Ceroid Lipofuscinosis Research Articles

Phenotypic/Genotypic Profile of Children with Neuronal Ceroid Lipofuscinosis in Southern Brazil

Abstract Introduction Neuronal ceroid lipofuscinoses (CLNs) are a group of lysosomal storage disorders of genetic origin, characterized by progressive neurodegeneration and intracellular accumulation of autofluorescent lipopigment. Thirteen genes related to CLNs are currently described, showing genetic and allelic heterogeneity, most of them with an autosomal recessive pattern. Due to the few descriptions of cases related to CLNs in Brazil, it is necessary to describe the phenotypic and genotypic characteristics of these patients. This study aims to evaluate the genotypic profile and correlate it with the phenotypic characteristics of patients with CLN in a children's hospital. Methods This study was performed as a descriptive cross-sectional study with analysis of medical records, imaging, and laboratory tests of patients who had a confirmed molecular diagnosis of CLN. Results The sample consisted of 11 patients from nine families with different subtypes of CLNs (CLN2, 5, 6, 7, and 8), with CLN2 being the most prevalent in the study. A total of 16 mutation variants were identified in genes associated with the five CLNs described in this study, with typical and atypical clinical phenotypes depending on the subtype and its variants. Conclusion Novel mutations identified in the patients in this study showed phenotypes of rapid and severe progression in the CLN2 patient and similar characteristics in CLN6 and CLN7 patients, as previously described in the literature.

Ceroid Lipofuscinosis in Children: Author's Reply.

Ceroid Lipofuscinosis in Children: Author's Reply.

CLN6's luminal tail-mediated functional interference between CLN6 mutants as a novel pathomechanism for the neuronal ceroid lipofuscinoses.

CLN6 (Ceroid Lipofuscinosis, Neuronal, 6) is a 311-amino acid protein spanning the endoplasmic reticulum membrane. Mutations in CLN6 are linked to CLN6 disease, a hereditary neurodegenerative disorder categorized into the neuronal ceroid lipofuscinoses. CLN6 disease is an autosomal recessive disorder and individuals affected with this disease have two identical (homozygous) or two distinct (compound heterozygous) CLN6 mutant alleles. Little has been known about CLN6's physiological roles and the disease mechanism. We recently found that CLN6 prevents protein aggregate formation, pointing to impaired CLN6's anti-aggregate activity as a cause for the disease. To comprehensively understand the pathomechanism, overall anti-aggregate activity derived from two different CLN6 mutants needs to be investigated, considering patients compound heterozygous for CLN6 alleles. We focused on mutant combinations involving the S132CfsX18 (132fsX) prematurely terminated protein, produced from the most frequent mutation in CLN6. The 132fsX mutant nullified anti-aggregate activity of the P299L CLN6 missense mutant but not of wild-type CLN6. Wild-type CLN6's resistance to the 132fsX mutant was abolished by replacement of amino acids 297-301, including Pro297 and Pro299, with five alanine residues. Given that removal of CLN6's C-terminal fifteen amino acids 297-311 (luminal tail) did not affect the resistance, we suggested that CLN6's luminal tail, when unleashed from Pro297/299-mediated conformational constraints, is improperly positioned by the 132fsX mutant, thereby blocking the induction of anti- aggregate activity. We here reveal a novel mechanism for dissipating CLN6 mutants' residual functions, providing an explanation for the compound heterozygosity-driven pathogenesis.

C subunit of the ATP synthase is an amyloidogenic calcium dependent channel-forming peptide with possible implications in mitochondrial permeability transition

The c subunit is an inner mitochondrial membrane (IMM) protein encoded by three nuclear genes. Best known as an integral part of the F0 complex of the ATP synthase, the c subunit is also present in other cytoplasmic compartments in ceroid lipofuscinoses. Under physiological conditions, this 75 residue-long peptide folds into an α-helical hairpin and forms oligomers spanning the lipid bilayer. In addition to its physiological role, the c subunit has been proposed as a key participant in stress-induced IMM permeabilization by the mechanism of calcium-induced permeability transition. However, the molecular mechanism of the c subunit participation in IMM permeabilization is not completely understood. Here we used fluorescence spectroscopy, atomic force microscopy and black lipid membrane methods to gain insights into the structural and functional properties of unmodified c subunit protein that might make it relevant to mitochondrial toxicity. We discovered that c subunit is an amyloidogenic peptide that can spontaneously fold into β-sheets and self-assemble into fibrils and oligomers in a Ca2+-dependent manner. C subunit oligomers exhibited ion channel activity in lipid membranes. We propose that the toxic effects of c subunit might be linked to its amyloidogenic properties and are driven by mechanisms similar to those of neurodegenerative polypeptides such as Aβ and α-synuclein.

Neuronal Ceroid Lipofuscinoses in Children.

Background:The neuronal ceroid lipofuscinoses (NCL) constitute a group of gray matter neurodegenerative disorders characterized by the accumulation of ceroid lipopigment in lysosomes in neurons and other cell types. There are very few published studies on NCL from India, especially in children.Methods:A retrospective study of confirmed patients of NCL diagnosed over a period of 10 years from January 2019 to December 2019.Results:Fifty children had a definitive diagnosis of NCL based on enzymatic studies or genetic testing using next-generation sequencing. Around 15 children were diagnosed to have CLN-1 (ceroid lipofuscinoses, neuronal-1) based on palmitoyl protein thioesterase-1 deficiency; 24 children were diagnosed with CLN2 (ceroid lipofuscinoses, neuronal-2) based on deficient tripeptidyl-peptidase-1 activity; three patients were diagnosed as CLN6, five patients as CLN7, one case each of CLN8, CLN11, and CLN14 based on genetic testing. Clinical presentation was quite varied and included refractory seizures, developmental delay/regression, and abnormal movements. Visual failure was not common in the present case series. Neuroimaging patterns in different types of NCL were different. All children had a progressive downhill course resulting in death in many over a period of 5–10 years of disease onset.Conclusion:NCL is not uncommon and diagnosis can be suspected based on clinical investigations and neuroimaging findings. Diagnosis can be confirmed by enzymatic assays or genetic testing.

Inflammatory bowel disease in Hermansky-Pudlak syndrome: a retrospective single-centre cohort study.

Knowledge about inflammatory bowel disease (IBD) in patients with Hermansky-Pudlak syndrome (HPS), a rare autosomal recessive disorder characterized by defective biogenesis of lysosome-related organelles, could provide insights into IBD in general. To expand the understanding of IBD in patients with HPS. Retrospective review of records from patients with HPS evaluated at the National Institutes of Health Clinical Center from 1995 to 2019 was conducted. Clinical features of IBD, genotyping results and histologic findings of colectomy specimens were analysed. IBD affected 37 (14.2%; 12 male, 25 female) of 261 patients with HPS. Median age of onset was 17years; range was 1 to 52years. The most common symptoms of HPS IBD were hematochezia, abdominal pain and loose stools. Fistulae or extra-intestinal manifestations developed in 30% or 22%, respectively. Genotyping showed that patients with biallelic variants in HPS1, HPS3, HPS4 or HPS6 were diagnosed with IBD. Six children had very early-onset IBD. Patients with HPS-3 had mild manifestations of IBD. Medical therapy and bowel resection were utilized to treat 73% and 35% of patients with HPS IBD, respectively; 7 of 13 patients receiving anti-tumor necrosis factor alpha therapy had prolonged clinical responses. Active cryptitis, chronic inflammatory changes, granulomas and ceroid lipofuscinosis were histopathologic findings in three colectomy specimens. IBD resembling Crohn's disease affects some patients with HPS; genetic heterogeneity is a feature of HPS IBD. HPS3 is a new gene associated with human IBD. Very early-onset IBD can develop in HPS.

Expanding the Neuroimaging Phenotype of Neuronal Ceroid Lipofuscinoses.

Neuronal ceroid lipofuscinoses are a group of neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigments in neuronal cells. As a result of storage material in the brain and retina, clinical manifestations include speech delay, cognitive dysfunction, motor regression, epilepsy, vision loss, and early death. At present, 14 different ceroid lipofuscinosis (CLN) genes are known. Recently, the FDA approved the use of recombinant human proenzyme of tripeptidyl-peptidase 1 for CLN2 disease, while phase I/IIa clinical trials for gene therapy in CLN3 and CLN6 are ongoing. Early diagnosis is, therefore, key to initiating treatment and arresting disease progression. Neuroimaging features of CLN1, CLN2, CLN3, and CLN5 diseases are well-described, with sparse literature on other subtypes. We aimed to investigate and expand the MR imaging features of genetically proved neuronal ceroid lipofuscinoses subtypes at our institution and also to report the time interval between the age of disease onset and the diagnosis of neuronal ceroid lipofuscinoses. We investigated and analyzed the age of disease onset and neuroimaging findings (signal intensity in periventricular, deep, and subcortical white matter, thalami, basal ganglia, posterior limb of the internal capsule, insular/subinsular regions, and ventral pons; and the presence or absence of supratentorial and/or infratentorial atrophy) of patients with genetically proved neuronal ceroid lipofuscinoses at our institution. This group consisted of 24 patients who underwent 40 brain MR imaging investigations between 1993 and 2019, with a male preponderance (male/female ratio = 15:9). The mean ages of disease onset, first brain MR imaging, and diagnosis of neuronal ceroid lipofuscinoses were 4.70 ± 3.48 years, 6.76 ± 4.49 years, and 7.27 ± 4.78 years, respectively. Findings on initial brain MR imaging included T2/FLAIR hypointensity in the thalami (n = 22); T2/FLAIR hyperintensity in the periventricular and deep white matter (n = 22), posterior limb of the internal capsule (n = 22), ventral pons (n = 19), and insular/subinsular region (n = 18); supratentorial (n = 21) and infratentorial atrophy (n = 20). Eight of 9 patients who had follow-up neuroimaging showed progressive changes. We identified reported classic neuroimaging features in all except 1 patient with neuronal ceroid lipofuscinoses in our study. CLN2, CLN5, and CLN7 diseases showed predominant cerebellar-over-cerebral atrophy. We demonstrate that abnormal signal intensity in the deep white matter, posterior limb of the internal capsule, and ventral pons is more common than previously reported in the literature. We report abnormal signal intensity in the insular/subinsular region for the first time. The difference in the median time from disease onset and diagnosis was 1.5 years.

A CLN6-CLN8 complex recruits lysosomal enzymes at the ER for Golgi transfer.

Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transferred to the Golgi complex by interaction with the Batten disease protein CLN8 (ceroid lipofuscinosis, neuronal, 8). Here we investigated the relationship of this pathway with CLN6, an ER-associated protein of unknown function that is defective in a different Batten disease subtype. Experiments focused on protein interaction and trafficking identified CLN6 as an obligate component of a CLN6-CLN8 complex (herein referred to as EGRESS: ER-to-Golgi relaying of enzymes of the lysosomal system), which recruits lysosomal enzymes at the ER to promote their Golgi transfer. Mutagenesis experiments showed that the second luminal loop of CLN6 is required for the interaction of CLN6 with the enzymes but dispensable for interaction with CLN8. In vitro and in vivo studies showed that CLN6 deficiency results in inefficient ER export of lysosomal enzymes and diminished levels of the enzymes at the lysosome. Mice lacking both CLN6 and CLN8 did not display aggravated pathology compared with the single deficiencies, indicating that the EGRESS complex works as a functional unit. These results identify CLN6 and the EGRESS complex as key players in lysosome biogenesis and shed light on the molecular etiology of Batten disease caused by defects in CLN6.

Outcomes of Umbilical Cord Blood Transplantation in Children with Batten Disease

Introduction Neuronal ceroid lipofuscinoses (NCL, Batten Disease) are a class of lysosomal storage diseases caused by mutations in ceroid lipofuscinoses (CLN) genes. Neurological deterioration is influenced by the CLN subtype, but death typically occurs in childhood. Symptoms include visual impairment, behavioral changes, loss of motor and/or cognitive skills, and seizures. Umbilical cord blood transplant (UCBT) is effective in treating many lysosomal storage disorders but there is very limited experience in BD. Methods We retrospectively studied 6 patients (pt) with BD receiving UCBT between the ages of 4 months and 8.6 years at our center. All received myeloablative conditioning with busulfan, cyclophosphamide and ATG. Cyclosporine and mycophenolate were given for prophylaxis of GVHD. Pts 5 and 6 were concomitantly enrolled on clinical trial (NCT02254863) and also received intra-thecal injection of DUOC-01 cells after hematologic engraftment. Selected patient and donor characteristics are shown in Table 1: Results Patients received UCB grafts matching at 4/6-6/6 loci with a median pre-cryo cell dose of 7.2 (range, 5.1-35.1) x107 cells/kg. All pts engrafted neutrophil and platelets in a median of 25 (range, 12-34) and 39 days (range, 32-105), respectively. Four patients developed Grade 2-4 acute GvHD and 2 developed extensive chronic GvHD. Pt 1, the only patient transplanted after severe neurologic decline with advanced disease, died from progressive BD and aseptic shock 12 months post-UCBT. All others are alive at a median of 3.8 years (range, 0.5-5.5). Pt 2 has normal motor function, is nonverbal and was diagnosed with autism 2 years post UCBT. Her brain MRI is normal to date. Pt 3 is blind, has had occasional seizures, requires educational support and has no MRI evidence of BD. Pts 4 and 5 have age appropriate neurocognition, no seizures, and receive only visual support in school. Both have normal MRIs. It is too early to assess neurologic outcomes in Pt 6 (7 months post-UCBT). Conclusion UCBT is feasible in children with BD. At a median follow up of 3.8 years (range, 0.5-5.5), 5/6 patients are alive and 4/5 are neuro-cognitively stable and one developed new onset seizures post-transplant. All 5 surviving pts have no MRI evidence of disease. With the inherent variability of the different forms of BD, additional pts and continued follow-up is needed to fully understand the effect of UCBT on the natural history of BD.

Outcomes of Umbilical Cord Blood Transplantation in Children with Batten Disease

Introduction Neuronal ceroid lipofuscinoses (NCL, Batten Disease) are a class of lysosomal storage diseases caused by mutations in ceroid lipofuscinoses (CLN) genes. Neurological deterioration is influenced by the CLN subtype, but death typically occurs in childhood. Symptoms include visual impairment, behavioral changes, loss of motor and/or cognitive skills, and seizures. Umbilical cord blood transplant (UCBT) is effective in treating many lysosomal storage disorders but there is very limited experience in BD. Methods We retrospectively studied 6 patients (pt) with BD receiving UCBT between the ages of 4 months and 8.6 years at our center. All received myeloablative conditioning with busulfan, cyclophosphamide and ATG. Cyclosporine and mycophenolate were given for prophylaxis of GVHD. Pts 5 and 6 were concomitantly enrolled on clinical trial (NCT02254863) and also received intra-thecal injection of DUOC-01 cells after hematologic engraftment. Selected patient and donor characteristics are shown in Table 1: Results Patients received UCB grafts matching at 4/6-6/6 loci with a median pre-cryo cell dose of 7.2 (range, 5.1-35.1) x107 cells/kg. All pts engrafted neutrophil and platelets in a median of 25 (range, 12-34) and 39 days (range, 32-105), respectively. Four patients developed Grade 2-4 acute GvHD and 2 developed extensive chronic GvHD. Pt 1, the only patient transplanted after severe neurologic decline with advanced disease, died from progressive BD and aseptic shock 12 months post-UCBT. All others are alive at a median of 3.8 years (range, 0.5-5.5). Pt 2 has normal motor function, is nonverbal and was diagnosed with autism 2 years post UCBT. Her brain MRI is normal to date. Pt 3 is blind, has had occasional seizures, requires educational support and has no MRI evidence of BD. Pts 4 and 5 have age appropriate neurocognition, no seizures, and receive only visual support in school. Both have normal MRIs. It is too early to assess neurologic outcomes in Pt 6 (7 months post-UCBT). Conclusion UCBT is feasible in children with BD. At a median follow up of 3.8 years (range, 0.5-5.5), 5/6 patients are alive and 4/5 are neuro-cognitively stable and one developed new onset seizures post-transplant. All 5 surviving pts have no MRI evidence of disease. With the inherent variability of the different forms of BD, additional pts and continued follow-up is needed to fully understand the effect of UCBT on the natural history of BD.

Three siblings with neuronal ceroid lipofuscinosis type 7 and 2 novel mutations in their MFSD8 gene

Objective The neuronal ceroid lipofuscinosis (CLN) are a group of severe lysosomal storage diseases.The patients present with clinically and genetically heterogeneous neurodegenerative disorders.This study aims to investigate the clinical characteristics and the gene mutations of a rare Chinese family with 3 siblings affected by CLN7. Methods The proband, a 5-year-old girl, visited us because of intermittently seizures and mental retardation for 2 years and a half in December, 2015.Clinical investigation, brain magnetic resonance imaging(MRI), biochemical and the gene analysis were performed for the etiological study. Results The proband had seizures at the age of 2 and a half years, with the progressive motor deterioration, speech disturbance, mental regression and vision loss.Her brain MRI showed diffusive cerebral atrophy.The blood aminoacids, acylcarnitine and urine organic acid profiles were normal.Lysosomal palmitoyl protein thioesterase and tripeptidyl peptidase activities of peripheral leukocytes were normal.A compound heterozygous mutation of c. 1351-1G>A and c. 300T>G was detected on her MFSD8 gene, supporting the diagnosis of CLN7.Both of the 2 mutations were novel.Each of her parents carried one of the mutations.Two brothers of the proband had similar clinical process.Her elder brother died at the age of 7 due to severe encephalopathy of unknown etiology.The younger brother showed dyskinesia from the age of 2 years and seizures from the age of 4 years.A compound heterozygous mutation on MFSD8 gene, c.1351-1G>A and c. 300T>G, was found from the younger brother, as same as the proband. Conclusions CLN7 is a rare disorder of CLN.In this study, the diagnosis of the 3 siblings with similar clinical process were much delayed.Gene analysis was key for the diagnosis.Two novel mutations were found on MFSD8 of the family.There is still no effective treatment for neurol ceroid lipofuscinosis.The prognosis is poor.Based on the mutation diagnosis, prenatal diagnosis for the next sibling is possible to the prevention of the disease. Key words: Neuronal ceroid lipofuscinosis; Neurodegenerative disorders; Lysosomal storage diseases; MFSD8 gene

An Alzheimer's Disease-Linked Loss-of-Function CLN5 Variant Impairs Cathepsin D Maturation, Consistent with a Retromer Trafficking Defect.

In a whole-exome sequencing study of multiplex Alzheimer's disease (AD) families, we investigated three neuronal ceroid lipofuscinosis genes that have been linked to retromer, an intracellular trafficking pathway associated with AD: ceroid lipofuscinosis 3 (CLN3), ceroid lipofuscinosis 5 (CLN5), and cathepsin D (CTSD). We identified a missense variant in CLN5 c.A959G (p.Asn320Ser) that segregated with AD. We find that this variant causes glycosylation defects in the expressed protein, which causes it to be retained in the endoplasmic reticulum with reduced delivery to the endolysosomal compartment, CLN5's normal cellular location. The AD-associated CLN5 variant is shown here to reduce the normal processing of cathepsin D and to decrease levels of full-length amyloid precursor protein (APP), suggestive of a defect in retromer-dependent trafficking.

A first CLN6 variant case of late infantile neuronal ceroid lipofuscinosis caused by a homozygous mutation in a boy from China: a case report

BackgroundNeuronal ceroid lipofuscinosis (NCLs) are lysosomal storage disorders characterized by seizures, motor impairment, and loss of vision. Ceroid lipofuscinosis (CLN) gene mutations are the cause, but NCL cases arising from CLN6 mutations have not been described in China to date. The CLN6 protein, which plays a role in lysosomal function, is an endoplasmic reticulum (ER) membrane protein with seven transmembrane (TM) domains. It has a cytosolic-facing amino terminal domain and a luminal-facing carboxyl terminal domain, with six loops between the TM domains.Case presentationHere we report a case involving a Chinese boy whose suspected diagnosis was a hereditary leukoencephalopathy, based on brain MRI imaging and epilepsy symptoms, language articulation disorders, ataxia, and unstable gait. The electroencephalogram showed epileptic discharges, and the brain MRI scan showed high signal intensity adjacent to the bilateral posterior horns of the lateral ventricles on T2-weighted images, along with cerebellar atrophy. Using next-generation sequencing for the genes in a panel for hereditary leukoencephalopathies, we detected a homozygous missense point mutation c.892G > A(p.Glu298Lys) in CLN6, and the variant was interpreted as pathogenic on in silico analysis. Absence of this mutation was confirmed in 259 controls. Late infantile NCL and secondary epilepsy were diagnosed, and oral sodium valproate was prescribed. The epilepsy was not well controlled, however, and the other signs had not improved at the 6-month follow-up. We also analyzed the loci of 31 CLN6 missense mutations, including those previously reported and the current one. We found that 22.6% (7/31) of the mutations are in the cytoplasmic domains, about 32.2% (10/31) are in the TM domains, and about 45.2% (14/31) are in the luminal domains. These mutations were mostly located in the TM3-TM4 loop (6/31), TM1-TM2 loop (4/31), and C-terminus (4/31), with none found in the TM4-TM5 loop, TM5-TM6 loop, or TM7.ConclusionsWe report the first case in China of NCL caused by a CLN6 mutation, expanding the genotype options for NCLs. In practice, NCLs generally are not the initial suspected diagnosis for such cases. Use of a gene sequencing panel for investigating unexplained seizures or leukoencephalopathies can help confirm the diagnosis.

Induced Pluripotent Stem Cells Derived from a CLN5 Patient Manifest Phenotypic Characteristics of Neuronal Ceroid Lipofuscinoses.

Neuronal ceroid lipofuscinoses (NCLs) are autosomal recessive progressive encephalopathies caused by mutations in at least 14 different genes. Despite extensive studies performed in different NCL animal models, the molecular mechanisms underlying neurodegeneration in NCLs remain poorly understood. To model NCL in human cells, we generated induced pluripotent stem cells (iPSCs) by reprogramming skin fibroblasts from a patient with CLN5 (ceroid lipofuscinosis, neuronal, 5) disease, the late infantile variant form of NCL. These CLN5 patient-derived iPSCs (CLN5Y392X iPSCs) harbouring the most common CLN5 mutation, c.1175_1176delAT (p.Tyr392X), were further differentiated into neural lineage cells, the most affected cell type in NCLs. The CLN5Y392X iPSC-derived neural lineage cells showed accumulation of autofluorescent storage material and subunit C of the mitochondrial ATP synthase, both representing the hallmarks of many forms of NCLs, including CLN5 disease. In addition, we detected abnormalities in the intracellular organelles and aberrations in neuronal sphingolipid transportation, verifying the previous findings obtained from Cln5-deficient mouse macrophages. Therefore, patient-derived iPSCs provide a suitable model to study the mechanisms of NCL diseases.

Novel mutation in two brothers with Hermansky Pudlak syndrome type 3

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder causing oculocutaneous albinism, bleeding disorder and ceroid lipofuscinosis. Platelets from HPS patients are characterized by impaired secretion of dense (δ)-bodies (CD63). Meanwhile, there are ten known human HPS genes, each leading to a particular clinical HPS subtype (HPS1-HPS10).We report on two Turkish brothers showing typical HPS phenotype comprising oculocutaneous albinism and bleeding symptoms. Pathological bleeding time as well as platelet aggregometry analyses revealed impaired platelet function. The brothers demonstrated absence of platelet δ-granule secretion as measured by flow cytometry. Using molecular genetic analyses, a novel homozygous 1bp-deletion in the HPS3 gene was identified in both brothers. In addition, the younger brother with HPS3 demonstrated psychomotoric retardation and cranial gliosis (magnetic resonance imaging, MRI). Array-CGH analysis revealed a de novo 0.482Mb deletion on chromosome 17 which is not present in his brother and parents.In this study, we identified a novel 1bp-deletion in the HPS3 gene causing HPS3 phenotype in two brothers. In patients with oculocutaneous albinism and increased bleeding symptoms platelet function should be analyzed. The identification of the molecular genetic defect allows the classification to a particular HPS subtype and is important for therapy and prognosis.

The Neuronal Ceroid Lipofuscinoses: A Case-Based Overview

The neuronal ceroid lipofuscinoses (NCLs) are a heterogeneous group of inherited, progressive neurodegenerative diseases. Manifestations may begin between the neonatal period and young adulthood, depending on the various subtypes. The different phenotypes are similar and include visual loss, seizures, loss of motor and cognitive function, and early death. At autopsy, there is massive neuronal loss with characteristic storage in the remaining neurons. Neurons appear to die because of increased rates of apoptosis and altered autophagy as occurs in lysosomal storage diseases. A total of 13 neuronal ceroid lipofuscinoses (CLN) genetic forms have been identified so far (CLN type 1–14). In the present review, we propose a classification scheme of the major forms and report the case of a familial recurrence of NCL type 10, with a mutation in the CLN10 gene coding for the cathepsin D protein.

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.

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.

δ-Tocopherol Reduces Lipid Accumulation in Niemann-Pick Type C1 and Wolman Cholesterol Storage Disorders

Niemann-Pick disease type C (NPC) and Wolman disease are two members of a family of storage disorders caused by mutations of genes encoding lysosomal proteins. Deficiency in function of either the NPC1 or NPC2 protein in NPC disease or lysosomal acid lipase in Wolman disease results in defective cellular cholesterol trafficking. Lysosomal accumulation of cholesterol and enlarged lysosomes are shared phenotypic characteristics of both NPC and Wolman cells. Utilizing a phenotypic screen of an approved drug collection, we found that δ-tocopherol effectively reduced lysosomal cholesterol accumulation, decreased lysosomal volume, increased cholesterol efflux, and alleviated pathological phenotypes in both NPC1 and Wolman fibroblasts. Reduction of these abnormalities may be mediated by a δ-tocopherol-induced intracellular Ca(2+) response and subsequent enhancement of lysosomal exocytosis. Consistent with a general mechanism for reduction of lysosomal lipid accumulation, we also found that δ-tocopherol reduces pathological phenotypes in patient fibroblasts from other lysosomal storage diseases, including NPC2, Batten (ceroid lipofuscinosis, neuronal 2, CLN2), Fabry, Farber, Niemann-Pick disease type A, Sanfilippo type B (mucopolysaccharidosis type IIIB, MPSIIIB), and Tay-Sachs. Our data suggest that regulated exocytosis may represent a potential therapeutic target for reduction of lysosomal storage in this class of diseases.

Patients with Hermansky-Pudlak Syndrome Show Various Phenotypes Caused by Novel Mutations,

Abstract 3286▪▪This icon denotes a clinically relevant abstract Background:Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder causing oculocutaneous albinism, bleeding disorder and ceroid lipofuscinosis. Platelets from HPS patients are characterized by impaired secretion of dense (δ)-bodies (CD63). Neutropenia and susceptibility to recurrent infections were exclusively observed in HPS2 patients so far. There are eight known human HPS genes (HPS1-HPS8), each leading to a particular clinical HPS subtype (HPS1-HPS8). Patients/Results:The patients show a typical HPS phenotype concerning oculocutaneous albinism and bleeding symptoms. In vivo-, in vitro bleeding time and platelet aggregometry analyses revealed impaired platelet function. We identified HPS3 in two Turkish brothers and HPS2 in a girl from the United Emirates. Both brothers with HPS3 demonstrated absence of platelet δ-granule secretion measured by flow cytometry analysis. A novel 1 bp-deletion in the HPS3 gene was identified in both brothers. In addition, one brother with HPS3 demonstrated psychomotoric retardation. MRI scan revealed cranial gliosis. Interestingly, array-CGH analysis revealed a 0.7 Mb deletion on chromosome 17 which had not been identified in the other brother and which seems to have caused the cranial gliosis.The girl with HPS2 suffered from life-threatening bleeding after tonsillectomy leading to severe asphyxia, resuscitation and finally, to mental retardation. Flow cytometry analysis demonstrated impaired platelet δ-granule secretion with a typical pattern for HPS2. CD63 expression was already increased on resting platelets, but there was only little increase after thrombin stimulation. Interestingly, only a NK-CD107 partial degranulation defect was diagnosed. So far, clinical symptoms of immunodeficiency are not obvious. Molecular genetic analyses revealed a novel 2 bp-deletion in the last exon of HPS2 leading to a frameshift and a prolonged altered protein. The location of the deletion at the very C-terminal end may prevent a complete loss of the HPS2 protein leading to a less pronounced severity of immunodeficiency as in other HPS2 patients. Conclusion:Patients with oculocutaneous albinism should be investigated for increased clinical bleeding symptoms. In case of increased bleeding symptoms, analyses of primary hemostasis should be initiated to confirm HPS. Using flow cytometry analyses HPS2 can be distinguished from the other subtypes of HPS. Molecular genetic investigations should be performed to differentiate the various subtypes of HPS which is important for therapy and prognosis. The HPS3 patient`s mental retardation seemed to be caused by an additional deletion. The identification of the molecular genetic defect helps to understand the patients` various clinical phenotypes. Disclosures:Zieger:Novo Nordisk: Research Funding.