Synergistic effects of treating the spinal cord and brain in CLN1 disease.

Infantile neuronal ceroid lipofuscinosis (INCL, or CLN1 disease) is an inherited neurodegenerative storage disorder caused by a deficiency of the lysosomal enzyme palmitoyl protein thioesterase 1 (PPT1). It was widely believed that the pathology associated with INCL was limited to the brain, but we have now found unexpectedly profound pathology in the human INCL spinal cord. Similar pathological changes also occur at every level of the spinal cord of PPT1-deficient (Ppt1-/- ) mice before the onset of neuropathology in the brain. Various forebrain-directed gene therapy approaches have only had limited success in Ppt1-/- mice. Targeting the spinal cord via intrathecal administration of an adeno-associated virus (AAV) gene transfer vector significantly prevented pathology and produced significant improvements in life span and motor function in Ppt1-/- mice. Surprisingly, forebrain-directed gene therapy resulted in essentially no PPT1 activity in the spinal cord, and vice versa. This leads to a reciprocal pattern of histological correction in the respective tissues when comparing intracranial with intrathecal injections. However, the characteristic pathological features of INCL were almost completely absent in both the brain and spinal cord when intracranial and intrathecal injections of the same AAV vector were combined. Targeting both the brain and spinal cord also produced dramatic and synergistic improvements in motor function with an unprecedented increase in life span. These data show that spinal cord pathology significantly contributes to the clinical progression of INCL and can be effectively targeted therapeutically. This has important implications for the delivery of therapies in INCL, and potentially in other similar disorders.

Proteomic Profiling in the Brain of CLN1 Disease Model Reveals Affected Functional Modules.

Neuronal ceroid lipofuscinoses (NCL) are the most commonly inherited progressive encephalopathies of childhood. Pathologically, they are characterized by endolysosomal storage with different ultrastructural features and biochemical compositions. The molecular mechanisms causing progressive neurodegeneration and common molecular pathways linking expression of different NCL genes are largely unknown. We analyzed proteome alterations in the brains of a mouse model of human infantile CLN1 disease-palmitoyl-protein thioesterase 1 (Ppt1) gene knockout and its wild-type age-matched counterpart at different stages: pre-symptomatic, symptomatic and advanced. For this purpose, we utilized a combination of laser capture microdissection-based quantitative liquid chromatography tandem mass spectrometry (MS) and matrix-assisted laser desorption/ionization time-of-flight MS imaging to quantify/visualize the changes in protein expression in disease-affected brain thalamus and cerebral cortex tissue slices, respectively. Proteomic profiling of the pre-symptomatic stage thalamus revealed alterations mostly in metabolic processes and inhibition of various neuronal functions, i.e., neuritogenesis. Down-regulation in dynamics associated with growth of plasma projections and cellular protrusions was further corroborated by findings from RNA sequencing of CLN1 patients' fibroblasts. Changes detected at the symptomatic stage included: mitochondrial functions, synaptic vesicle transport, myelin proteome and signaling cascades, such as RhoA signaling. Considerable dysregulation of processes related to mitochondrial cell death, RhoA/Huntington's disease signaling and myelin sheath breakdown were observed at the advanced stage of the disease. The identified changes in protein levels were further substantiated by bioinformatics and network approaches, immunohistochemistry on brain tissues and literature knowledge, thus identifying various functional modules affected in the CLN1 childhood encephalopathy.

Quantitative analysis of PPT1 interactome in human neuroblastoma cells.

Mutations in the CLN1 gene that encodes Palmitoyl protein thioesterase 1 (PPT1) or CLN1, cause Infantile NCL (INCL, MIM#256730). PPT1 removes long fatty acid chains such as palmitate from modified cysteine residues of proteins. The data shown here result from isolated protein complexes from PPT1-expressing SH-SY5Y stable cells that were subjected to single step affinity purification coupled to mass spectrometry (AP-MS). Prior to the MS analysis, we utilised a modified filter-aided sample preparation (FASP) protocol. Based on label free quantitative analysis of the data by SAINT, 23 PPT1 interacting partners (IP) were identified. A dense connectivity in PPT1 network was further revealed by functional coupling and extended network analyses, linking it to mitochondrial ATP synthesis coupled protein transport and thioester biosynthetic process. Moreover, the terms: inhibition of organismal death, movement disorders and concentration of lipid were predicted to be altered in the PPT1 network. Data presented here are related to Scifo et al. (J. Proteomics, 123 (2015) 42-53).

Proteomic analysis of the palmitoyl protein thioesterase 1 interactome in SH-SY5Y human neuroblastoma cells.

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited progressive childhood disorders, characterized by early accumulation of autofluorescent storage material in lysosomes of neurons or other cells. Clinical symptoms of NCL include: progressive loss of vision, mental and motor deterioration, epileptic seizures and premature death. CLN1 disease (MIM#256730) is caused by mutations in the CLN1 gene, which encodes palmitoyl protein thioesterase 1 (PPT1). In this study, we utilised single step affinity purification coupled to mass spectrometry (AP-MS) to unravel the in vivo substrates of human PPT1 in the brain neuronal cells. Protein complexes were isolated from human PPT1 expressing SH-SY5Y stable cells, subjected to filter-aided sample preparation (FASP) and analysed on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. A total of 23 PPT1 interacting partners (IP) were identified from label free quantitation of the MS data by SAINT platform. Three of the identified PPT1 IP, namely CRMP1, DBH, and MAP1B are predicted to be palmitoylated. Our proteomic analysis confirmed previously suggested roles of PPT1 in axon guidance and lipid metabolism, yet implicates the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway. BIOLOGICAL SIGNIFICANCE: The significance of this work lies in the unravelling of putative in vivo substrates of human CLN1 or PPT1 in brain neuronal cells. Moreover, the PPT1 IP implicate the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway.

Gene expression alterations in the cerebellum and granule neurons of Cstb(-/-) mouse are associated with early synaptic changes and inflammation.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited neurodegenerative disease, manifesting with myoclonus, seizures and ataxia, caused by mutations in the cystatin B (CSTB) gene. With the aim of understanding the molecular basis of pathogenetic events in EPM1 we characterized gene expression changes in the cerebella of pre-symptomatic postnatal day 7 (P7) and symptomatic P30 cystatin B -deficient (Cstb(-/-) ) mice, a model for the disease, and in cultured Cstb(-/-) cerebellar granule cells using a pathway-based approach. Differentially expressed genes in P7 cerebella were connected to synaptic function and plasticity, and in cultured cerebellar granule cells, to cell cycle, cytoskeleton, and intracellular transport. In particular, the gene expression data pinpointed alterations in GABAergic pathway. Electrophysiological recordings from Cstb(-/-) cerebellar Purkinje cells revealed a shift of the balance towards decreased inhibition, yet the amount of inhibitory interneurons was not declined in young animals. Instead, we found diminished number of GABAergic terminals and reduced ligand binding to GABAA receptors in Cstb(-/-) cerebellum. These results suggest that alterations in GABAergic signaling could result in reduced inhibition in Cstb(-/-) cerebellum leading to the hyperexcitable phenotype of Cstb(-/-) mice. At P30, the microarray data revealed a marked upregulation of immune and defense response genes, compatible with the previously reported early glial activation that precedes neuronal degeneration. This further implies the role of early-onset neuroinflammation in the pathogenesis of EPM1.

Potential role of subunit c of F0F1-ATPase and subunit c of storage body in the mitochondrial permeability transition. Effect of the phosphorylation status of subunit c on pore opening.

Phosphorylated and non-phosphorylated forms of the F0F1-ATPase subunit c from rat liver mitochondria (RLM) were purified and their effect on the opening of the permeability transition pore (mPTP) was investigated. Addition of dephosphorylated subunit c to RLM induced mitochondrial swelling, decreased the membrane potential and reduced the Ca2+ uptake capacity, which was prevented by cyclosporin A. The same effect was observed in the presence of storage subunit c purified from livers of sheep affected with ceroid lipofuscinosis. In black-lipid bilayer membranes subunit c increased the conductance due to formation of single channels with fast and slow kinetics. The dephosphorylated subunit c formed channels with slow kinetics, i.e. the open state being of significantly longer duration than in the case of channels formed by the phosphorylated form that had short life spans and fast kinetics. The channels formed were cation-selective more so with the phosphorylated form. Subunit c of rat liver mitochondria was able to bind Ca2+. Collectively, the data allowed us to suppose that subunit c F0F1-ATPase might be a structural/regulatory component of mPTP exerting its role in dependence on phosphorylation status.

NCL disease mechanisms.

Despite the identification of a large number of disease-causing genes in recent years, it is still unclear what disease mechanisms operate in the neuronal ceroid lipofuscinoses (NCLs, Batten disease). As a group they are defined by the specific accumulation of protein, either subunit c of mitochondrial ATP synthase or SAPs A and D in lysosome-derived organelles, and regionally specific neurodegeneration. Evidence from biochemical and cell biology studies indicates related lesions in intracellular vesicle trafficking and lysosomal function. There is also extensive immunohistological evidence of a causative role of disease associated neuroinflammation. However the nature of these lesions is not clear nor is it clear why they lead to the defining pathology. Several different theories have proposed a range of potential mechanisms, but it remains to be determined which are central to pathogenesis, and whether there is a mechanism consistent across the group, or if it differs between disease forms. This review summarises the evidence that is currently available and the progress that has been made in understanding these profoundly disabling disorders. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.

Drafting the CLN3 protein interactome in SH-SY5Y human neuroblastoma cells: a label-free quantitative proteomics approach.

Neuronal ceroid lipofuscinoses (NCL) are the most common inherited progressive encephalopathies of childhood. One of the most prevalent forms of NCL, Juvenile neuronal ceroid lipofuscinosis (JNCL) or CLN3 disease (OMIM: 204200), is caused by mutations in the CLN3 gene on chromosome 16p12.1. Despite progress in the NCL field, the primary function of ceroid-lipofuscinosis neuronal protein 3 (CLN3) remains elusive. In this study, we aimed to clarify the role of human CLN3 in the brain by identifying CLN3-associated proteins using a Tandem Affinity Purification coupled to Mass Spectrometry (TAP-MS) strategy combined with Significance Analysis of Interactome (SAINT). Human SH-SY5Y-NTAP-CLN3 stable cells were used to isolate native protein complexes for subsequent TAP-MS. Bioinformatic analyses of isolated complexes yielded 58 CLN3 interacting partners (IP) including 42 novel CLN3 IP, as well as 16 CLN3 high confidence interacting partners (HCIP) previously identified in another high-throughput study by Behrends et al., 2010. Moreover, 31 IP of ceroid-lipofuscinosis neuronal protein 5 (CLN5) were identified (18 of which were in common with the CLN3 bait). Our findings support previously suggested involvement of CLN3 in transmembrane transport, lipid homeostasis and neuronal excitability, as well as link it to G-protein signaling and protein folding/sorting in the ER.

Cell biology and function of neuronal ceroid lipofuscinosis-related proteins.

Neuronal ceroid lipofuscinoses (NCL) comprise a group of inherited lysosomal disorders with variable age of onset, characterized by lysosomal accumulation of autofluorescent ceroid lipopigments, neuroinflammation, photoreceptor- and neurodegeneration. Most of the NCL-related genes encode soluble and transmembrane proteins which localize to the endoplasmic reticulum or to the endosomal/lysosomal compartment and directly or indirectly regulate lysosomal function. Recently, exome sequencing led to the identification of four novel gene defects in NCL patients and a new NCL nomenclature currently comprising CLN1 through CLN14. Although the precise function of most of the NCL proteins remains elusive, comprehensive analyses of model organisms, particularly mouse models, provided new insight into pathogenic mechanisms of NCL diseases and roles of mutant NCL proteins in cellular/subcellular protein and lipid homeostasis, as well as their adaptive/compensatorial regulation at the transcriptional level. This review summarizes the current knowledge on the expression, function and regulation of NCL proteins and their impact on lysosomal integrity. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.

Exacerbated neuronal ceroid lipofuscinosis phenotype in Cln1/5 double-knockout mice.

Both CLN1 and CLN5 deficiencies lead to severe neurodegenerative diseases of childhood, known as neuronal ceroid lipofuscinoses (NCLs). The broadly similar phenotypes of NCL mouse models, and the potential for interactions between NCL proteins, raise the possibility of shared or convergent disease mechanisms. To begin addressing these issues, we have developed a new mouse model lacking both Cln1 and Cln5 genes. These double-knockout (Cln1/5 dko) mice were fertile, showing a slight decrease in expected Mendelian breeding ratios, as well as impaired embryoid body formation by induced pluripotent stem cells derived from Cln1/5 dko fibroblasts. Typical disease manifestations of the NCLs, i.e. seizures and motor dysfunction, were detected at the age of 3 months, earlier than in either single knockout mouse. Pathological analyses revealed a similar exacerbation and earlier onset of disease in Cln1/5 dko mice, which exhibited a pronounced accumulation of autofluorescent storage material. Cortical demyelination and more pronounced glial activation in cortical and thalamic regions was followed by cortical neuron loss. Alterations in lipid metabolism in Cln1/5 dko showed a specific increase in plasma phospholipid transfer protein (PLTP) activity. Finally, gene expression profiling of Cln1/5 dko cortex revealed defects in myelination and immune response pathways, with a prominent downregulation of α-synuclein in Cln1/5 dko mouse brains. The simultaneous loss of both Cln1 and Cln5 genes might enhance the typical pathological phenotypes of these mice by disrupting or downregulating shared or convergent pathogenic pathways, which could potentially include interactions of CLN1 and CLN5.

Cathepsin D deficiency induces cytoskeletal changes and affects cell migration pathways in the brain.

Cathepsin D deficiency is a fatal neurodegenerative disease characterized by extreme loss of neurons and myelin. Our previous studies have demonstrated that structural and functional alterations in synapses are central to the disease pathogenesis. Therefore, we took a systematic approach to examine the synaptic proteome in cathepsin D knock-out mice, where the synaptic pathology resembles that of human patients. We applied quantitative mass spectrometry analysis on synaptosomal fractions isolated from cathepsin D knock-out and control mice at the age of 24 days. From the approximately 600 identified proteins, 43 were present in different amounts (P<0.05, measured in triple biological replicates) in cathepsin D knock-out mice compared to controls. We connected and bridged these 43 proteins using protein interaction data, and overlaid the network with brain specific gene expression information. Subsequently, we superimposed the network with Gene Ontology, pathway, phenotype and disease involvement, allowing construction of a dynamic, disease-protein centered network and prediction of functional modules. The measured changes in the protein levels, as well as some of the bioinformatically predicted ones, were confirmed by quantitative Western blotting or qualitative immunohistochemistry. This combined approach indicated alterations in distinct cellular entities, previously not associated with the disease, and including microtubule associated cytoskeleton and cell projection organization. Cell spreading and wound healing assays confirmed strongly compromised spatial orientation, associated with changes in distribution of focal adhesions and integrin assembly, in cathepsin D deficient cells. These changes might contribute to commencement of synaptic alterations and neuronal degeneration observed in cathepsin D deficiency.

Morphologic and functional correlates of synaptic pathology in the cathepsin D knockout mouse model of congenital neuronal ceroid lipofuscinosis.

Mutations in the cathepsin D (CTSD) gene cause an aggressive neurodegenerative disease (congenital neuronal ceroid lipofuscinosis) that leads to early death. Recent evidence suggests that presynaptic abnormalities play a major role in the pathogenesis of CTSD deficiencies. To identify the early events that lead to synaptic alterations, we investigated synaptic ultrastructure and function in presymptomatic CTSD knockout (Ctsd) mice. Electron microscopy revealed that there were significantly greater numbers of readily releasable synaptic vesicles present in Ctsd mice than in wild-type control mice as early as postnatal day 16. The size of this synaptic vesicle pool continued to increase with disease progression in the hippocampus and thalamus of the Ctsd mice. Electrophysiology revealed a markedly decreased frequency of miniature excitatory postsynaptic currents (mEPSCs) with no effect on paired-pulse modulation of the evoked excitatory post synaptic potentials in the hippocampus of Ctsd mice. The reduced mEPSCs frequency was observed before the appearance of epilepsy or any morphologic sign of synaptic degeneration. Taken together, these data indicate that CTSD is required for normal synaptic function and that a failure in synaptic trafficking or recycling may bean early and important pathologic mechanism in Ctsd mice; these presynaptic abnormalities may initiate synaptic degeneration in advance of subsequent neuronal loss.

Mutations in DNAJC5, encoding cysteine-string protein alpha, cause autosomal-dominant adult-onset neuronal ceroid lipofuscinosis.

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is characterized by accumulation of autofluorescent storage material in neural tissues and neurodegeneration and has an age of onset in the third decade of life or later. The genetic and molecular basis of the disease has remained unknown for many years. We carried out linkage mapping, gene-expression analysis, exome sequencing, and candidate-gene sequencing in affected individuals from 20 families and/or individuals with simplex cases; we identified in five individuals one of two disease-causing mutations, c.346_348delCTC and c.344T>G, in DNAJC5 encoding cysteine-string protein alpha (CSPα). These mutations-causing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectively-are located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSPα in neuronal cells. The resulting depletion of functional CSPα might cause in parallel the presynaptic dysfunction and the progressive neurodegeneration observed in affected individuals and lysosomal accumulation of misfolded and proteolysis-resistant proteins in the form of characteristic ceroid deposits in neurons. Our work represents an important step in the genetic dissection of a genetically heterogeneous group of ANCLs. It also confirms a neuroprotective role for CSPα in humans and demonstrates the need for detailed investigation of CSPα in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation.

Distinct neuropathologic phenotypes after disrupting the chloride transport proteins ClC-6 or ClC-7/Ostm1.

The proteins ClC-6 and ClC-7 are expressed in the endosomal-lysosomal system. Because Clcn6-deficient mice display some features of neuronal ceroid lipofuscinosis (NCL), CLCN6 may be a candidate gene for novel forms of NCL. Using landmarks of disease progression from NCL mouse models as a guide, we examined neuropathologic alterations in the central nervous system of Clcn6(-/-), Clcn7(-/-), andgl mice. gl mice bear a mutation in Ostm1, the ß-subunit critical for Clcn7 function. Severely affected Clcn7(-/-) and gl mice have remarkably similar neuropathologic phenotypes, with pronounced reactive changes and neuron loss in the thalamocortical system, similar to findings in early-onset forms of NCL. In contrast, Clcn6(-/-) mice display slowly progressive, milder neuropathologic features with very little thalamic involvement or microglial activation. These findings detail for the first time the markedly different neuropathologic consequences of mutations in these two CLC genes. Clcn7(-/-) and gl mice bear a close resemblance to the progressive neuropathologic phenotypes of early onset forms of NCL, whereas the distinct phenotype of Clcn6-deficient mice suggests that this gene could be a candidate for a later-onset form of mild neurologic dysfunction with some NCL-like features.

Lysosomal dysfunction promotes cleavage and neurotoxicity of tau in vivo.

Expansion of the lysosomal system, including cathepsin D upregulation, is an early and prominent finding in Alzheimer's disease brain. Cell culture studies, however, have provided differing perspectives on the lysosomal connection to Alzheimer's disease, including both protective and detrimental influences. We sought to clarify and molecularly define the connection in vivo in a genetically tractable model organism. Cathepsin D is upregulated with age in a Drosophila model of Alzheimer's disease and related tauopathies. Genetic analysis reveals that cathepsin D plays a neuroprotective role because genetic ablation of cathepsin D markedly potentiates tau-induced neurotoxicity. Further, generation of a C-terminally truncated form of tau found in Alzheimer's disease patients is significantly increased in the absence of cathepsin D. We show that truncated tau has markedly increased neurotoxicity, while solubility of truncated tau is decreased. Importantly, the toxicity of truncated tau is not affected by removal of cathepsin D, providing genetic evidence that modulation of neurotoxicity by cathepsin D is mediated through C-terminal cleavage of tau. We demonstrate that removing cathepsin D in adult postmitotic neurons leads to aberrant lysosomal expansion and caspase activation in vivo, suggesting a mechanism for C-terminal truncation of tau. We also demonstrate that both cathepsin D knockout mice and cathepsin D-deficient sheep show abnormal C-terminal truncation of tau and accompanying caspase activation. Thus, caspase cleavage of tau may be a molecular mechanism through which lysosomal dysfunction and neurodegeneration are causally linked in Alzheimer's disease.

Murine cathepsin D deficiency is associated with dysmyelination/myelin disruption and accumulation of cholesteryl esters in the brain.

Cathepsin D (CTSD) deficiencies are fatal neurological diseases that in human infants and in sheep are characterized by extreme loss of neurons and myelin. To date, similar morphological evidence for myelin disruption in CTSD knockout mice has not been reported. Here, we show that CTSD deficiency leads to pronounced myelin changes in the murine brain: myelin-related proteolipid protein and myelin basic protein were both markedly reduced at postnatal day 24, and the amount of lipids characteristically high in myelin (e.g. plasmalogen-derived alkenyl chains and glycosphingolipid-derived 20- and 24-carbon acyl chains) were significantly lowered compared with controls. These changes were accompanied by ultrastructural alterations of myelin, including significant thinning of myelin sheaths. Furthermore, in CTSD knockout brains there was a pronounced accumulation of cholesteryl esters and abnormal levels of proteins related to cholesterol transport, with an increased content of apolipoprotein E and a reduced content of ATP-binding cassette transporter A1. These results provide evidence for dysmyelination and altered trafficking of cholesterol in brains of CTSD knockout mice, and warrant further studies on the role of lipid metabolism in the pathogenesis of CTSD deficiencies.

Cathepsin D expression level affects alpha-synuclein processing, aggregation, and toxicity in vivo.

BACKGROUND: Elevated SNCA gene expression and intracellular accumulation of the encoded alpha-synuclein (aSyn) protein are associated with the development of Parkinson disease (PD). To date, few enzymes have been examined for their ability to degrade aSyn. Here, we explore the effects of CTSD gene expression, which encodes the lysosomal protease cathepsin D (CathD), on aSyn processing. RESULTS: Over-expression of human CTSD cDNA in dopaminergic MES23.5 cell cultures induced the marked proteolysis of exogenously expressed aSyn proteins in a dose-dependent manner. Unexpectedly, brain extractions, Western blotting and ELISA quantification revealed evidence for reduced levels of soluble endogenous aSyn in ctsd knock-out mice. However, these CathD-deficient mice also contained elevated levels of insoluble, oligomeric aSyn species, as detected by formic acid extraction. In accordance, immunohistochemical studies of ctsd-mutant brain from mice, sheep and humans revealed selective synucleinopathy-like changes that varied slightly among the three species. These changes included intracellular aSyn accumulation and formation of ubiquitin-positive inclusions. Furthermore, using an established Drosophila model of human synucleinopathy, we observed markedly enhanced retinal toxicity in ctsd-null flies. CONCLUSION: We conclude from these complementary investigations that: one, CathD can effectively degrade excess aSyn in dopaminergic cells; two, ctsd gene mutations result in a lysosomal storage disorder that includes microscopic and biochemical evidence of aSyn misprocessing; and three, CathD deficiency facilitates aSyn toxicity. We therefore postulate that CathD promotes 'synucleinase' activity, and that enhancing its function may lower aSyn concentrations in vivo.

Novel mutation and the first prenatal screening of cathepsin D deficiency (CLN10).

The neuronal ceroid lipofuscinoses (NCLs) are autosomal recessively inherited disorders collectively considered to be one among the most common pediatric neurodegenerative lysosomal storage diseases. Four main clinical subtypes have been described based on the age at presentation: infantile, late infantile, juvenile and adult types. In addition, rare congenital cases of NCL have been reported in the literature. Previously, a homozygous mutation in the cathepsin D gene has been shown to cause congenital NCL in a patient of Pakistani origin. We report a case of a 39-week estimated gestational age female infant with severe microcephaly and hypertonia, whereas MRI showed generalized hypoplasia of the cerebral and cerebellar hemispheres. The infant died on day two after birth. Postmortem examination revealed a small, firm brain with extensive neuronal loss and gliosis. Remaining neurons, astrocytes and macrophages contained PAS-positive storage material with granular ultrastructure and immunoreactivity against sphingolipid activator protein D. A diagnosis of congenital NCL was rendered with a novel mutation, c.299C > T (p.Ser100Phe) in exon 3 of the cathepsin D gene. In the patient fibroblasts, cathepsin D activity was marginal, but the protein appeared stable and normally processed. This was confirmed in overexpression studies. Importantly, by identification of the mutation in the family, we were able to confirm the first prenatal diagnosis excluding cathepsin D deficiency in the younger sibling of the patient.

Accumulation of bis(monoacylglycero)phosphate and gangliosides in mouse models of neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses comprise a group of inherited severe neurodegenerative lysosomal disorders characterized by lysosomal dysfunction and massive accumulation of fluorescent lipopigments and aggregated proteins. To examine the role of lipids in neurodegenerative processes of these diseases, we analysed phospho- and glycolipids in the brains of ctsd-/- and nclf mice, disease models of cathepsin D and CLN6 deficiency, respectively. Both ctsd-/- and nclf mice exhibited increased levels of GM2 and GM3 gangliosides. Immunohistochemically GM2 and GM3 staining was found preferentially in neurons and glial cells, respectively, of ctsd-/- mice. Of particular note, a 20-fold elevation of the unusual lysophospholipid bis(monoacylglycero)phosphate was specifically detected in the brain of ctsd-/- mice accompanied with sporadic accumulation of unesterified cholesterol in distinct cells. The impaired processing of the sphingolipid activator protein precursor, an in vitro cathepsin D substrate, in the brain of ctsd-/- mice may provide the mechanistic link to the storage of lipids. These studies show for the first time that cathepsin D regulates the lysosomal phospho- and glycosphingolipid metabolism suggesting that defects in the composition, trafficking and/or recycling of membrane components along the late endocytic pathway may be critical for the pathogenesis of early onset neuronal ceroid lipofuscinoses.