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The variant late infantile form of the inherited neurodegenerative Batten disease (BD) is caused by mutations in the CLN7/MFSD8 gene and represents a strong candidate for gene therapy. Post-natal intracerebral administration of AAV9-hCLN7 to Cln7Δex2 knockout mice resulted in extended lifespan but dose escalation resulted in reduced acuity in neurophysiology tests, cerebral atrophy and elevated neuroinflammation. Comparing patient and control iPSC-derived neural progenitor cells (iNPC) we discovered that CLN7 localizes to the nucleus as well as the endolysosomal network and is differentially distributed in BD iNPC. Proteomics identified a profound nuclear defect in BD iNPC that compounds with mitochondrial and lysosomal metabolic defects resulting in elevated apoptosis. We further identified a 50kDa common nuclear CLN7 isoform and a 37kDa isoform that accumulates only in BD iNPC nuclei. Our findings suggest that successful treatment of CLN7 BD will require combinatorial therapies addressing both loss and aberrant gain of protein function.

PSENEN/PEN2 is the smallest subunit of the γ-secretase complex, an intramembrane protease that cleaves proteins within their transmembrane domains. Mutations in components of the γ-secretase underlie familial Alzheimer disease. In addition to its proteolytic activity, supplementary, γ-secretase independent, functions in the macroautophagy/autophagy-lysosome system have been proposed. Here, we screened for PSENEN-interacting proteins and identified CLN3. Mutations in

Biallelic MFSD8 variants are an established cause of severe late-infantile subtype of neuronal ceroid lipofuscinosis (v-LINCL), a severe lysosomal storage disorder, but have also been associated with nonsyndromic adult-onset maculopathy. Here, we functionally characterized two novel MFSD8 variants found in a child with juvenile isolated maculopathy, in order to establish a refined prognosis. ABCA4 locus resequencing was followed by the analysis of other inherited retinal disease genes by whole exome sequencing (WES). Minigene assays and cDNA sequencing were used to assess the effect of a novel MFSD8 splice variant. MFSD8 expression was quantified with qPCR and overexpression studies were analyzed by immunoblotting. Transmission electron microscopy (TEM) was performed on a skin biopsy and ophthalmological and neurological re-examinations were conducted. WES revealed two novel MFSD8 variants: c.[590del];[439+3A>C] p.[Gly197Valfs*2];[Ile67Glufs*3]. Characterization of the c.439+3A>C variant via splice assays showed exon-skipping (p.Ile67Glufs*3), while overexpression studies of the corresponding protein indicated expression of a truncated polypeptide. In addition, a significantly reduced MFSD8 RNA expression was noted in patient's lymphocytes. TEM of a skin biopsy revealed typical v-LINCL lipopigment inclusions while neurological imaging of the proband displayed subtle cerebellar atrophy. Functional characterization demonstrated the pathogenicity of two novel MFSD8 variants, found in a child with an initial diagnosis of juvenile isolated maculopathy but likely evolving to v-LINCL with a protracted disease course. Our study allowed a refined neurological prognosis in the proband and expands the natural history of MFSD8-associated disease.

The neuronal ceroid lipofuscinoses comprise a group of neurodegenerative lysosomal storage disorders caused by mutations in at least 13 different genes and primarily affect the brain and the retina of children or young adults. The disorders are characterized by progressive neurological deterioration with dementia, epilepsy, loss of vision, motor disturbances, and early death. While various therapeutic strategies are currently being explored as treatment options for these fatal disorders, there is presently only one clinically approved drug that has been shown to effectively attenuate the progression of a specific form of neuronal ceroid lipofuscinosis, CLN2 disease (cerliponase alfa, a lysosomal enzyme infused into the brain ventricles of patients with CLN2 disease). Therapeutic approaches for the treatment of other forms of neuronal ceroid lipofuscinosis include the administration of immunosuppressive agents to antagonize neuroinflammation associated with neurodegeneration, the use of various small molecules, stem cell therapy, and gene therapy. An important aspect of future work aimed at developing therapies for neuronal ceroid lipofuscinoses is the need for treatments that effectively attenuate neurodegeneration in both the brain and the retina.

Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent‐conjugated bacterial toxins to label Gb3 to develop a cell‐based high content imaging (HCI) screening assay for the repurposing of FDA‐approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient‐derived induced pluripotent stem cells (iPSC). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease., The neuronal ceroid lipofuscinoses (NCL), commonly known as Batten disease (BD), are a group of recessively inherited fatal diseases of the nervous system that typically arise in childhood. There is neither cure nor drugs to revert the course of these diseases.

Trabajo presentado al 20th Biennial Meeting of The Society for Free Radical Research International (SFRR-I) del 15 al 18 de marzo de forma virtual, Neuronal ceroid lipofuscinoses (NCLs), known as Batten disease, are the most common of the rare neurodegenerative disorders in children. These disorders are grouped together based on clinical similarities and uniform neuropathological features, including accumulation of lipofuscin in lysosomes and widespread gliosis. CLN7 disease is one of these NCLs that present in late infancy and is caused by mutations in the CLN7/MFSD8 gene, which encodes a lysosomal membrane glycoprotein of unknown function, hence the biochemical processes affected by CLN7-loss of function are not understood. Here, we found in the Cln7Δex2 mouse model of CLN7 disease that failure in the autophagy-lysosomal pathway causes aberrant accumulation of reactive oxygen species (ROS)-producing brain mitochondria. Metabolic profile analysis of Cln7Δex2 neurons revealed a decrease in the basal oxygen consumption rate (OCR), ATP-linked and maximal OCR and proton leak, indicating bioenergetically impaired mitochondria. To assess the impact of ROS on CLN7 disease progression, Cln7Δex2 mice were crossed with mice expressing a mitochondrial-tagged form of catalase (mCAT) governed by a neuron-specific promoter (Cln7Δex2-CAMKIIaCre-mCAT). The increased mROS observed in Cln7Δex2 neurons was abolished in Cln7Δex2- CAMKIIaCre-mCAT neurons, verifying the efficacy of this approach. The brain mitochondrial swelling and mitochondrial cristae profile widening observed in Cln7Δex2 mice were abolished in Cln7Δex2-CAMKIIaCre-mCAT mice. Notably, Cln7Δex2 brain accumulation of subunit C-ATPase and lysosomal lipofuscin, as well as gliosis, which are hallmarks of the disease, were ameliorated in Cln7Δex2- CAMKIIaCre-mCAT mice. Altogether, these findings indicate that the generation of ROS by bioenergetically-impaired mitochondria in Cln7Δex2 neurons contributes to the histopathological symptoms of CLN7 disease.

The neuronal ceroid lipofuscinoses (NCLs) are a family of monogenic life-limiting pediatric neurodegenerative disorders collectively known as Batten disease1. Although genetically heterogeneous2, NCLs share several clinical symptoms and pathological hallmarks such as lysosomal accumulation of lipofuscin and astrogliosis2,3. CLN7 disease belongs to a group of NCLs that present in late infancy4–6 and, whereas CLN7/MFSD8 gene is known to encode a lysosomal membrane glycoprotein4,7–9, the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments1,10. Here, we found in the Cln7Δex2 mouse model11 of CLN7 disease that failure in the autophagy-lysosomal pathway causes accumulation of structurally and bioenergetically impaired, reactive oxygen species (ROS)-producing neuronal mitochondria that contribute to CLN7 pathogenesis. Cln7Δex2 neurons exhibit a metabolic shift mediated by pro-glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3). PFKFB3 inhibition in Cln7Δex2 mice in vivo and in CLN7 patients-derived cells rectified key disease hallmarks. Thus, specifically targeting glycolysis may alleviate CLN7 pathogenesis.

Voltage-gated potassium (Kv) channels of the Kv4 subfamily associate with Kv channel–interacting proteins (KChIPs), which leads to enhanced surface expression and shapes the inactivation gating of these channels. KChIP3 has been reported to also interact with the late endosomal/lysosomal membrane glycoprotein CLN3 (ceroid lipofuscinosis neuronal 3), which is modified because of gene mutation in juvenile neuronal ceroid lipofuscinosis (JNCL). The present study was undertaken to find out whether and how CLN3, by its interaction with KChIP3, may indirectly modulate Kv4.2 channel expression and function. To this end, we expressed KChIP3 and CLN3, either individually or simultaneously, together with Kv4.2 in HEK 293 cells. We performed co-immunoprecipitation experiments and found a lower amount of KChIP3 bound to Kv4.2 in the presence of CLN3. In whole-cell patch-clamp experiments, we examined the effects of CLN3 co-expression on the KChIP3-mediated modulation of Kv4.2 channels. Simultaneous co-expression of CLN3 and KChIP3 with Kv4.2 resulted in a suppression of the typical KChIP3-mediated modulation; i.e. we observed less increase in current density, less slowing of macroscopic current decay, less acceleration of recovery from inactivation, and a less positively shifted voltage dependence of steady-state inactivation. The suppression of the KChIP3-mediated modulation of Kv4.2 channels was weaker for the JNCL-related missense mutant CLN3(R334C) and for a JNCL-related C-terminal deletion mutant (CLN3ΔC). Our data support the notion that CLN3 is involved in Kv4.2/KChIP3 somatodendritic A-type channel formation, trafficking, and function, a feature that may be lost in JNCL.

Mutations in the CLN7/MFSD8 gene encoding the lysosomal membrane protein CLN7 are causative of CLN7 disease, an inherited neurodegenerative disorder that typically affects children. To gain insight into the pathomechanisms of CLN7 disease, we established an immortalized cell line based on cerebellar (Cb) granule neuron precursors isolated from Cln7-/- mice. Here, we demonstrate that Cln7-deficient neuron-derived Cb cells display an abnormal phenotype that includes increased size and defective outward movement of late endosomes and lysosomes as well as impaired lysosomal exocytosis. Whereas Cln7-/- Cb cells appeared to be autophagy-competent, loss of Cln7 resulted in enhanced cell death under prolonged nutrient deprivation. Furthermore, reduced cell survival of Cln7-deficient cells was accompanied by a significantly impaired protein kinase B/Akt phosphorylation at Ser473 during long-term starvation. In summary, our data demonstrate for the first time that the putative lysosomal transporter CLN7 is relevant for lysosome motility and plays an important role for neuronal cell survival under conditions of starvation.

Background One of the most important steps taken by Beyond Batten Disease Foundation in our quest to cure juvenile Batten (CLN3) disease is to understand the State of the Science. We believe that a strong understanding of where we are in our experimental understanding of the CLN3 gene, its regulation, gene product, protein structure, tissue distribution, biomarker use, and pathological responses to its deficiency, lays the groundwork for determining therapeutic action plans. Objectives To present an unbiased comprehensive reference tool of the experimental understanding of the CLN3 gene and gene product of the same name. Methods BBDF compiled all of the available CLN3 gene and protein data from biological databases, repositories of federally and privately funded projects, patent and trademark offices, science and technology journals, industrial drug and pipeline reports as well as clinical trial reports and with painstaking precision, validated the information together with experts in Batten disease, lysosomal storage disease, lysosome/endosome biology. Results The finished product is an indexed review of the CLN3 gene and protein which is not limited in page size or number of references, references all available primary experiments, and does not draw conclusions for the reader. Conclusions Revisiting the experimental history of a target gene and its product ensures that inaccuracies and contradictions come to light, long‐held beliefs and assumptions continue to be challenged, and information that was previously deemed inconsequential gets a second look. Compiling the information into one manuscript with all appropriate primary references provides quick clues to which studies have been completed under which conditions and what information has been reported. This compendium does not seek to replace original articles or subtopic reviews but provides an historical roadmap to completed works., The CLN3 gene, its regulation, gene product, CLN3 protein structure, tissue distribution, biomarker use, and pathological responses to its deficiency.

Defects in the MFSD8 gene encoding the lysosomal membrane protein CLN7 lead to CLN7 disease, a neurodegenerative lysosomal storage disorder belonging to the group of neuronal ceroid lipofuscinoses. Here, we have performed a SILAC-based quantitative analysis of the lysosomal proteome using Cln7-deficient mouse embryonic fibroblasts (MEFs) from a Cln7 knockout (ko) mouse model. From 3335 different proteins identified, we detected 56 soluble lysosomal proteins and 29 highly abundant lysosomal membrane proteins. Quantification revealed that the amounts of 12 different soluble lysosomal proteins were significantly reduced in Cln7 ko MEFs compared with wild-type controls. One of the most significantly depleted lysosomal proteins was Cln5 protein that underlies another distinct neuronal ceroid lipofuscinosis disorder. Expression analyses showed that the mRNA expression, biosynthesis, intracellular sorting and proteolytic processing of Cln5 were not affected, whereas the depletion of mature Cln5 protein was due to increased proteolytic degradation by cysteine proteases in Cln7 ko lysosomes. Considering the similar phenotypes of CLN5 and CLN7 patients, our data suggest that depletion of CLN5 may play an important part in the pathogenesis of CLN7 disease. In addition, we found a defect in the ability of Cln7 ko MEFs to adapt to starvation conditions as shown by impaired mammalian target of rapamycin complex 1 reactivation, reduced autolysosome tubulation and increased perinuclear accumulation of autolysosomes compared with controls. In summary, depletion of multiple soluble lysosomal proteins suggest a critical role of CLN7 for lysosomal function, which may contribute to the pathogenesis and progression of CLN7 disease.

Summary: Lysine glutarylation (Kglu) of mitochondrial proteins is associated with glutaryl-CoA dehydrogenase (GCDH) deficiency, which impairs lysine/tryptophan degradation and causes destruction of striatal neurons during catabolic crisis with subsequent movement disability. By investigating the role of Kglu modifications in this disease, we compared the brain and liver glutarylomes of Gcdh-deficient mice. In the brain, we identified 73 Kglu sites on 37 mitochondrial proteins involved in various metabolic degradation pathways. Ultrastructural immunogold studies indicated that glutarylated proteins are heterogeneously distributed in mitochondria, which are exclusively localized in glial cells. In liver cells, all mitochondria contain Kglu-modified proteins. Glutarylation reduces the catalytic activities of the most abundant glutamate dehydrogenase (GDH) and the brain-specific carbonic anhydrase 5b and interferes with GDH-protein interactions. We propose that Kglu contributes to the functional heterogeneity of mitochondria and may metabolically adapt glial cells to the activity and metabolic demands of neighboring GCDH-deficient neurons. : Schmiesing et al. show that the lack of GCDH results in glutarylation of mitochondrial proteins in glial cells affecting amino acid metabolism and the tricarboxylic acid cycle. They identify glutamate dehydrogenase as a target suppressed by glutarylation that is linked to glial glutamate metabolism and anaplerosis in GCDH-deficient neuronal cells. Keywords: glutaryl-CoA-dehydrogenase, glutaryl-proteome, glutamate dehydrogenase, carboanhydrase 5B, protein-protein interaction, immunogold electron microscopy, glutaric aciduria type 1/brain

Mutations in the major facilitator superfamily domain containing 8 (MFSD8) gene coding for the lysosomal CLN7 membrane protein result in CLN7 disease, a lysosomal storage disease of childhood. CLN7 disease belongs to a group of inherited disorders, called neuronal ceroid lipofuscinoses (NCL), which are characterized by the accumulation of autofluorescent ceroid lipopigments, neuroinflammation, photoreceptor- and neurodegeneration. We have disrupted the Mfsd8 gene by insertion of a lacZ gene-trap cassette between exons 1 and 2 in mice and have analyzed the impact of Cln7 depletion on neuronal and visceral tissues. Analysis of lacZ reporter gene activity in heterozygous Mfsd8((wt/tm1a)) mice showed strong Mfsd8 mRNA expression in the cerebral cortex, in the hippocampus and in the kidney. Homozygous Mfsd8((tm1a/tm1a)) mice were viable and fertile and resembled biochemically the NCL-phenotype of human CLN7 patients including the accumulation of autofluorescent material in the brain and peripheral tissues and of subunit c of mitochondrial ATP synthase in the cerebellum and nuclei of distinct brain regions, and the degeneration of photoreceptor cells in the retina. Lysosomal storage was found in large neurons of the medulla, the hippocampus and in Purkinje cells of the cerebellum in mutant mice. The ultrastructure of the storage material revealed dense lamellar bodies with irregular forms within cerebellar and hippocampal neurons. In the brain loss of Cln7 was accompanied by mild reactive microgliosis and subtle astrogliosis by 10months of age, respectively. In summary we have generated a mouse model which is partly valuable as some but not all neuropathological features of human CLN7 disease are recapitulated thus representing an animal model to study CLN7-specific disease mechanisms.

The Golgi-resident N-acetylglucosamine-1-phosphotransferase (PT) complex is composed of two α-, β-, and γ-subunits and represents the key enzyme for the biosynthesis of mannose 6-phosphate recognition marker on soluble lysosomal proteins. Mutations in the PT complex cause the lysosomal storage diseases mucolipidosis II and III. A prerequisite for the enzymatic activity is the site-1 protease-mediated cleavage of the PT α/β-subunit precursor protein in the Golgi apparatus. Here, we have investigated structural requirements of the PT α/β-subunit precursor protein for its efficient export from the endoplasmic reticulum (ER). Both wild-type and a cleavage-resistant type III membrane PT α/β-subunit precursor protein are exported whereas coexpressed separate α- and β-subunits failed to reach the cis-Golgi compartment. Mutational analyses revealed combinatorial, non-exchangeable dileucine and dibasic motifs located in a defined sequence context in the cytosolic N- and C-terminal domains that are required for efficient ER exit and subsequent proteolytic activation of the α/β-subunit precursor protein in the Golgi. In the presence of a dominant negative Sar1 mutant the ER exit of the PT α/β-subunit precursor protein is inhibited indicating its transport in coat protein complex II-coated vesicles. Expression studies of missense mutations identified in mucolipidosis III patients that alter amino acids in the N- and C-terminal domains demonstrated that the substitution of a lysine residue in close proximity to the dileucine sorting motif impaired ER-Golgi transport and subsequent activation of the PT α/β-subunit precursor protein. The data suggest that the oligomeric type III membrane protein PT complex requires a combinatorial sorting motif that forms a tertiary epitope to be recognized by distinct sites within the coat protein complex II machinery.

CLN7 is a polytopic lysosomal membrane glycoprotein of unknown function and is deficient in variant late infantile neuronal ceroid lipofuscinosis. Here we show that full-length CLN7 is proteolytically cleaved twice, once proximal to the used N-glycosylation sites in lumenal loop L9 and once distal to these sites. Cleavage occurs by cysteine proteases in acidic compartments and disruption of lysosomal targeting of CLN7 results in inhibition of proteolytic cleavage. The apparent molecular masses of the CLN7 fragments suggest that both cleavage sites are located within lumenal loop L9. The known disease-causing mutations, p.T294K and p.P412L, localized in lumenal loops L7 and L9, respectively, did not interfere with correct lysosomal targeting of CLN7 but enhanced its proteolytic cleavage in lysosomes. Incubation of cells with selective cysteine protease inhibitors and expression of CLN7 in gene-targeted mouse embryonic fibroblasts revealed that cathepsin L is required for one of the two proteolytic cleavage events. Our findings suggest that CLN7 is inactivated by proteolytic cleavage and that enhanced CLN7 proteolysis caused by missense mutations in selected luminal loops is associated with disease.

CLN7 is a polytopic lysosomal membrane protein deficient in variant late infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder. In this study fluorescence protease protection assays and mutational analyses revealed the N- and C-terminal tails of CLN7 in the cytosol and two N-glycosylation sites at N371 and N376. Both partially and non-glycosylated CLN7 were correctly transported to lysosomes. To identify lysosomal targeting motifs, we generated CD4-chimera fused to the N- and C-terminal domains of CLN7. Lysosomal localization of the chimeric proteins requires a consensus acidic dileucine-based motif in the N-terminus and two tandem tyrosine-based signals in the C-terminus. Mutation of these sorting motifs resulted in cell surface redistribution of CD4 chimeras. However, the dileucine-based motif is of critical importance for lysosomal localization of the full-length CLN7 in different cell lines. Cell surface biotinylation revealed that at equilibrium 22% of total CLN7 is localized at the plasma membrane. Mutation of the dileucine motif or the co-expression of dominant-negative mutant dynamin K44A led to a further increase of CLN7 at the plasma membrane. Our data demonstrate that CLN7 contains several cytoplasmic lysosomal targeting signals of which the N-terminal dileucine-based motif is required for the predominant lysosomal targeting along the indirect pathway and clathrin-mediated endocytosis of CLN7.

Lysosomes contain more than 50 soluble hydrolases that are targeted to lysosomes in a mannose 6-phosphate (Man6P)-dependent manner. The phosphorylation of man- nose residues on high mannose-type oligosaccharides of newly synthesized lysosomal enzymes is catalyzed by two multimeric enzymes, GlcNAc-1-phosphotransferase and GlcNAc-1-phosphodiester-α-N-acetylglucosaminidase, allowing the binding to two distinct Man6P receptors in the Golgi apparatus. Inherited defects in the GlcNAc-1-phosphotransferase complex result in missorting and cellular loss of lysosomal enzymes, and the subsequent lysosomal dysfunction causes the lysosomal storage disorders mucolipidosis types II and III. Biosynthetic studies and the availability of Man6P receptor-deficient mouse models have provided new insights into the structural requirements for preferential binding of subsets of lysosomal enzymes to Man6P receptors as well as the identification of alternative targeting pathways.

The neuronal ceroid lipofuscinoses (NCLs) form a group of autosomal recessively inherited neurodegenerative disorders that mainly affect children. Ten NCL forms can be distinguished by age at onset, clinicopathologic features, and genetics. In eight of these forms, the underlying genes have been identified. At present, approximately 10% of all patients do not fall into one of the eight known genetic forms of NCL. We have identified two Asian families with two novel homozygous mutations in the CLN5 gene. In the first Pakistani family, two children developed symptoms of an early juvenile NCL. After exclusion of mutations in genes known to be associated with this age of onset in families from many different countries (CLN1, CLN2, CLN3, CLN6, CLN8 and CLN10) SNP array-based homozygosity mapping led to the identification of a novel homozygous mutation c.1072_1073delTT (p.Leu358AlafsX4) in CLN5. In the second Afghan family, two children developed symptoms of a late infantile NCL. The mutation c.1137G>T (p.Trp379Cys) in CLN5 was identified. The affected children in these families represent the first reported CLN5 patients originating in Asian sibships. Expression analysis showed that mutant p.Leu358AlafsX4 CLN5 is truncated and lacks a used N-glycosylation site at Asn401. The missense mutation p.Trp379Cys affected neither the size nor glycosylation of the CLN5 protein. Double immunofluorescence microscopy showed that while the wild-type CLN5 protein is localized in lysosomes, both mutant CLN5 proteins are retained in the endoplasmic reticulum rather than reaching the lysosome.

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a neurodegenerative disorder caused by defective function of the lysosomal membrane glycoprotein CLN3. The activity of the lysosomal acid phosphatase (LAP/ACP2) was found to be significantly increased in the cerebellum and brain stem of Cln3-targeted mice during the early stages of postnatal life. Histochemical localization studies revealed an increased LAP/ACP2 staining intensity in neurons of the cerebral cortex of 48-week-old Cln3-targeted mice as compared with controls. Additionally, the expression of another lysosomal membrane protein LAMP-2 was increased in all brain areas. Knockdown of CLN3 expression in HeLa cells by RNA interference also resulted in increased LAP/ACP2 and LAMP-2 expression. Finally in fibroblasts of two juvenile neuronal ceroid lipofuscinosis patients elevated levels of LAP/ACP2 were found. Both activation of gene transcription and increased protein half-life appear to contribute to increased LAP/ACP2 protein expression in CLN3-deficient cells. The data suggest that lysosomal dysfunction and accumulation of storage material require increased biogenesis of LAP/ACP2 and LAMP-2 positive membranes which makes LAP/ACP2 suitable as biomarker of Batten disease.

Mutations in the polytopic lysosomal membrane glycoprotein CLN3 result in a severe neurodegenerative disorder. Previous studies identified two cytosolic signal structures contributing to lysosomal targeting. We now examined the role of glycosylation and the C-terminal CAAX motif in lysosomal transport of CLN3 in non-neuronal and neuronal cells. Mutational analysis revealed that in COS7 cells, CLN3 is glycosylated at asparagine residues 71 and 85. Both partially and non-glycosylated CLN3 were transported correctly to lysosomes. Mevalonate incorporation and farnesyltransferase inhibitor studies indicate that CLN3 is prenylated most likely at cysteine 435. Substitution of cysteine 435 reduced the steady-state level of CLN3 in lysosomes most likely because of impaired sorting in early endosomal structures, particularly in neuronal cells. Additionally, the cell surface expression of CLN3 was increased in the presence of farnesyltransferase inhibitors. Alteration of the spacing between the transmembrane domain and the CAAX motif or the substitution of the entire C-terminal domain of CLN3 with cytoplasmic tails of mannose 6-phosphate receptors have demonstrated the importance of the C-terminal domain of proper length and composition for exit of the endoplasmic reticulum. The data suggest that co-operative signal structures in different cytoplasmic domains of CLN3 are required for efficient sorting and for transport to the lysosome.

CLN7 disease is an autosomal recessive, childhood-onset neurodegenerative lysosomal storage disorder caused by the defective lysosomal membrane protein CLN7. We have disrupted the Cln7/Mfsd8 gene in mice by targeted deletion of exon 2 generating a novel knockout (KO) mouse model for CLN7 disease, which recapitulates key features of human CLN7 disease pathology. Cln7 KO mice showed increased mortality and a neurological phenotype including hind limb clasping and myoclonus. Lysosomal dysfunction in the brain of mutant mice was shown by the storage of autofluorescent lipofuscin-like lipopigments, subunit c of mitochondrial ATP synthase and saposin D and increased expression of lysosomal cathepsins B, D and Z. By immunohistochemical co-stainings, increased cathepsin Z expression restricted to Cln7-deficient microglia and neurons was found. Ultrastructural analyses revealed large storage bodies in Purkinje cells of Cln7 KO mice containing inclusions composed of irregular, curvilinear and rectilinear profiles as well as fingerprint profiles. Generalized astrogliosis and microgliosis in the brain preceded neurodegeneration in the olfactory bulb, cerebral cortex and cerebellum in Cln7 KO mice. Increased levels of LC3-II and the presence of neuronal p62- and ubiquitin-positive protein aggregates suggested that impaired autophagy represents a major pathomechanism in the brain of Cln7 KO mice. The data suggest that loss of the putative lysosomal transporter Cln7 in the brain leads to lysosomal dysfunction, impaired constitutive autophagy and neurodegeneration late in disease.

Mucolipidosis II (ML II) is a fatal lysosomal storage disorder resulting from defects in the multimeric GlcNAc-1-phosphotransferase responsible for the initial step in the generation of the mannose 6-phosphate (M6P) recognition marker. M6P residues on oligosaccharides of newly synthesized lysosomal enzymes are essential for efficient receptor-mediated transport to lysosomes. We used the recombinant GlcNAc-1-phosphotransferase gamma subunit as an affinity matrix to purify an unknown protein identified as the product of GNPTA (encoding GNPTA, previously known as MGC4170). The cDNA encodes a protein of 1,256 amino acids with two putative transmembrane domains and a complex preserved modular structure comprising at least six domains. The N-terminal domain of GNPTA, interrupted by a long insertion, shows similarities to bacterial capsule biosynthesis proteins. We identified seven mutations in GNPTA that lead to premature translational termination in six individuals with ML II. Retroviral transduction of fibroblasts from an individual with ML II resulted in the expression and localization of GNPTA in the Golgi apparatus, accompanied by the correction of hypersecretion of lysosomal enzymes. Our results provide evidence that GNPTA encodes a subunit of GlcNAc-1-phosphotransferase defective in individuals with ML II.

The juvenile form of ceroid lipofuscinosis (Batten disease) is a neurodegenerative lysosomal storage disorder caused by mutations in the CLN3 gene. CLN3 encodes a multimembrane-spanning protein of unknown function, which is mainly localized in lysosomes in non-neuronal cells and in endosomes in neuronal cells. For this study we constructed chimeric proteins of three CLN3 cytoplasmic domains fused to the lumenal and transmembrane domains of the reporter proteins LAMP-1 and lysosomal acid phosphatase to identify lysosomal targeting motifs and to determine the intracellular transport and subcellular localization of the chimera in transfected cell lines. We report that a novel type of dileucine-based sorting motif, EEEX(8)LI, present in the second cytoplasmic domain of CLN3, is sufficient for proper targeting to lysosomes. The first cytoplasmic domain of CLN3 and the mutation of the dileucine motif resulted in a partial missorting of chimeric proteins to the plasma membrane. At equilibrium, 4-13% of the different chimera are present at the cell surface. Analysis of lysosome-specific proteolytic processing revealed that lysosomal acid phosphatase chimera containing the second cytoplasmic domain of CLN3 showed the highest rate of lysosomal delivery, whereas the C terminus of CLN3 was found to be less efficient in lysosomal targeting. However, none of these cytosolic CLN3 domains was able to interact with AP-1, AP-3, or GGA3 adaptor complexes. These data revealed that lysosomal sorting motifs located in an intramolecular cytoplasmic domain of a multimembrane-spanning protein have different structural requirements for adaptor binding than sorting signals found in the C-terminal cytoplasmic domains of single- or dual-spanning lysosomal membrane proteins.

In the circulation, most of the insulin-like growth factors (IGFs) are bound to a ternary 150 kDa complex with IGF-binding protein (IGFBP)-3 and the acid labile subunit. In the current study, we identify transferrin (Tf) by mass spectrometry, and immunoprecipitation as a component of a major IGF-binding fraction separated from human plasma. IGF ligand blotting, cross-linkage experiments and surface plasmon resonance spectrometry have been used to demonstrate the capability of Tf to bind IGFs specifically. In combination with Tf, IGFBP-3 showed a 5-fold higher affinity for IGF-II than IGFBP-3 alone. The data suggest that Tf may play an important role in regulating IGF/IGFBP-3 functions.

Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha-ras and c-myc, in transfected Rat1 fibroblasts. While c-myc overexpression did not modify clusterin gene activity, the Ha-ras oncogene produced a seven to tenfold repression of clusterin mRNA; this down-regulation was also observed in the presence of c-myc. Since no induction of the clusterin gene was observed by the two oncogenes, we tested the alternative mechanism involving apoptosis. Growth factor withdrawal induced apoptosis, as shown by DNA degradation and micronuclei formation in the floating cells. Concomittantly we observed a three to tenfold increase in the amount of clusterin mRNA in the adhering cells of Rat1 and the c-myc transformed cell lines, and a weaker induction in the Ha-ras transformed cell line. On the basis of our results, we suggest that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells. We conclude that apoptotic processes, not oncogenic mutations, are responsible for increased clusterin expression in tumors.

Mutations in the CLN3 gene lead to juvenile neuronal ceroid lipofuscinosis, a pediatric neurodegenerative disorder characterized by visual loss, epilepsy and psychomotor deterioration. Although most CLN3 patients carry the same 1-kb deletion in the CLN3 gene, their disease phenotype can be variable. The aims of this study were to (i) study the clinical phenotype in CLN3 patients with identical genotype, (ii) identify genes that are dysregulated in CLN3 disease regardless of the clinical course that could be useful as biomarkers, and (iii) find modifier genes that affect the progression rate of the disease. A total of 25 CLN3 patients homozygous for the 1-kb deletion were classified into groups with rapid, average or slow disease progression using an established clinical scoring system. Genome-wide expression profiling was performed in eight CLN3 patients with different disease progression and matched controls. The study showed high phenotype variability in CLN3 patients. Five genes were dysregulated in all CLN3 patients and present candidate biomarkers of the disease. Of those, dual specificity phosphatase 2 (DUSP2) was also validated in acutely CLN3-depleted cell models and in CbCln3(Δex7/8) cerebellar precursor cells. A total of 13 genes were upregulated in patients with rapid disease progression and downregulated in patients with slow disease progression; one gene showed dysregulation in the opposite way. Among these potential modifier genes, guanine nucleotide exchange factor 1 for small GTPases of the Ras family (RAPGEF1) and transcription factor Spi-B (SPIB) were validated in an acutely CLN3-depleted cell model. These findings indicate that differential perturbations of distinct signaling pathways might alter disease progression and provide insight into the molecular alterations underlying neuronal dysfunction in CLN3 disease and neurodegeneration in general.

Lysosomal hydrolases catalyze the degradation of a variety of macromolecules including proteins, carbohydrates, nucleic acids and lipids. The biogenesis of lysosomes or lysosome-related organelles requires a continuous substitution of soluble acid hydrolases and lysosomal membrane proteins. The targeting of lysosomal hydrolases depends on mannose 6-phosphate residues (M6P) that are recognized by specific receptors mediating their transport to an endosomal/prelysosomal compartment. The key role in the formation of M6P residues plays the GlcNAc-1-phosphotransferase localized in the Golgi apparatus. Two genes have been identified recently encoding the type III alpha/beta-subunit precursor membrane protein and the soluble gamma-subunit of GlcNAc-1-phosphotransferase. Mutations in these genes result in two severe diseases, mucolipidosis type II (MLII) and III (MLIII), biochemically characterized by the missorting of multiple lysosomal hydrolases due to impaired formation of the M6P recognition marker, and general lysosomal dysfunction. This review gives an update on structural properties, localization and functions of the GlcNAc-1-phosphotransferase subunits and improvements of pre- and postnatal diagnosis of ML patients. Further, the generation of recombinant single-chain antibody fragments against M6P residues and of new mouse models of MLII and MLIII will have considerable impact to provide deeper insight into the cell biology of lysosomal dysfunctions and the pathomechanisms underlying these lysosomal disorders. (C) 2009 Elsevier GmbH. All rights reserved

The neuronal ceroid lipofuscinoses (NCLs) form a group of autosomal recessively inherited neurodegenerative disorders that mainly affect children. Ten NCL forms can be distinguished by age at onset, clinicopathologic features, and genetics. In eight of these forms, the underlying genes have been identified. At present, approximately 10% of all patients do not fall into one of the eight known genetic forms of NCL. We have identified two Asian families with two novel homozygous mutations in the CLN5 gene. In the first Pakistani family, two children developed symptoms of an early juvenile NCL. After exclusion of mutations in genes known to be associated with this age of onset in families from many different countries (CLN1, CLN2, CLN3, CLN6, CLN8 and CLN10) SNP array-based homozygosity mapping led to the identification of a novel homozygous mutation c.1072_1073delTT (p.Leu358AlafsX4) in CLN5. In the second Afghan family, two children developed symptoms of a late infantile NCL. The mutation c.1137GT (p.Trp379Cys) in CLN5 was identified. The affected children in these families represent the first reported CLN5 patients originating in Asian sibships. Expression analysis showed that mutant p.Leu358AlafsX4 CLN5 is truncated and lacks a used N-glycosylation site at Asn401. The missense mutation p.Trp379Cys affected neither the size nor glycosylation of the CLN5 protein. Double immunofluorescence microscopy showed that while the wild-type CLN5 protein is localized in lysosomes, both mutant CLN5 proteins are retained in the endoplasmic reticulum rather than reaching the lysosome.

Insulin-like growth factors (IGFs), IGF receptors and IGF binding proteins (IGFBPs) participate in the regulation of proliferation and differentiation of epithelial cells. Expression of the growth-inhibitory murine IGFBP-6 in epithelial Madin–Darby canine kidney (MDCK) cells followed by 2D analysis revealed the presence of multiple isoforms. Metabolic labelling experiments showed that several IGFBP-6 isoforms are modified by phosphate and sulfate groups. Expression analysis of mutant IGFBP-6 further demonstrated that serine residue 143 is O-glycosylated. Substitution of serine 143 by alanine did slightly reduce the preferential sorting of mIGFBP-6 to the apical site in MDCK cells grown on semipermeable filters. Both the presence of multiple and heterogeneously modified isoforms of murine IGFBP-6 in MDCK cells, and the preferential secretion of non-glycosylated IGFBP-6 mutants to the apical side suggest that the major apical sorting signal is the protein moiety.

Modification of the carbohydrate chains of soluble lysosomal enzymes with mannose 6-phosphate residues is a prerequisite for their mannose 6-phosphate receptor-dependent transport to lysosomes. GlcNac-1-phosphotransferase localized in the Golgi apparatus represents a hexameric alpha(2)beta(2)gamma(2) subunit complex and plays a key role in the formation of the mannose 6-phosphate recognition marker. Defects in the GlcNac-1-phosphotransferase complex cause two diseases, mucolipidosis type II and III, which are characterized by missorting and cellular loss of lysosomal enzymes, and lysosomal accumulation of storage material. The recent identification of two genes, GNPTAB and GNPTG, encoding the three subunits of GlcNac-1-phosphotransferase leads to an improvement of both pre- and postnatal diagnosis of affected individuals, and permits the analysis of structural requirements for efficient formation of mannose 6-phosphate residues on lysosomal enzymes. The alpha/beta subunits precursor matures by proteolytic cleavage and contains the catalytic activity as well as the capability to recognize lysosomal enzymes. The role of the gamma-subunits for activity, stability and oligomerization of the GlcNac-1-phosphotransferase subunits is still unclear.

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.

More than 50 acid hydrolases involved in the ordered lysosomal degradation of a variety of proteins, lipids, carbohydrates, and nucleic acids have been identified. The hydrolases are enclosed by a membrane containing a set of highly glycosylated lysosomal membrane proteins. Lysosomal enzymes are also components of cell type-specific compartments referred to as lysosome-related organelles which include melanosomes, lytic gran-ules, MHC class II compartments, platelet-dense granules, and synaptic-like microvesicles. The biogenesis of new lysosomes or lysosome-related organelles requires a continuous substitu-tion with newly synthesized components. The targeting of acid hydrolases depends on the presence of mannose 6-phosphate (M6P) residues that are recognized by specific receptors mediating the intracellular transport to an endosomal/ prelysosomal compartment. The acidi-fication of endosomes, lysosomes, and lysosome-related organelles facilitates not only the dis-sociation of the receptor-ligand complexes, but also the proteolytic processing required for the enzymatic activation of several hydrolases as well as the denaturation of proteins as prerequisite for lysosomal proteolysis.

Mucopolysaccharidosis type IIIA (MPSIIIA) is an autosomal recessive lysosomal storage disease caused by mutations in the N-sulfoglucosamine sulfohydrolase gene (SGSH; encoding sulfamidase, also sulphamidase) leading to the lysosomal accumulation and urinary excretion of heparan sulfate. Considerable variation in the onset and severity of the clinical phenotype is observed. We report here on expression studies of four novel mutations: c.318C>A (p.Ser106Arg), c.488T>C (p.Leu163Pro), c.571G>A (p.Gly191Arg), and c.1207_1209delTAC (p.Tyr403del), and five previously known mutations: c.220C>T (p.Arg74Cys), c.697C>T (p.Arg233X), c.1297C>T (p.Arg433Trp), c.1026dupC (p.Leu343fsX158), and c.1135delG (p.Val379fsX33) identified in MPSIIIA patients. Transient expression of mutant sulfamidases in BHK or CHO cells revealed that all the mutants were enzymatically inactive with the exception of c.318C>A (p.Ser106Arg), which showed 3.3% activity of the expressed wild-type enzyme. Western blot analysis demonstrated that the amounts of expressed mutant sulfamidases were significantly reduced compared with cells expressing wild type. No polypeptides were immunodetectable in extracts of cells transfected with the cDNA carrying the c.697C>T (p.Arg233X) nonsense mutation. In vitro translation and pulse-chase experiments showed that rapid degradation rather than a decrease in synthesis is responsible for the low, steady-state level of the mutant proteins in cells. The amounts of secreted mutant precursor forms, the cellular stability, the proteolytic processing, and data from double-label immunofluorescence microscopy suggest that the degradation of the majority of newly synthesized c.220C>T (p.Arg74Cys), c.571G>A (p.Gly191Arg), c.1297C>T (p.Arg433Trp), c.1026dupC (p.Leu343fsX158), and c.1135delG (p.Val379fsX33) mutant proteins probably occurs in the ER, whereas c.488T>C (p.Leu163Pro) mutant protein showed instability in the lysosomes.

Variant late infantile neuronal ceroid lipofuscinosis, a lysosomal storage disorder characterized by progressive mental deterioration and blindness, is caused by mutations in a polytopic membrane protein (CLN6) with unknown intracellular localization and function. In this study, transient transfection of BHK21 cells with CLN6 cDNA and immunoblot analysis using peptide-specific CLN6 antibodies demonstrated the expression of a approximately 27-kDa protein that does not undergo proteolytic processing. Cross-linking experiments revealed the presence of CLN6 dimers. Using double immunofluorescence microscopy, epitope-tagged CLN6 was shown to be retained in the endoplasmic reticulum (ER) with no colocalization with the cis-Golgi or lysosomal markers. The translocation into the ER and proper folding were confirmed by the N-linked glycosylation of a mutant CLN6 polypeptide. Pulse-chase labeling of fibroblasts from CLN6 patients and from sheep (OCL6) and mouse (nclf) models of the disease followed by immunoprecipitation of cathepsin D indicated that neither the synthesis, sorting nor the proteolytic processing of this lysosomal enzyme was affected in CLN6-defective cells. However, the degradation of the endocytosed index protein arylsulfatase A was strongly reduced in all of the mutant CLN6 cell lines compared with controls. These data suggest that defects in the ER-resident CLN6 protein lead to lysosomal dysfunctions, which may result in lysosomal accumulation of storage material.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by the deficiency of the lysosomal tripeptidyl peptidase-I encoded by CLN2. We previously detected in two LINCL patients a homozygous missense mutation, p.Asn286Ser, that affects a potential N-glycosylation site. We introduced the p.Asn286Ser mutation into the wild-type CLN2 cDNA and performed transient expression analysis to determine the effect on the catalytic activity, intracellular targeting, and glycosylation of the CLN2 protein. Expression of mutant p.Asn286Ser CLN2 in HEK293 cells revealed that the mutant was enzymatically inactive. Western blot analysis demonstrated that at steady state the amounts of expressed p.Asn286Ser CLN2 were reduced compared with wild-type expressing cells. The rate of synthesis and the sorting of the newly synthesized p.Asn286Ser CLN2 in the Golgi was not affected compared with wild-type CLN2 protein. The electrophoretic mobility of the immunoprecipitated mutant p.Asn286Ser CLN2 was increased by approximately 2 kDa compared with the wild-type CLN2 protein, whereas deglycosylation led to the generation of polypeptides of the same apparent size. The data suggest that mutant p.Asn286Ser CLN2 lacks one oligosaccharide chain resulting in enzymatic inactivation.

The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynelä, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19, 2786—2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme.

The interaction of adaptor protein (AP) complexes with signal structures in the cytoplasmic domains of membrane proteins is required for intracellular sorting. Tyrosine- or dileucine-based motifs have been reported to bind to medium chain subunits (mu) of AP-1, AP-2, or AP-3. In the present study, we have examined the interaction of the entire 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR46-CT) containing tyrosine- as well as dileucine-based motifs with mu2 and mu3A chains using the yeast two-hybrid system. Both mu2 and mu3A bind specifically to the MPR46-CT. In contrast, mu3A fails to bind to the cytoplasmic domain of the 300-kDa mannose 6-phosphate receptor. Mutational analysis of the MPR46-CT revealed that the tyrosine-based motif and distal sequences rich in acidic amino acid residues are sufficient for effective binding to mu2. However, the dileucine motif was found to be one part of a consecutive complex C-terminal structure comprising tyrosine and dileucine motifs as well as clusters of acidic residues necessary for efficient binding of mu3A. Alanine substitution of 2 or 4 acidic amino acid residues of this cluster reduces the binding to mu3A much more than to mu2. The data suggest that the MPR46 is capable of interacting with different AP complexes using multiple partially overlapping sorting signals, which might depend on posttranslational modifications or subcellular localization of the receptor.

Purpose Neuronal ceroid lipofuscinoses comprise a genetically heterogeneous group of mainly childhood-onset neurodegenerative lysosomal storage disorders. Progressive loss of vision is among the typical clinical symptoms of these fatal disorders. Here, we performed a detailed analysis of retinal degeneration in mice deficient in the lysosomal membrane protein CLN7, a novel animal model of CLN7 disease. Methods Immunohistochemical analyses of retinas at different ages were performed to qualitatively and quantitatively characterize retinal degeneration in CLN7-deficient mice. Storage material in mutant retinas was analyzed by electron microscopy, and expression levels of various lysosomal proteins were studied using immunohistochemistry, immunoblot analyses, and quantitative real-time PCR. Results We observed an early onset and rapidly progressing degeneration of photoreceptor cells in CLN7-deficient mice, resulting in the loss of more than 70% rod photoreceptors in 4-month-old animals. The number of cone photoreceptors was not detectably altered at this age. Loss of rod photoreceptors was accompanied by reactive astrogliosis and microgliosis. Immunohistochemical and immunoblot analyses revealed accumulation of subunit c of mitochondrial ATP synthase and saposin D in mutant retinas, and electron microscopic analyses demonstrated the presence of curvilinear bodies or fingerprint-like profiles in various cell types of CLN7-deficient retinas. We also found a marked dysregulation of various lysosomal proteins in mutant retinas. Conclusions We conclude that the retina of CLN7-deficient mice represents a useful model to elucidate the pathomechanisms ultimately leading to neurodegeneration in CLN7 disease, and to evaluate the efficacy of strategies aimed at developing treatments for this fatal neurodegenerative lysosomal storage disorder.