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1.
Nat Struct Mol Biol ; 27(2): 192-201, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32042150

RESUMO

Point mutations in cysteine string protein-α (CSPα) cause dominantly inherited adult-onset neuronal ceroid lipofuscinosis (ANCL), a rapidly progressing and lethal neurodegenerative disease with no treatment. ANCL mutations are proposed to trigger CSPα aggregation/oligomerization, but the mechanism of oligomer formation remains unclear. Here we use purified proteins, mouse primary neurons and patient-derived induced neurons to show that the normally palmitoylated cysteine string region of CSPα loses palmitoylation in ANCL mutants. This allows oligomerization of mutant CSPα via ectopic binding of iron-sulfur (Fe-S) clusters. The resulting oligomerization of mutant CSPα causes its mislocalization and consequent loss of its synaptic SNARE-chaperoning function. We then find that pharmacological iron chelation mitigates the oligomerization of mutant CSPα, accompanied by partial rescue of the downstream SNARE defects and the pathological hallmark of lipofuscin accumulation. Thus, the iron chelators deferiprone (L1) and deferoxamine (Dfx), which are already used to treat iron overload in humans, offer a new approach for treating ANCL.


Assuntos
Proteínas de Choque Térmico HSP40/genética , Proteínas de Membrana/genética , Lipofuscinoses Ceroides Neuronais/genética , Mutação Puntual , Agregação Patológica de Proteínas/genética , Animais , Células Cultivadas , Feminino , Células HEK293 , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Quelantes de Ferro/metabolismo , Lipoilação , Proteínas de Membrana/metabolismo , Camundongos , Lipofuscinoses Ceroides Neuronais/metabolismo , Neurônios/metabolismo , Agregação Patológica de Proteínas/metabolismo , Ligação Proteica , Multimerização Proteica
2.
Elife ; 82019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31663851

RESUMO

The autosomal dominant neuronal ceroid lipofuscinoses (NCL) CLN4 is caused by mutations in the synaptic vesicle (SV) protein CSPα. We developed animal models of CLN4 by expressing CLN4 mutant human CSPα (hCSPα) in Drosophila neurons. Similar to patients, CLN4 mutations induced excessive oligomerization of hCSPα and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most CLN4 mutant hCSPα accumulated abnormally, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructural examination revealed frequent abnormal membrane structures in axons and neuronal somata. The lethality, oligomerization and prelysosomal accumulation induced by CLN4 mutations was attenuated by reducing endogenous wild type (WT) dCSP levels and enhanced by increasing WT levels. Furthermore, reducing the gene dosage of Hsc70 also attenuated CLN4 phenotypes. Taken together, we suggest that CLN4 alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking.


Assuntos
Drosophila melanogaster/genética , Mutação com Ganho de Função , Lipofuscinoses Ceroides Neuronais/genética , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Drosophila melanogaster/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Lipofuscinoses Ceroides Neuronais/metabolismo , Neurônios/ultraestrutura , Vesículas Sinápticas/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo
3.
Hum Mol Genet ; 28(19): 3309-3322, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31294445

RESUMO

The Finnish-variant late infantile neuronal ceroid lipofuscinosis, also known as CLN5 disease, is caused by mutations in the CLN5 gene. Cln5 is strongly expressed in the developing brain and expression continues into adulthood. CLN5, a protein of unknown function, is implicated in neurodevelopment but detailed investigation is lacking. Using Cln5-/- embryos of various ages and cells harvested from Cln5-/- brains we investigated the hitherto unknown role of Cln5 in the developing brain. Loss of Cln5 results in neuronal differentiation deficits and delays in interneuron development during in utero period. Specifically, the radial thickness of dorsal telencephalon was significantly decreased in Cln5-/- mouse embryos at embryonic day 14.5 (E14.5), and expression of Tuj1, an important neuronal marker during development, was down-regulated. An interneuron marker calbindin and a mitosis marker p-H3 showed down-regulation in ganglionic eminences. Neurite outgrowth was compromised in primary cortical neuronal cultures derived from E16 Cln5-/- embryos compared with WT embryos. We show that the developmental deficits of interneurons may be linked to increased levels of the repressor element 1-silencing transcription factor, which we report to bind to glutamate decarboxylase (Gad1), which encodes GAD67, a rate-limiting enzyme in the production of gamma-aminobutyric acid (GABA). Indeed, adult Cln5-/- mice presented deficits in hippocampal parvalbumin-positive interneurons. Furthermore, adult Cln5-/- mice presented deficits in hippocampal parvalbumin-positive interneurons and showed age-independent cortical hyper excitability as measured by electroencephalogram and auditory-evoked potentials. This study highlights the importance of Cln5 in neurodevelopment and suggests that in contrast to earlier reports, CLN5 disease is likely to develop during embryonic stages.


Assuntos
Encéfalo/crescimento & desenvolvimento , Glutamato Descarboxilase/genética , Interneurônios/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Lipofuscinoses Ceroides Neuronais/genética , Animais , Encéfalo/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Lipofuscinoses Ceroides Neuronais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Parvalbuminas/metabolismo , Proteínas Repressoras/genética , Tubulina (Proteína)/metabolismo
4.
PLoS One ; 14(7): e0212382, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31291241

RESUMO

Mutation in the GRN gene, encoding the progranulin (PGRN) protein, shows a dose-dependent disease correlation, wherein haploinsufficiency results in frontotemporal lobar degeneration (FTLD) and complete loss results in neuronal ceroid lipofuscinosis (NCL). Although the exact function of PGRN is unknown, it has been increasingly implicated in lysosomal physiology. Here we report that PGRN interacts with the lysosomal enzyme, glucocerebrosidase (GCase), and is essential for proper GCase activity. GCase activity is significantly reduced in tissue lysates from PGRN-deficient mice. This is further evidence that reduced lysosomal hydrolase activity may be a pathological mechanism in cases of GRN-related FTLD and NCL.


Assuntos
Glucosilceramidase/metabolismo , Progranulinas/deficiência , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Glucosilceramidase/genética , Células HEK293 , Haploinsuficiência , Humanos , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Progranulinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
Hum Mutat ; 40(11): 1924-1938, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31283065

RESUMO

Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is an autosomal recessive condition caused by variants in the TPP1 gene, leading to deficient activity of the lysosomal enzyme tripeptidyl peptidase I (TPP1). We update on the spectrum of TPP1 variants associated with CLN2 disease, comprising 131 unique variants from 389 individuals (717 alleles) collected from the literature review, public databases, and laboratory communications. Previously unrecorded individuals were added to the UCL TPP1-specific database. Two known pathogenic variants, c.509-1 G>C and c.622 C>T (p.(Arg208*)), collectively occur in 60% of affected individuals in the sample, and account for 50% of disease-associated alleles. At least 86 variants (66%) are private to single families. Homozygosity occurs in 45% of individuals where both alleles are known (87% of reported individuals). Atypical CLN2 disease, TPP1 enzyme deficiency with disease onset and/or progression distinct from classic late-infantile CLN2, represents 13% of individuals recorded with associated phenotype. NCBI ClinVar currently holds records for 37% of variants collected here. Effective CLN2 disease management requires early diagnosis; however, irreversible neurodegeneration occurs before a diagnosis is typically reached at age 5. Timely classification and public reporting of TPP1 variants is essential as molecular testing increases in use as a first-line diagnostic test for pediatric-onset neurological disease.


Assuntos
Aminopeptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Predisposição Genética para Doença , Mutação , Lipofuscinoses Ceroides Neuronais/genética , Serina Proteases/genética , Alelos , Aminopeptidases/química , Animais , Biomarcadores , Bases de Dados Genéticas , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Modelos Animais de Doenças , Estudos de Associação Genética , Genótipo , Humanos , Simulação de Dinâmica Molecular , Lipofuscinoses Ceroides Neuronais/diagnóstico , Lipofuscinoses Ceroides Neuronais/metabolismo , Fenótipo , Conformação Proteica , Serina Proteases/química , Relação Estrutura-Atividade
6.
Cells ; 8(5)2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100984

RESUMO

The neuronal ceroid lipofuscinoses comprise a group of neurodegenerative disorders with similar clinical manifestations whose precise mechanisms of disease are presently unknown. We created multiple cell lines each with different levels of reduction of expression of the gene coding for the type 2 variant of the disease, Tripeptidyl peptidase (Tpp1), in the cellular slime mould Dictyostelium discoideum. Knocking down Tpp1 in Dictyostelium resulted in the accumulation of autofluorescent material, a characteristic trait of Batten disease. Phenotypic characterisation of the mutants revealed phenotypic deficiencies in growth and development, whilst endocytic uptake of nutrients was enhanced. Furthermore, the severity of the phenotypes correlated with the expression levels of Tpp1. We propose that the phenotypic defects are due to altered Target of Rapamycin (TOR) signalling. We show that treatment of wild type Dictyostelium cells with rapamycin (a specific TOR complex inhibitor) or antisense inhibition of expression of Rheb (Ras homologue enriched in the brain) (an upstream TOR complex activator) phenocopied the Tpp1 mutants. We also show that overexpression of Rheb rescued the defects caused by antisense inhibition of Tpp1. These results suggest that the TOR signalling pathway is responsible for the cytopathological outcomes in the Dictyostelium Tpp1 model of Batten disease.


Assuntos
Aminopeptidases/genética , Dictyostelium/genética , Dictyostelium/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Serina Proteases/genética , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , DNA Antissenso , DNA de Protozoário , Dictyostelium/citologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Lisossomos/enzimologia , Mutação , Imagem Óptica , Fagocitose/efeitos dos fármacos , Fenótipo , Fototaxia , RNA Mensageiro/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores
7.
Biochim Biophys Acta Biomembr ; 1861(10): 182993, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31132336

RESUMO

Mutations in the ATP13A2 gene (PARK9, CLN12, OMIM 610513) were initially associated with a form of Parkinson's Disease (PD) known as Kufor Rakeb Syndrome (KRS). However, the genetic spectrum of ATP13A2-associated disorders was expanded in the last years, because it has been found to underlay variants of neuronal ceroid-lipofuscinoses (NCLs) and hereditary spastic paraplegia. As ATP13A2 seems to be a key component of the endo-lysosome pathway, the fact that these pathologies are commonly characterized by endo-lysosomal dysfunction is not surprising. Here we report that increasing the level of functional ATP13A2 in a stable SH-SY5Y cell line disrupts lipid homeostasis. ATP13A2 overexpression increases the fluorescence intensity of the fluorescent analog phosphatidylethanolamine (NBD-PE) and the formation of multilamellar bodies, resembling the so-called "drug-induced phospholipidosis". We also found that expression of ATP13A2 reduces the ceramide-fluorescence intensity and the content of bis(monoacylglyceryl)phosphate (BMP). BMP is required for lipid degradation and exosome biogenesis inside acidic compartments, so this result suggests that ATP13A2 may be modifying the lipid digestion capacity and/or the redistribution of lipids in these subcellular organelles. In addition, ATP13A2-overexpression decreased the total content of triglycerides (TGs), cholesterol and lipid droplets. As TGs are necessary for the synthesis of new membranes, this observation suggests that increasing the function of ATP13A2 switches the endo-lysosomal system towards vesicle secretion.


Assuntos
Fosfolipídeos/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Linhagem Celular Tumoral , Colesterol/metabolismo , Endossomos/metabolismo , Homeostase , Humanos , Metabolismo dos Lipídeos , Lisossomos/metabolismo , Monoglicerídeos/metabolismo , Mutação , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/metabolismo , Fosfatidiletanolaminas/metabolismo , Triglicerídeos/metabolismo
8.
Neurobiol Dis ; 127: 362-373, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30928643

RESUMO

The late-infantile Batten disease or late-infantile neuronal ceroid lipofuscinosis (LINCL) is an autosomal recessive lysosomal storage disorder caused by mutations in the Cln2 gene leading to deficiency of lysosomal enzyme tripeptidyl peptidase 1 (TPP1). At present, available options for this fatal disorder are enzyme replacement therapy and gene therapy, which are extensively invasive and expensive. Our study demonstrates that 3-hydroxy-(2,2)-dimethyl butyrate (HDMB), a brain endogenous molecule, is capable of stimulating TPP1 expression and activity in mouse primary astrocytes and a neuronal cell line. HDMB activated peroxisome proliferator-activated receptor-α (PPARα), which, by forming heterodimer with Retinoid X receptor-α (RXRα), transcriptionally upregulated the Cln2 gene. Moreover, by using primary astrocytes from wild type, PPARα-/- and PPARß-/- mice, we demonstrated that HDMB specifically required PPARα for inducing TPP1 expression. Finally, oral administration of HDMB to Cln2 heterozygous (Cln2+/-) mice led to a marked upregulation of TPP1 expression in the motor cortex and striatum in a PPARα-dependent fashion. Our study suggests that HDMB, a brain endogenous ligand of PPARα, might have therapeutic importance for LINCL treatment.


Assuntos
Aminopeptidases/metabolismo , Astrócitos/efeitos dos fármacos , Butiratos/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Neurônios/efeitos dos fármacos , PPAR alfa/metabolismo , Serina Proteases/metabolismo , Aminopeptidases/genética , Animais , Astrócitos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Butiratos/uso terapêutico , Linhagem Celular , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Lipofuscinoses Ceroides Neuronais/tratamento farmacológico , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Neurônios/metabolismo , Serina Proteases/genética , Regulação para Cima
9.
ASN Neuro ; 11: 1759091419843393, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31003587

RESUMO

In humans, homozygous mutations in the TPP1 gene results in loss of tripeptidyl peptidase 1 (TPP1) enzymatic activity, leading to late infantile neuronal ceroid lipofuscinoses disease. Using a mouse model that targets the Tpp1 gene and recapitulates the pathology and clinical features of the human disease, we analyzed end-stage (4 months) transcriptional changes associated with lack of TPP1 activity. Using RNA sequencing technology, Tpp1 expression changes in the forebrain/midbrain and cerebellum of 4-month-old homozygotes were compared with strain-related controls. Transcriptional changes were found in 510 and 1,550 gene transcripts in forebrain/midbrain and cerebellum, respectively, from Tpp1-deficient brain tissues when compared with age-matched controls. Analysis of the differentially expressed genes using the Ingenuity™ pathway software, revealed increased neuroinflammation activity in microglia and astrocytes that could lead to neuronal dysfunction, particularly in the cerebellum. We also observed upregulation in the production of nitric oxide and reactive oxygen species; activation of leukocyte extravasation signals and complement pathways; and downregulation of major transcription factors involved in control of circadian rhythm. Several of these expression changes were confirmed by independent quantitative polymerase chain reaction and histological analysis by mRNA in situ hybridization, which allowed for an in-depth anatomical analysis of the pathology and provided independent confirmation of at least two of the major networks affected in this model. The identification of differentially expressed genes has revealed new lines of investigation for this complex disorder that may lead to novel therapeutic targets.


Assuntos
Aminopeptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Regulação da Expressão Gênica/fisiologia , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Serina Proteases/genética , Transcriptoma/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Camundongos , Mutação , Lipofuscinoses Ceroides Neuronais/patologia
10.
Brain Dev ; 41(8): 726-730, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31029456

RESUMO

INTRODUCTION: Neuronal ceroid lipofuscinoses (NCLs; CLN) are mainly autosomal recessive neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigments in neuronal and other cells. Symptoms include visual disabilities, motor decline, and epilepsy. Causative genes are CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN8, CLN10, CLN11, CLN12, CLN13, and CLN14. We present the fourth Japanese case with a CLN6 mutation. CASE PRESENTATION: At 3 years of age, our patient became clumsy and fell down easily. He developed focal seizures with impaired consciousness and was started on carbamazepine. He showed ataxic walking and dysarthria with increased deep tendon reflexes. Interictal electroencephalogram revealed slow waves in the left temporal and occipital areas. Brain magnetic resonance imaging showed cerebellar atrophy and ventriculomegaly. In optical coherence tomography (OCT), the inner layer of the retina was thick and highly reflective. Exome sequencing revealed a known homozygous mutation, C.794_976del, p. (Ser265del) in CLN6. DISCUSSION: A total of 130 cases of NCL with CLN6 mutations have been reported globally, of which only four were from Japan including the current patient. The deletion of serine at position 265 has been reported in six cases. Ser265 is located in a region of short repeated sequences that is susceptible to mutation. Clinical trials of gene therapy using adeno-associated virus serotype 9 have started for NCL6, making early diagnosis crucial. OCT examination might be helpful in achieving a diagnosis.


Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Lipofuscinoses Ceroides Neuronais/genética , Pré-Escolar , Progressão da Doença , Epilepsia/genética , Homozigoto , Humanos , Japão , Masculino , Limitação da Mobilidade , Mutação , Lipofuscinoses Ceroides Neuronais/metabolismo , Convulsões/genética , Caminhada/fisiologia
11.
Cells ; 8(2)2019 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-30717401

RESUMO

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating neurological disorders that have a global distribution and affect people of all ages. Commonly known as Batten disease, this form of neurodegeneration is linked to mutations in 13 genetically distinct genes. The precise mechanisms underlying the disease are unknown, in large part due to our poor understanding of the functions of NCL proteins. The social amoeba Dictyostelium discoideum has proven to be an exceptional model organism for studying a wide range of neurological disorders, including the NCLs. The Dictyostelium genome contains homologs of 11 of the 13 NCL genes. Its life cycle, comprised of both single-cell and multicellular phases, provides an excellent system for studying the effects of NCL gene deficiency on conserved cellular and developmental processes. In this review, we highlight recent advances in NCL research using Dictyostelium as a biomedical model.


Assuntos
Dictyostelium/metabolismo , Modelos Biológicos , Proteínas de Protozoários/metabolismo , Humanos , Lipofuscinoses Ceroides Neuronais/metabolismo , Osmorregulação , Fenótipo
12.
Sci Rep ; 9(1): 151, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30655561

RESUMO

Neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN5 deficiency causes a subtype of NCL, referred to as CLN5 disease. CLN5 is a soluble lysosomal protein with an unclear function in the cell. Increased levels of the autophagy marker protein LC3-II have been reported in several subtypes of NCLs. In this report, we examine whether autophagy is altered in CLN5 disease. We found that the basal level of LC3-II was elevated in both CLN5 disease patient fibroblasts and CLN5-deficient HeLa cells. Further analysis using tandem fluorescent mRFP-GFP-LC3 showed the autophagy flux was increased. We found the alpha-synuclein (α-syn) gene SNCA was highly up-regulated in CLN5 disease patient fibroblasts. The aggregated form of α-syn is well known for its role in the pathogenicity of Parkinson's disease. Higher α-syn protein levels confirmed the SNCA up-regulation in both patient cells and CLN5 knockdown HeLa cells. Furthermore, α-syn was localized to the vicinity of lysosomes in CLN5 deficient cells, indicating it may have a lysosome-related function. Intriguingly, knocking down SNCA reversed lysosomal perinuclear clustering caused by CLN5 deficiency. These results suggest α-syn may affect lysosomal clustering in non-neuronal cells, similar to its role in presynaptic vesicles in neurons.


Assuntos
Fibroblastos/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , alfa-Sinucleína/metabolismo , Autofagia , Fibroblastos/patologia , Células HeLa , Humanos , Lisossomos/metabolismo , Regulação para Cima
13.
Orphanet J Rare Dis ; 14(1): 19, 2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30665444

RESUMO

BACKGROUND: CLN6-Batten disease is a rare neurodevelopmental disorder characterized pathologically by the accumulation of lysosomal storage material, glial activation and neurodegeneration, and phenotypically by loss of vision, motor coordination, and cognitive ability, with premature death occurring in the second decade of life. In this study, we investigate whether sex differences in a mouse model of CLN6-Batten disease impact disease onset and progression. RESULTS: A number of noteworthy differences were observed including elevated accumulation of mitochondrial ATP synthase subunit C in the thalamus and cortex of female Cln6 mutant mice at 2 months of age. Moreover, female mutant mice showed more severe behavioral deficits. Beginning at 9 months of age, female mice demonstrated learning and memory deficits and suffered a more severe decline in motor coordination. Further, compared to their male counterparts, female animals succumbed to the disease at a slightly younger age, indicating an accelerated disease progression. Conversely, males showed a marked increase in microglial activation at 6 months of age in the cortex relative to females. CONCLUSIONS: Thus, as female Cln6 mutant mice exhibit cellular and behavioral deficits that precede similar pathologies in male mutant mice, our findings suggest the need for consideration of sex-based differences in CLN6 disease progression during development of preclinical and clinical studies.


Assuntos
Proteínas de Membrana/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Lipofuscinoses Ceroides Neuronais/genética , Doenças Raras/genética , Doenças Raras/metabolismo
14.
J Mol Biol ; 431(5): 1038-1047, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30690031

RESUMO

Single-copy loss-of-function mutations in the progranulin gene (PGRN) underlie the neurodegenerative disease frontotemporal lobar degeneration, while homozygous loss-of-function of PGRN results in the lysosomal storage disorder neuronal ceroid lipofuscinosis. Despite evidence that normal PGRN levels are critical for neuronal health, the function of this protein is not yet understood. Here, we show that PGRN stimulates the in vitro maturation of the lysosomal aspartyl protease cathepsin D (CTSD). CTSD is delivered to the endolysosomal system as an inactive precursor (proCTSD) and requires sequential cleavage steps via intermediate forms to achieve the mature state (matCTSD). In co-immunoprecipitation experiments, PGRN interacts predominantly with immature pro- and intermediate forms of CTSD. PGRN enhances in vitro conversion of proCTSD to matCTSD in a concentration-dependent manner. Differential scanning fluorimetry shows a destabilizing effect induced by PGRN on proCTSD folding (∆Tm = -1.7 °C at a 3:1 molar ratio). We propose a mechanism whereby PGRN binds to proCTSD, destabilizing the propeptide from the enzyme catalytic core and favoring conversion to mature forms of the enzyme. Further understanding of the role of PGRN in CTSD maturation will assist in the development of targeted therapies for neurodegenerative disease.


Assuntos
Catepsina D/metabolismo , Precursores Enzimáticos/metabolismo , Progranulinas/metabolismo , Catepsina D/genética , Linhagem Celular , Precursores Enzimáticos/genética , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Mutação/genética , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Progranulinas/genética
15.
J Neurochem ; 148(5): 612-624, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29964296

RESUMO

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a lysosomal storage disease caused by autosomal recessive mutations in ceroid lipofuscinosis 3 (CLN3). Children with JNCL experience progressive visual, cognitive, and motor deterioration with a decreased life expectancy (late teens-early 20s). Neuronal loss is thought to occur, in part, via glutamate excitotoxicity; however, little is known about astrocyte glutamate regulation in JNCL. Spontaneous Ca2+ oscillations were reduced in murine Cln3Δex7/8 astrocytes, which were also observed following glutamate or cytokine exposure. Astrocyte glutamate transport is an energy-demanding process and disruptions in metabolic pathways could influence glutamate homeostasis in Cln3Δex7/8 astrocytes. Indeed, basal mitochondrial respiration and ATP production were significantly reduced in Cln3Δex7/8 astrocytes. These changes were not attributable to reduced mitochondria, since mitochondrial DNA levels were similar between wild type and Cln3Δex7/8 astrocytes. Interestingly, despite these functional deficits in Cln3Δex7/8 astrocytes, glutamate transporter expression and glutamate uptake were not dramatically affected. Concurrent with impaired astrocyte metabolism and Ca2+ signaling, murine Cln3Δex7/8 neurons were hyper-responsive to glutamate, as reflected by heightened and prolonged Ca2+ signals. These findings identify intrinsic metabolic and Ca2+ signaling defects in Cln3Δex7/8 astrocytes that may contribute to neuronal dysfunction in CLN3 disease. This article is part of the Special Issue "Lysosomal Storage Disorders".


Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Lipofuscinoses Ceroides Neuronais/metabolismo , Neurônios/metabolismo , Animais , Feminino , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Chaperonas Moleculares/genética
16.
Biochim Biophys Acta Mol Basis Dis ; 1865(2): 322-328, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30453012

RESUMO

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN8 deficiency causes a subtype of NCL, referred to as CLN8 disease. CLN8 is an ER resident protein with unknown function; however, a role in ceramide metabolism has been suggested. In this report, we identified PP2A and its biological inhibitor I2PP2A as interacting proteins of CLN8. PP2A is one of the major serine/threonine phosphatases in cells and governs a wide range of signaling pathways by dephosphorylating critical signaling molecules. We showed that the phosphorylation levels of several substrates of PP2A, namely Akt, S6 kinase, and GSK3ß, were decreased in CLN8 disease patient fibroblasts. This reduction can be reversed by inhibiting PP2A phosphatase activity with cantharidin, suggesting a higher PP2A activity in CLN8-deficient cells. Since ceramides are known to bind and influence the activity of PP2A and I2PP2A, we further examined whether ceramide levels in the CLN8-deficient cells were changed. Interestingly, the ceramide levels were reduced by 60% in CLN8 disease patient cells compared to controls. Furthermore, we observed that the conversion of ER-localized NBD-C6-ceramide to glucosylceramide and sphingomyelin in the Golgi apparatus was not affected in CLN8-deficient cells, indicating transport of ceramides from ER to the Golgi apparatus was normal. A model of how CLN8 along with ceramides affects I2PP2A and PP2A binding and activities is proposed.


Assuntos
Ceramidas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Proteína Fosfatase 2/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Glucosilceramidase/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/deficiência , Modelos Biológicos , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingolipídeos/metabolismo
17.
Nat Cell Biol ; 20(12): 1370-1377, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30397314

RESUMO

Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment1-3. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system4-6, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease)7. ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Animais , Células HeLa , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Ligação Proteica , Transporte Proteico/genética
18.
Invest Ophthalmol Vis Sci ; 59(12): 5082-5097, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30372735

RESUMO

Purpose: Vision loss caused by photoreceptor death represents one of the first symptoms in neuronal ceroid lipofuscinosis, a condition characterized by accumulation of intracellular waste. Cln6nclf mice have a naturally occurring mutation in ceroid-lipofuscinosis neuronal (CLN) protein 6 and are a model of this disorder. In order to identify the effect intracellular waste (lipofuscin) accumulation plays in driving retinal degeneration, the time course of degeneration was carefully characterized functionally using the electroretinogram and structurally using histology. Methods: Cln6nclf and C57BL/6J, wild-type, mice were studied at postnatal day 18 (P18), P30, P60, P120, and P240, and retinal degeneration was correlated with changes in the retinal pigment epithelial (RPE) and neuronal autophagy-lysosomal pathways using super-resolution microscopy. Results: In Cln6nclf mice there was significant loss of rod photoreceptor function at P18, prior to photoreceptor nuclei loss at P60. In contrast, cone pathway function was not affected until P240. The loss of rod photoreceptor function correlated with significant disruption of the autophagy-lysosomal degradation pathways within photoreceptors, but not in the RPE or other retinal neurons. Additionally, there was cytosolic accumulation of P62 and undigested mitochondrial-derived, ATP synthase subunit C in the photoreceptor layers of Cln6nclf mice at P30. Conclusions: These results suggest that rod photoreceptors have an increased sensitivity to disturbances in the autophagy-lysosomal pathway and the subsequent failure of mitochondrial turnover, relative to other retinal cells. It is likely that primary failure of the rod photoreceptors rather than the RPE or other retinal neurons underlies the early visual dysfunction that occurs in the Cln6nclf mouse model.


Assuntos
Autofagia/fisiologia , Lisossomos/fisiologia , Proteínas de Membrana/metabolismo , Lipofuscinoses Ceroides Neuronais/fisiopatologia , Degeneração Retiniana/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Animais , Western Blotting , Contagem de Células , Modelos Animais de Doenças , Eletrorretinografia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Lipofuscinoses Ceroides Neuronais/metabolismo , Fenótipo , Estimulação Luminosa , Retina/fisiopatologia , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais
19.
Sci Rep ; 8(1): 15229, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30323181

RESUMO

Late infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a rare lysosomal storage disorder caused by a monogenetic deficiency of tripeptidyl peptidase-1 (TPP1). Despite knowledge that lipofuscin is the hallmark disease product, the relevant TPP1 substrate and its role in neuronal physiology/pathology is unknown. We hypothesized that untargeted metabolite profiling of cerebrospinal fluid (CSF) could be used as an effective tool to identify disease-associated metabolic disruptions in CLN2 disease, offering the potential to identify biomarkers that inform on disease severity and progression. Accordingly, a mass spectrometry-based untargeted metabolite profiling approach was employed to differentiate CSF from normal vs. CLN2 deficient individuals. Of 1,433 metabolite features surveyed, 29 linearly correlated with currently employed disease severity scores. With tandem mass spectrometry 8 distinct metabolite identities were structurally confirmed based on retention time and fragmentation pattern matches, vs. standards. These putative CLN2 biomarkers include 7 acetylated species - all attenuated in CLN2 compared to controls. Because acetate is the major bioenergetic fuel for support of mitochondrial respiration, deficient acetylated species in CSF suggests a brain energy defect that may drive neurodegeneration. Targeted analysis of these metabolites in CSF of CLN2 patients offers a powerful new approach for monitoring CLN2 disease progression and response to therapy.


Assuntos
Biomarcadores/líquido cefalorraquidiano , Encéfalo/metabolismo , Metaboloma/genética , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Acetatos/metabolismo , Adolescente , Adulto , Idoso , Aminopeptidases/líquido cefalorraquidiano , Aminopeptidases/genética , Animais , Encéfalo/patologia , Criança , Pré-Escolar , Dipeptidil Peptidases e Tripeptidil Peptidases/líquido cefalorraquidiano , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Metabolômica , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Lipofuscinoses Ceroides Neuronais/líquido cefalorraquidiano , Lipofuscinoses Ceroides Neuronais/patologia , Neurônios/metabolismo , Neurônios/patologia , Serina Proteases/líquido cefalorraquidiano , Serina Proteases/genética , Índice de Gravidade de Doença , Adulto Jovem
20.
Anal Chem ; 90(20): 12168-12171, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30204428

RESUMO

A new tandem mass spectrometry (MS/MS)-based approach for measurement of the enzymatic activity of palmitoyl protein thioesterase I (PPT1) in dried blood spots (DBS) is presented. Deficiency in this enzyme leads to infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten disease, CLN1). The assay could distinguish between 80 healthy newborns and three previously diagnosed INCL patients. Unlike the fluorimetric PPT1 assay, the MS/MS assay does not require recombinant ß-glucosidase. Furthermore, the assay could be easily combined with a TPP1 enzyme assay (for CLN2 disease) and can be potentially multiplexed with a large panel of additional lysosomal enzyme assays by MS/MS for newborn screening and postscreening analysis.


Assuntos
Teste em Amostras de Sangue Seco , Proteínas de Membrana/sangue , Lipofuscinoses Ceroides Neuronais/diagnóstico , Tioléster Hidrolases/sangue , Humanos , Lactente , Proteínas de Membrana/metabolismo , Estrutura Molecular , Lipofuscinoses Ceroides Neuronais/sangue , Lipofuscinoses Ceroides Neuronais/metabolismo , Espectrometria de Massas em Tandem , Tioléster Hidrolases/metabolismo
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