of which result in the introduction of a premature stop codon Sequencing of the orthologous gene in the nclf mouse revealed a frameshift truncation, which is replicated in three families of Pakistani origin [13 ] CLN6 is also predicted to be orthologous to NCLcausing genes in both South Hampshire and Merino sheep [14,15], although the precise mutation in each sheep model remains unidentified At present, the gene loci of the two remaining unidentified forms of NCL remain elusive The gene locus for Turkish variant LINCL (CLN7) remains unidentified, but may be allelic to CLN8 [16] Efforts to isolate the proposed CLN4 gene mutated in ANCL or Kufs disease have been unsuccessful However, mutations in other NCL-causing genes that leave some functionally active gene product may be responsible for certain delayed onset forms of NCL [17,18] However, it is unlikely that this explanation holds true for all forms of ANCL, especially the rarer autosomal dominant form or Parry disease [19] The cell biology of neuronal ceroid lipofuscinoses involving soluble lysosomal enzymes The CLN1 gene encodes a palmitoyl-protein thioesterase-1 (PPT1), a soluble lysosomal hydrolase that cleaves long-chain fatty acid side chains from proteins [6,10] Several lines of evidence support the lysosomal nature of PPT1 function, most recently via the effects of lysosomotropic agents upon the accumulation of lipid thioesters in PPT1-deficient lymphoblasts [20 ] The appearance of lipid thioesters in the lysosomal fraction is blocked by inhibitors of lysosomal proteolysis, including cysteamine, suggesting that the effects of this proposed therapeutic agent may be more complex than previously thought [20 ] In contrast, a non-lysosomal localization of transfected PPT1 has been reported in mouse primary neurons and fractionated mouse brain, co-localizing with markers of synaptosomes and synaptic vesicles [21 ] and PPT1 is targeted preferentially to axons, colocalizing with microtubule associated protein-1 [22] These data have been interpreted as evidence that PPT1 may play a crucial role outside lysosomes in the brain and may be associated with synaptic function Interestingly, the subcellular localization of PPT1 is redistributed with kainic acid-induced status epilepticus, not only enhancing co-localization with presynaptic membrane markers but also inducing the prolonged localized upregulation of PPT1 expression [23] Taken together, these data suggest further roles for PPT1 in protecting neurons from excitotoxicity and in synaptic plasticity The CLN2 gene encodes tripeptidyl peptidase-1 (TPP- 1), which cleaves tripeptides from the N-terminus of small proteins before their degradation by other lysosomal proteases [10] The biochemical and immunohistochemical mapping of TPP-1 distribution reveals variable, but widespread, expression in both somatic and nervous tissue, although TPP-1 activity and protein levels are not always correlated [24,25] Until recently very little was known about the natural substrates of TPP-1, but emerging evidence indicates that TPP-1 is essential for

3 Progress towards understanding the neurobiology of Batten disease Cooper 123 the degradation of cholecystokinin-8 in the mouse brain [26] Failure to degrade neuropeptides may have deleterious effects for neuronal function and survival It is significant that outside the CNS a second peptidase can compensate for the loss of TPP-1, potentially explaining why non-neuronal tissues are spared [27 ] The cell biology of neuronal ceroid lipofuscinoses involving transmembrane proteins The genes mutated in JNCL (CLN3) and Finnish variant LINCL (CLN5) both encode proteins that are predicted to reside in the lysosomal membrane [28], although emerging evidence suggests additional extralysosomal roles As with PPT1, the expression of transfected and endogenous CLN3 is not exclusively lysosomal in neurons The targeting of CLN3 to synaptosomes, although not synaptic vesicles themselves, implies that specific routes exist for the intracellular trafficking of CLN3 within neurons [29] Characterization of the biosynthesis and intracellular location of transiently transfected CLN5 appears to confirm its lysosomal localization, with mutant forms of CLN5 retained within the Golgi complex [30] The studies also revealed the secretion of a MW glycosylated CLN5 peptide, suggesting that the use of different start methionines may produce both membrane-bound and soluble forms of this protein [30,31 ] The presence of each CLN protein within the endosomal lysosomal system (CLN1, CLN2, CLN3, CLN5) and evidence that each are trafficked through, or are present in (CLN8), the endoplasmic reticulum Golgi system, suggests that these gene products may participate in a common biological process [32] Although the precise nature of this process is open to speculation, similarities between the pathological profiles of each form of NCL lend further weight to the hypothesis that its disruption is a key event The first evidence for direct interactions between the transmembrane form of CLN5 with both CLN2 and CLN3 has been suggested by the results of co-precipitation and in vitro binding assays [31 ], although the function of such interactions is not yet established Cell death mechanisms Recent evidence from other neurodegenerative disorders argues against exclusively apoptotic versus necrotic cell death, instead favouring a variety of cell death forms [33] It is likely that the NCLs follow this pattern with a spectrum of cell death events, although mechanistic observations have only been made in JNCL [34] and the mechanisms that trigger cell death remain unclear A proposed anti-apoptotic role for CLN3 has now been localized to domains clustered around the c terminus, with specific deletion mutants in highly conserved regions, resulting in slowed growth and susceptibility to etoposide-induced apoptosis in lymphoblasts [34] The non-opioid analgesic flupirtine also prevents etoposide-induced apoptotic cell death in CLN3- and CLN2- deficient teratocarcinoma-derived cell lines [35], but remains untested in appropriate animal models The observed upregulation of CLN3 in some forms of cancer [36] may lead to the discovery of other potential roles for CLN3 in regulating cell death, as has been suggested for PPT1 [37] New mouse models of neuronal ceroid lipofuscinosis PPT1 null mutant mice exhibit an NCL-like phenotype with widespread accumulation of autofluorescent lipopigments, a shortened lifespan, progressive motor abnormalities and spontaneous myoclonic seizures [38 ] These mice also exhibit regionally specific cortical atrophy, laminar disruption and profound loss of subpopulations of inhibitory interneurons in the cortex and hippocampus, with persisting neurons exhibiting a range of dendritic abnormalities and the loss of synaptic spines [39] Although it has been suggested that PPT1 plays an essential role in neuronal development and growth, initial studies suggest little effect of a lack of PPT1 on the developing CNS A more detailed analysis may reveal subtle effects, but PPT1 appears to play a role in the maintenance of certain neuronal populations rather than during their development A third mouse model of JNCL has also become available, using a knock-in strategy to replicate the approximately 1 kb deletion in the CLN3 gene present in the majority of JNCL patients [40 ] These CLN3 Dex7/8 mice exhibit an NCL-like phenotype at an earlier age than previous CLN3 null mutant models, with widespread accumulation of autofluorescent lipopigments, motor abnormalities and shortened lifespan [40 ] Pathological changes within the CNS of CLN3 Dex7/8 mice are present prenatally, challenging the notion that JNCL only exerts its effect after a period of normal development A detailed characterization of CLN3 Dex7/8 mice may uncover more subtle JNCL phenotypes, but it is already apparent that the retina of these mice exhibit the loss of cone-associated markers [40 ], whereas CLN3 null mutants exhibit remarkably mild retinal pathology [41], certainly compared with mnd and nclf mice [42] As with other murine JNCL models, CLN3 Dex7/8 mice do not exhibit spontaneous seizures, but may show an altered threshold to seizure induction as described in CLN3 null mutant mice [43] The discovery that the mnd mouse bears a mutation in the CLN8 gene [8], has seen a resurgence of studies in this model Mnd mice exhibit a complex series of changes in the expression of glutamate receptor expres-

4 124 Developmental disorders sion [44] Most notable is the increased expression of the GluR2 subunit of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor, suggesting a compensatory change in response to elevated glutamate levels, which has been demonstrated by metabolic profiling [45 ] Progress has also been made towards providing functional and behavioural landmarks of disease progression Presymptomatic mnd mice are hyperactive, more aggressive than controls, and display poor contextual and cued memory [46], recapitulating at least some of the behaviours seen in patients Investigations of seizure susceptibility in both mnd and nclf mice revealed findings similar to CLN3 null mutant mice, but with seizure phenotypes strikingly different from controls (Pearce, personal communication) Clues about pathogenesis from other mutant mice Inactivation of a second lysosomal thioesterase, PPT2, produces a mild form of the PPT1 null mutant phenotype [38 ], suggesting that PPT2 plays a role in the brain not carried out by PPT1 Surprisingly, chloride channel-3 null mutant mice also exhibit an aggressive NCL-like phenotype, which closely resembles that of cathepsin-d-deficient mice [47 ], including retinal degeneration and reduced brain size [48] Among other roles, chloride channels act to regulate the ionic composition of intracellular compartments and thus their ph [49] Certainly, chloride channel-3 mutants also exhibit impaired acidification of synaptic vesicles, which has direct implications for neurotransmitter uptake [48] As such, it may be significant that mutations of all CLN proteins except CLN2 and CLN8 have demonstrable effects upon ph within the endosomal lysosomal system [50] Furthermore, compounds that affect the ph of acidic intracellular compartments also appear to alter maturation of the TPP-1 proenzyme within lysosomes [51] Applying new methodology and learning new perspectives Novel methodologies that permit the simultaneous assessment of multiple gene or protein expression can provide powerful insights into disease mechanisms High-density oligonucleotide microarrays have been used to examine changes in gene expression in the cerebellum of CLN3 null mutant mice [52 ] Bioinformatic analysis revealed more than twofold changes in the gene expression of 330 genes, which can be ascribed to a variety of functional groups (full data set available at wwwurmcrochester edu/research/fgc/resourcehtml), most notably the upregulation of genes associated with an immune or inflammatory response and in the regulation and utilization of glutamate [52 ] Metabonomic screening [53] has recently been used to generate a metabolic profile of mnd mice, revealing deficits of vitamin E in both serum and brain and abnormal levels of low-weight metabolites, suggesting their redistribution across intracellular compartments [45 ] Significantly, dietary vitamin E supplementation reversed some of the metabolic abnormalities, especially increasing phenylalanine in the CNS, but had no effect upon brain pathology [45 ] Lipid chromatography and quantitative mass spectroscopy reveal profound changes in the composition of membrane phospholipids in INCL, but not JNCL postmortem brains [54] These changes reflect pathological changes in membrane composition that could contribute directly to disease progression, but it will be crucial to determine when in pathogenesis these events first occur Glial and neuroimmune responses in neuronal ceroid lipofuscinosis There is considerable evidence for the activation of glial cell populations in different forms of NCL Autopsy material derived from individuals with different forms of NCL shows a consistent and regionally specific pattern of astrocytosis and microglial activation (Tyynelä et al, unpublished data) Similar data can be obtained from null mutant mouse models, although the timing of glial reactivity in relation to neuronal pathology remains unclear However, activated microglia in cathepsin-d null mutant mice appear to play an active pathological role via the release of nitric oxide [55] As these mice exhibit an aggressive INCL-like phenotype, it will be important to determine whether similar mechanisms operate in models of NCL Reactive changes in astrocyte populations are likely to exert a profound effect upon the local environment around neurons and directly influence neuronal activity and synaptic efficacy [56] Individuals with JNCL and CLN3 null mutant mice have a circulating autoantibody to glutamic acid decarboxylase (GAD65) that inhibits this enzyme s ability to convert glutamic acid to a-aminobutyric acid (GABA) and results in the presynaptic elevation of glutamate [52 ] GAD65 autoantibodies are also present in an extended series of JNCL patients, but not in individuals with other lysosomal storage disorders [57] These findings may have significant implications for excitotoxic mechanisms, and could explain the specific targeting of inhibitory interneurons in this disorder However, it remains unclear whether the autoimmune response contributes to JNCL pathogenesis or is one of several epiphenomena Nevertheless, the lysosome plays an active role in antigen presentation, a process that is apparently abnormal in other storage disorders [58] Although CNS levels of IgGs are significantly elevated in CLN3 null mutant mice, it remains unclear how these macromolecules gain access to the CNS

5 Progress towards understanding the neurobiology of Batten disease Cooper 125 Large animal models of neuronal ceroid lipofuscinosis Accurate large animal models will prove essential for assaying the biodistribution and efficacy of potential therapies as these become available Naturally occurring sheep strains that exhibit recessive NCL-like disorders are the best characterized examples, especially at a biochemical level [14,15] At least two of these strains appear to bear mutations in ONCL6, the ovine orthologue of CLN6; including the Merino flock that bears close resemblance to the more established South Hampshire model [14] These ovine models also exhibit a characteristic loss of GABAergic interneurons [15] A form of NCL has also been reported in Borderdale sheep that appears to be distinct from ONCL6, but still exhibits the accumulation of subunit c of mitochondrial ATPase [59], in contrast to the Swedish Landrace sheep that have a deficiency of cathepsin D and exhibit INCL-like storage material [60] Introduction of the point mutation that causes this ovine form of NCL into the mouse cathepsin D molecule severely affects its activity, stability, processing and secretion [61] Human neuronal ceroid lipofuscinosis pathology Different forms of NCL share a distinct pattern of neuronal degeneration in the hippocampus, unlike that associated with temporal lobe epilepsies, with the heavy involvement of sectors CA2 CA4 but relative sparing of CA1 [62] This regional specificity also extends to activated glia and immunohistochemically identified interneurons, which exhibit a graded severity of loss with the relative sparing of calretinin-positive interneurons across all regions (Tyynelä et al, unpublished data) The selective vulnerability of interneuron populations may reflect the differential buffering capacities of calcium-binding proteins [63], or a more fundamental and regionally specific property, as yet unidentified These data closely mirror findings in murine and sheep models, further emphasizing the existence of common pathological themes maintained across species boundaries and different forms of NCL There is also considerable value in combining modern morphological and molecular methods to revisit archival patient-derived material [64], although robust quantitative data from human material remain sorely lacking Towards therapy: following the clinical progression of the neuronal ceroid lipofuscinoses Once the focus ultimately shifts towards testing therapies it will be essential that rigorously defined landmarks exist for following disease progression and judging therapeutic efficacy Ongoing efforts have provided considerable information, with recent reports detailing altered visual performance [7,65], somatosensory evoked responses [66], sleep patterns [67] and hyperandrogenism [68] in different forms of NCL Imaging studies will be particularly informative, especially when combined with functional markers, and represent a relatively noninvasive means to assay progressive and selective effects of the disorder upon the CNS [69,70] The provision of readily quantifiable scales to score the clinical course of different forms of NCL will prove particularly valuable in defining specific endpoints in the design of clinical trials [71 ] At present, the therapeutic outlook for the NCLs remains bleak, and many ethical and technical challenges remain to be surmounted With the advent of appropriate animal models the systematic testing of therapeutic interventions can now begin, although suitable models do not yet exist for all forms of NCL Replacement of the missing enzyme may be an option, in INCL and LINCL, but problems of enzyme production and the method of delivery will need to be overcome The recent encouraging results of gene therapy in reversing functional and pathological deficits in a mouse model of mucopolyaccharidosis type VII [72 ] suggest that similar approaches may be appropriate in the NCLs [73] Indeed, recent findings suggest that such viral mediated delivery may be feasible in LINCL [74 ] However, this approach will necessarily be limited to forms of NCL with deficits in soluble lysosomal enzymes Hematopoietic stem cell transplantation is now not recommended for the treatment of INCL [75], but there is hope that transplanted neural stem cells may eventually be capable of replacing damaged neurons However, as with other approaches, appropriate reagents and cell lines will need to be generated and rigorously tested before this potential may be realized Conclusion Without a detailed understanding of the precise mechanisms that operate within each form of NCL, successful treatments for these fatal disorders will remain a distant prospect The identification of gene products is now complete for the majority of different forms and has enabled the investigation of normal and pathological cell biology Many issues remain to be resolved, but several lines of evidence point to the critical involvement of the lysosomal endosomal system Nevertheless, it will be vital to establish whether CLN gene products play other specific roles within the nervous system The development of new animal models and the application of novel technologies is gradually creating a detailed picture of the progressive effects of disease These efforts must be mirrored by the continued development of appropriate means to follow the clinical course in affected individuals

6 126 Developmental disorders Acknowledgements The author would like to thank colleagues in the Pediatric Storage Disorders Laboratory (PSDL) for their invaluable contributions, Lara Alden for secretarial support, Nadeem Khan, Jill Weimer and Drs David Pearce, Jaana Tyynelä, Jean-Pierre Lin and Alison Barnwell for their constructive comments on this manuscript Studies from the PSDL cited in this review were supported by grants from the NIH (R21 NS A1), the Wellcome Trust (Biomedical Research Collaboration grant ), the Natalie Fund, the Remy Fund, the Batten Disease Support and Research Association, the Batten Disease Family Association and the Batten Disease Support and Research Trust References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1 Progressive Intellectual and Neurological Deterioration Survey 16th Annual Report, September 2002, and Autumn 2002 newsletter of the Royal College British Paediatric Surveillance Unit, Royal College of Paediatrics and Child Health, UK In this latest update to an ongoing survey of the causes underlying intellectual and neurological deterioration in childhood, out of a total of 406 cases the NCLs represent the largest disease category with 67 cases (165%), and the combined total for all storage disorders is 257 cases (633%) 2 Wisniewski KE, Zhong N, Philippart M Pheno/genotypic correlations of neuronal ceroid lipofuscinoses Neurology 2001; 57: Mitchison HM, Mole SE Neurodegenerative disease: the neuronal ceroid lipofuscinoses (Batten disease) Curr Opin Neurol 2001; 14: Labbe EE, Lopez I, Murphy L, O Brien C Optimism and psychosocial functioning in caring for children with Battens and other neurological diseases Psychol Rep 2002; 90: The effects of a long-term progressive and ultimately fatal disorder such as the NCLs upon parents and carers have not previously been assessed quantitatively In comparison with parents of children with epilepsy or cerebral palsy, the parents or carers of NCL sufferers were found to be less optimistic, and have significantly higher depression and anxiety scores 5 Gardiner RM Clinical features and molecular genetic basis of the neuronal ceroid lipofuscinoses Adv Neurol 2002; 89: Hofmann SL, Atashband A, Cho SK, et al Neuronal ceroid lipofuscinoses caused by defects in soluble lysosomal enzymes (CLN1 and CLN2) Curr Mol Med 2002; 2: Case Records of the Massachusetts General Hospital Weekly clinicopathological exercises Case A 5-year-old boy with seizures and progressive deterioration of cognitive and motor function N Engl J Med 2002; 347: Mole S Gene table: neuronal ceroid lipofuscinoses Eur J Paediatr Neurol 2002; 6: Lavrov AY, Ilyna ES, Zakharova EY, et al The first three Russian cases of classical, late-infantile, neuronal ceroid lipofuscinosis Eur J Paediatr Neurol 2002; 6: Gupta P, Hofmann SL Neuronal ceroid lipofuscinosis/batten disease: the lysosomal proteinoses Mol Psychiatry 2002; 7: Ju W, Zhong R, Moore S, et al Identification of novel CLN2 mutations shows Canadian specific NCL2 alleles J Med Genet 2002; 39: Gao H, Boustany RM, Espinola JA, et al Mutations in a novel CLN6-encoded transmembrane protein cause variant neuronal ceroid lipofuscinosis in man and mouse Am J Hum Genet 2002; 70: See Ref [13 ] 13 Wheeler RB, Sharp JD, Schultz RA, et al The gene mutated in variant lateinfantile neuronal ceroid lipofuscinosis (CLN6) and in nclf mutant mice encodes a novel predicted transmembrane protein Am J Hum Genet 2002; 70: These two studies report the cloning and initial mutational analysis of the CLN6 gene, which is mutated in a form of variant late infantile NCL that is particularly prominent in southern Mediterranean and South American families Both groups provide evidence that the candidate gene FLJ20561 on chromosome 15q21 23 represents CLN6 Armed with this information, it will be possible to generate tools for investigating CLN6 function Importantly, these findings also validate nclf mice, Merino and South Hampshire sheep as animal models of variant ILNCL 14 Cook RW, Jolly RD, Palmer DN, et al Neuronal ceroid lipofuscinosis in Merino sheep Aust Vet J 2002; 80: Oswald MJ, Kay GW, Palmer DN Changes in GABAergic neuron distribution in situ and in neuron cultures in ovine (OCL6) Batten disease Eur J Paediatr Neurol 2001; 5 (Suppl A): Mitchell WA, Wheeler RB, Sharp JD, et al Turkish variant late infantile neuronal ceroid lipofuscinosis (CLN7) may be allelic to CLN8 Eur J Paediatr Neurol 2001; 5 (Suppl A): Van Diggelen OP, Keulemans JL, Kleijer WJ, et al Pre- and postnatal enzyme analysis for infantile, late infantile and adult neuronal ceroid lipofuscinosis (CLN1 and CLN2) Eur J Paediatr Neurol 2001; 5 (Suppl A): Mazzei R, Conforti FL, Magariello A, et al A novel mutation in the CLN1 gene in a patient with juvenile neuronal ceroid lipofuscinosis J Neurol 2002; 249: Nijssen PC, Brusse E, Leyten AC, et al Autosomal dominant adult neuronal ceroid lipofuscinosis: parkinsonism due to both striatal and nigral dysfunction Movement Disord 2002; 17: Lu JY, Verkruyse LA, Hofmann SL The effects of lysosomotropic agents on normal and INCL cells provide further evidence for the lysosomal nature of palmitoyl-protein thioesterase function Biochim Biophys Acta 2002; 1583:35 44 See Ref [21 ] 21 Lehtovirta M, Kyttala A, Eskelinen EL, et al Palmitoyl protein thioesterase (PPT) localizes into synaptosomes and synaptic vesicles in neurons: implications for infantile neuronal ceroid lipofuscinosis (INCL) Hum Mol Genet 2001; 10:69 75 These two papers present alternative, but not mutually exclusive, views on PPT1 localization and function Ref [20 ] provides compelling evidence for lysosomal function in lymphoblasts and also reveals data that suggest that cysteamine may not act directly to cleave thioester bonds In contrast, the data in Ref [21 ] suggest that PPT1 has a synaptic localization in primary neurons This may represent an example of cell-type specific sub-cellular localization for PPT1, and it will have significant implications for pathogenesis if PPT1 also proves to play a distinct functional role in neurons 22 Ahtiainen L, Van Diggelen OP, Jalanko A, Kopra O Palmitoyl protein thioesterase 1 is targeted to the axons in neurons J Comp Neurol 2003; 455: Suopanki J, Lintunen M, Lahtinen H Status epilepticus induces changes in the expression and localization of endogenous palmitoyl-protein thioesterase 1 Neurobiol Dis 2002; 10: Koike M, Shibata M, Ohsawa Y, et al The expression of tripeptidyl peptidase I in various tissues of rats and mice Arch Histol Cytol 2002; 65: Kurachi Y, Oka A, Itoh M, et al Distribution and development of CLN2 protein, the late-infantile neuronal ceroid lipofuscinosis gene product Acta Neuropathol (Berl) 2001; 102: Warburton MJ, Bernardini F Tripeptidyl peptidase-i is essential for the degradation of sulphated cholecystokinin-8 (CCK-8S) by mouse brain lysosomes Neurosci Lett 2002; 11: Bernardini F, Warburton MJ Lysosomal degradation of cholecystokinin-(29-33)-amide in mouse brain is dependent on tripeptidyl peptidase-i: implications for the degradation and storage of peptides in classical late-infantile neuronal ceroid lipofuscinosis Biochem J 2002; 366: It remains to be seen if neuropeptide accumulation plays a direct role in the disease process, but the identification of a role for TPP-1 in cholecystokinin degradation provides further insight into the neuron-specific nature of TPP-1 deficiency In somatic tissues dipeptidyl peptidase-1 is able to compensate for the lack of TPP function, but does not perform this role in the brain in which the expression of dipeptidyl peptidase-1 is very low 28 Vesa J, Peltonen L Mutated genes in juvenile and variant late infantile neuronal ceroid lipofuscinoses encode lysosomal proteins Curr Mol Med 2002; 2: Luiro K, Kopra O, Lehtovirta M, Jalanko A CLN3 protein is targeted to neuronal synapses but excluded from synaptic vesicles: new clues to Batten disease Hum Mol Genet 2001; 10: Isosomppi J, Vesa J, Jalanko A, et al Lysosomal localization of the neuronal ceroid lipofuscinosis CLN5 protein Hum Mol Genet 2002;

7 Progress towards understanding the neurobiology of Batten disease Cooper Vesa J, Chin MH, Oelgeschlager K, et al Neuronal ceroid lipofuscinoses are connected at molecular level: interaction of CLN5 protein with CLN2 and CLN3 Mol Biol Cell 2002; 13: Several lines of evidence point to a critical role for the lysosomal endosomal system in the pathogenesis of several forms of NCL These authors provide the first evidence of direct interactions of NCL genes at a molecular level, but the functional significance of these interactions is presently unknown As cell typespecific localizations and functions for NCL gene products have been proposed (Refs [28] and [29]), it will be crucial to re-examine these interactions in primary neuronal cultures or cell lines with neuronal properties 32 Weimer JM, Kriscenski-Perry E, Elshatory Y, Pearce DA The neuronal ceroid lipofuscinoses: mutations in different proteins result in similar disease Neuromol Med 2002; 1: Graeber MB, Moran LB Mechanisms of cell death in neurodegenerative disease: fashion, fiction, and facts Brain Pathol 2002; 12: Persaud-Sawin DA, VanDongen A, Boustany RM Motifs within the CLN3 protein: modulation of cell growth rates and apoptosis Hum Mol Genet 2002; 11: Dhar S, Bitting RL, Rylova SN, et al Flupirtine blocks apoptosis in Batten patient lymphoblasts and in human postmitotic CLN3- and CLN2-deficient neurons Ann Neurol 2002; 51: Rylova SN, Amalfitano A, Persaud-Sawin DA, et al The CLN3 gene is a novel molecular target for cancer drug discovery Cancer Res 2002; 62: Dawson G, Dawson S, Marinzi C, Dawson P Anti-tumor promoting effects of palmitoyl: protein thioesterase inhibitors against a human neurotumor cell line Cancer Lett 2002; 187: Gupta P, Soyombo AA, Atashband A, et al Disruption of PPT1 or PPT2 causes neuronal ceroid lipofuscinosis in knockout mice Proc Natl Acad Sci U S A 2001; 98: The provision of a mouse model for INCL represents an important step forwards to investigate the effects of PPT1 mutation in vivo PPT1 null mutant mice present with an aggressive NCL-like phenotype with pathological and behavioural abnormalities and spontaneous seizures Together with more detailed pathological studies (Ref [39]) these findings set the scene for testing therapeutic strategies, including gene therapy, enzyme replacement or substrate depletion 39 Cooper JD, Gupta P, Bible E, Hofmann S Profound loss of GABAergic interneurons in the PPT1 knockout mouse model of infantile neuronal ceroid lipofuscinosis Neuropathol Appl Neurobiol 2002; 28: Cotman SL, Vrbanac V, Lebel LA, et al Cln3 Dex7/8 knock-in mice with the common JNCL mutation exhibit progressive neurologic disease that begins before birth Hum Mol Genet 2002; 11: Although two CLN3 null mutant mice already exist, Cln3 Dex7/8 knock-in mice represent an accurate model of the most common mutation in JNCL These mice present with a more pronounced NCL-like phenotype than previous models, with detectable pathological effects evident prenatally The authors also suggest that no direct link exists between pathological inclusions and neurodegeneration, reinforcing that the accumulated lipopigments are not themselves likely to be a toxic entity 41 Seigel GM, Lotery A, Kummer A, et al Retinal pathology and function in a Cln3 knockout mouse model of juvenile neuronal ceroid lipofuscinosis (Batten disease) Mol Cell Neurosci 2002; 19: Chang B, Hawes NL, Hurd RE, et al Retinal degeneration mutants in the mouse Vision Res 2002; 42: Kriscenski-Perry E, Applegate CD, Serour A, et al Altered flurothyl seizure induction latency, phenotype, and subsequent mortality in a mouse model of juvenile neuronal ceroid lipofuscinosis/batten disease Epilepsia 2002; 43: Mennini T, Bigini P, Ravizza T, et al Expression of glutamate receptor subtypes in the spinal cord of control and mnd mice, a model of motor neuron disorder J Neurosci Res 2002; 70: Griffin JL, Muller D, Woograsingh R, et al Vitamin E deficiency and metabolic deficits in neuronal ceroid lipofuscinosis described by bioinformatics Physiol Genom 2002; 11: Metabonomic profiling is a powerful new technique for exploring the metabolic consequences of gene mutation Application of these techniques to mnd mice reveals specific metabolic changes, including vitamin E deficiency Dietary supplementation with vitamin E has long been proposed as beneficial in the NCLs, but in this study demonstrated only incomplete reversal of metabolic defects and no detectable effect on pathology 46 Bolivar VJ, Scott Ganus J, Messer A The development of behavioral abnormalities in the motor neuron degeneration (mnd) mouse Brain Res 2002; 937: Yoshikawa M, Uchida S, Ezaki J, et al CLC-3 deficiency leads to phenotypes similar to human neuronal ceroid lipofuscinosis Genes Cell 2002; 7: Although the function of transmembrane CLN gene products remains elusive, the discovery of an NCL-like phenotype in CLC-3 null mutant mice may provide significant clues for pathogenesis At present ion channel or pump-like functions remain unproved for any of the NCL genes, but several lines of evidence converge upon ph regulation as a possible functional role for some of these proteins (see Ref [50] and previous studies in yeast) 48 Stobrawa SM, Breiderhoff T, Takamori S, et al Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus Neuron 2001; 29: Iyer R, Iverson TM, Accardi A, Miller C A biological role for prokaryotic ClC chloride channels Nature 2002; 419: Holopainen JM, Saarikoski J, Kinnunen PK, Jarvela I Elevated lysosomal ph in neuronal ceroid lipofuscinoses (NCLs) Eur J Biochem 2001; 268: Golabek AA, Kida E, Walus M, et al Human tripeptidyl-peptidase I: Biosynthesis, glycosylation and enzymatic processing in vivo J Biol Chem 2002; [epub ahead of print] 52 Chattopadhyay S, Ito M, Cooper JD, et al An autoantibody inhibitory to glutamic acid decarboxylase in the neurodegenerative disorder Batten disease Hum Mol Genet 2002; 11: The discovery of an autoimmune response in human and murine JNCL potentially has significant implications for pathogenesis However, it will be crucial to determine whether this autoimmune response contributes directly to disease processes or is secondary to other events It also remains to be demonstrated whether this response is JNCL specific or is also seen in other forms of NCL 53 Nicholson JK, Connelly J, Lindon JC, Holmes E Metabonomics: a platform for studying drug toxicity and gene function Nat Rev Drug Discov 2002; 1: Käkelä R, Somerharju P, Tyynelä J Analysis of phospholipid molecular species in brains of patients with infantile and juvenile neuronal ceroidlipofuscinosis using liquid chromatography and electrospray ionization mass spectrometry Journal of Neurochemistry 2003; (in press) 55 Nakanishi H, Zhang J, Koike M, et al Involvement of nitric oxide released from microglia-macrophages in pathological changes of cathepsin D-deficient mice J Neurosci 2001; 21: Piet R, Poulain DA, Oliet SH Modulation of synaptic transmission by astrocytes in the rat supraoptic nucleus J Physiol Paris 2002; 96: Chattopadhyay S, Kriscenski-Perry E, Wenger DA, Pearce DA An autoantibody to GAD65 in sera of patients with juvenile neuronal ceroid lipofuscinoses Neurology 2002; 59: Daly TM, Lorenz RG, Sands MS Abnormal immune function in vivo in a murine model of lysosomal storage disease Pediatr Res 2000; 47: Jolly RD, Arthur DG, Kay GW, Palmer DN Neuronal ceroid-lipofuscinosis in Borderdale sheep NZ Vet J 2002; 50: Tyynelä J, Sohar I, Sleat DE, et al Congenital ovine neuronal ceroid lipofuscinosis a cathepsin D deficiency with increased levels of the inactive enzyme Eur J Paediatr Neurol 2001; 5 (Suppl A): Partanen S, Storch S, Loffler HG, et al A replacement of the active site aspartic acid residue-293 in mouse cathepsin D affects its intracellular stability, processing, and transport in HEK 293 cells Biochem J 2003; 369: Haltia M, Herva R, Suopanki J, et al Hippocampal lesions in the neuronal ceroid lipofuscinoses Eur J Paediatr Neurol 2001; 5 (Suppl A): d Orlando C, Celio MR, Schwaller B Calretinin and calbindin D-28k, but not parvalbumin protect against glutamate-induced delayed excitotoxicity in transfected N18-RE 105 neuroblastoma-retina hybrid cells Brain Res 2002; 945: Wheeler RB, Schlie M, Kominami E, et al Neuronal ceroid lipofuscinosis: late infantile or Jansky Bielschowsky type re-revisited Acta Neuropathol (Berl) 2001; 102: Wilkinson ME Ceroid lipofuscinosis, neuronal 3, juvenile-batten disease: case report and literature review Optometry 2001; 72: Lauronen L, Huttunen J, Kirveskari E, et al Enlarged SI and SII somatosensory evoked responses in the CLN5 form of neuronal ceroid lipofuscinosis Clin Neurophysiol 2002; 113: Kirveskari E, Partinen M, Santavuori P Sleep and its disturbance in a variant form of late infantile neuronal ceroid lipofuscinosis (CLN5) J Child Neurol 2001; 16: Aberg LE, Tiitinen A, Autti TH, et al Hyperandrogenism in girls with juvenile neuronal ceroid lipofuscinosis Eur J Paediatr Neurol 2002; 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8 128 Developmental disorders 69 Rinne JO, Ruottinen HM, Nagren K, et al Positron emission tomography shows reduced striatal dopamine D1 but not D2 receptors in juvenile neuronal ceroid lipofuscinosis Neuropediatrics 2002; 33: Sayit E, Yorulmaz I, Bekis R, et al Comparison of brain perfusion SPECT and MRI findings in children with neuronal ceroid-lipofuscinosis and in their families Ann Nucl Med 2002; 16: Steinfeld R, Heim P, von Gregory H, et al Late infantile neuronal ceroid lipofuscinosis: quantitative description of the clinical course in patients with CLN2 mutations Am J Med Genet 2002; 112: These authors describe the development of a total disability score in LINCL patients, similar to their previous work in JNCL The scoring system combines quantitative measures of motor, visual and verbal functions plus seizure frequency Importantly, these measures do not depend on a clinical scale, but on a simple assessment system of reliable recordings made by the patients families 72 Brooks AI, Stein CS, Hughes SM, et al Functional correction of established central nervous system deficits in an animal model of lysosomal storage disease with feline immunodeficiency virus-based vectors Proc Natl Acad Sci U S A 2002; 99: A considerable body of literature exists examining the effects of adeno-associated viral-mediated gene therapy in a mouse model of mucopolysaccharidosis type VII The authors describe the significant effects of gene therapy in mucopolysaccharidosis type VII mice using feline immunodeficiency virus-based vectors in reversing spatial learning and memory deficits and pathological changes Significantly, these effects were found treating adult mice with an established lysosomal disease of the CNS, indicating that the efficacy of this form of therapy is not restricted to neonatal administration 73 Sondhi D, Hackett NR, Apblett RL, et al Feasibility of gene therapy for late neuronal ceroid lipofuscinosis [Review] Arch Neurol 2001; 58: Haskell RE, Hughes SM, Chiorini JA, et al, Viral-mediated delivery of the late-infantile neuronal ceroid lipofuscinosis gene, TPP-1 to the mouse central nervous system Gene Ther 2003; 10: This paper provides the first evidence that adenoviral, adeno-associated and lentiviral vectors are capable of producing long lasting expression of biologically active TPP-1 protein within the CNS This is an essential prerequisite for successful gene therapy and sets the scene for testing the efficacy of these viral delivery systems once a mouse model of LINCL has been developed 75 Lönnqvist T, Vanhanen SL, Vettenranta K, et al Hematopoietic stem cell transplantation in infantile neuronal ceroid lipofuscinosis Neurology 2001; 57: