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1.
Int J Mol Sci ; 20(20)2019 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-31635081

RESUMO

Niemann-Pick Type C (NP-C) is a rare disorder of lipid metabolism caused by mutations within the NPC1 and NPC2 genes. NP-C is a neurovisceral disease leading to a heterogeneous, multisystemic spectrum of symptoms in those affected. Until now, there is no investigative tool to demonstrate the significance of single variants within the NPC genes. Hence, the aim of the study was to establish a test that allows for an objective assessment of the pathological potential of NPC1 gene variants. Chinese hamster ovary cells defective in the NPC1 gene accumulate cholesterol in lysosomal storage organelles. The cells were transfected with NPC1-GFP plasmid vectors carrying distinct sequence variants. Filipin staining was used to test for complementation of the phenotype. The known variant p.Ile1061Thr showed a significantly impaired cholesterol clearance after 12 and 24 h compared to the wild type. Among the investigated variants, p.Ser954Leu and p.Glu1273Lys showed decelerated cholesterol clearance as well. The remaining variants p.Gln60His, p.Val494Met, and p.Ile787Val showed a cholesterol clearance indistinguishable from wild type. Further, p.Ile1061Thr acquired an enhanced clearance ability upon 25-hydroxycholesterol treatment. We conclude that the variants that caused an abnormal clearance phenotype are highly likely to be of clinical relevance. Moreover, we present a system that can be utilized to screen for new drugs.


Assuntos
Teste de Complementação Genética , Variação Genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Alelos , Sequência de Aminoácidos , Animais , Células CHO , Células Cultivadas , Colesterol/metabolismo , Mapeamento Cromossômico , Cricetulus , Frequência do Gene , Estudos de Associação Genética , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Doença de Niemann-Pick Tipo C/diagnóstico , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , Fenótipo , Conformação Proteica , Relação Estrutura-Atividade
2.
Nat Commun ; 10(1): 4276, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537798

RESUMO

Transport of dietary cholesterol from endocytic organelles to the endoplasmic reticulum (ER) is essential for cholesterol homoeostasis, but the mechanism and regulation of this transport remains poorly defined. Membrane contact sites (MCS), microdomains of close membrane apposition, are gaining attention as important platforms for non-vesicular, inter-organellar communication. Here we investigate the impact of ER-endocytic organelle MCS on cholesterol transport. We report a role for Niemann-Pick type C protein 1 (NPC1) in tethering ER-endocytic organelle MCS where it interacts with the ER-localised sterol transport protein Gramd1b to regulate cholesterol egress. We show that artificially tethering MCS rescues the cholesterol accumulation that characterises NPC1-deficient cells, consistent with direct lysosome to ER cholesterol transport across MCS. Finally, we identify an expanded population of lysosome-mitochondria MCS in cells depleted of NPC1 or Gramd1b that is dependent on the late endosomal sterol-binding protein STARD3, likely underlying the mitochondrial cholesterol accumulation in NPC1-deficient cells.


Assuntos
Transporte Biológico/fisiologia , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Animais , Células CHO , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Doença de Niemann-Pick Tipo C/genética , Interferência de RNA , RNA Interferente Pequeno/genética
3.
J Appl Genet ; 60(3-4): 357-365, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31485950

RESUMO

The Npc1nih/nih-null model and the Npc1nmf164/nmf164 hypomorph models of Niemann-Pick C1 (NPC1) disease show defects in olfaction. We have tested the effects of the life-prolonging treatment hydroxypropyl-beta-cyclodextrin (HPBCD) on olfaction and neural stem cell numbers when delivered either systemically or by nasal inhalation. Using the paradigm of finding a hidden cube of food after overnight food deprivation, Npc1nih/nih homozygous mice showed a highly significant delay in finding the food compared with wild-type mice. Npc1nmf164/nmf164 homozygous mice showed an early loss of olfaction which was mildly corrected by somatic delivery of HPBCD which also increased the number of neural stem cells in the mutant but did not change the number in wild-type mice. In contrast, nasal delivery of this drug, at 1/5 the dosage used for somatic delivery, to Npc1nmf164/nmf164 mutant mice delayed loss of olfaction but the control of nasal delivered saline did so as well. The nasal delivery of HPBCD to wild-type mice caused loss of olfaction but nasal delivery of saline did not. Neural stem cell counts were not improved by nasal therapy with HPBCD. We credit the delay in olfaction found with the treatment, a delay which was also found for time of death, to a large amount of stimulation the mice received with handling during the nasal delivery.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Olfato/efeitos dos fármacos , Administração Intranasal , Animais , Proliferação de Células/efeitos dos fármacos , Colesterol , Modelos Animais de Doenças , Humanos , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Olfato/genética
4.
BMC Med Genet ; 20(1): 123, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296176

RESUMO

BACKGROUND: Niemann-Pick disease type C (NP-C) is an inherited neurodegenerative disease (1 per 100 000 newborns) caused by NPC proteins impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. To date the NP-C diagnostics is usually based on cholesterol detection in fibroblasts using an invasive and time-consuming Filipin staining and we need more arguments to widely introduce oxysterols as a biomarkers in NP-C. METHODS: Insofar as NP-C represents about 8% of all infant cholestases, in this prospective observational study we tried to re-assess the specificity plasma oxysterol and chitotriosidase as a biochemical screening markers of NP-C in children with cholestasis syndrome of unknown origin. For 108 patients (aged from 2 weeks to 7 years) the levels of cholestane-3ß,5α,6ß-triol (C-triol) and chitotriosidase (ChT) were measured. For patients with elevated C-triol and/or ChT the NPC1 and NPC2 genes were Sanger-sequenced and 47 additional genes (from the custom liver damage panel) were NGS-sequenced. RESULTS: Increased C-triol level (> 50 ng/ml) was detected in 4 (of 108) infants with cholestasis syndrome of unknown origin, with following molecular genetic NP-C diagnosis for one patient. Plasma cholesterol significantly correlates with C-triol (p < 0.05). NGS of high C-triol infants identified three patients with mutations in JAG1 (Alagille syndrome) and ABCB11 (Byler disease) genes. Increased ChT activity was detected in 8 (of 108) patients with various aetiologies, including NP-C, Byler disease and biliary atresia. CONCLUSION: Combined analysis of ChT activity and C-triol levels is an effective method for identifying NP-C.


Assuntos
Colestase/complicações , Hexosaminidases/sangue , Doença de Niemann-Pick Tipo C/diagnóstico , Doença de Niemann-Pick Tipo C/genética , Oxisteróis/sangue , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Síndrome de Alagille/genética , Aminoacil-tRNA Sintetases/genética , Atresia Biliar/genética , Biomarcadores/sangue , Proteínas de Transporte/genética , Criança , Pré-Escolar , Colestase Intra-Hepática/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Glicoproteínas/genética , Hexosaminidases/metabolismo , Humanos , Lactente , Recém-Nascido , Proteína Jagged-1/genética , Fígado , Masculino , Glicoproteínas de Membrana/genética , Mutação , Doenças Neurodegenerativas , Oxisteróis/metabolismo , Estudos Prospectivos , Sensibilidade e Especificidade
5.
J Clin Neurosci ; 68: 266-267, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31221578

RESUMO

Niemann Pick type C (NP-C) is an autosomal recessive neurovisceral lysosomal storage disorder caused by NPC1 and NPC2 gene mutations. We screened for NP-C 24 patients with Progressive Supranuclear Palsy and 10 with Multiple System Atrophy cerebellar type (MSA-C). Among PSP patients, no NPC1 or NPC2 gene variants were detected. One patient with MSA-C (10%) resulted to carry a pathogenic missense NPC1 gene mutation (p.C184Y) in heterozygous state. NPC1 genes variants might represent a risk or susceptibility factor in the development of α-synucleinopathies such as MSA. The common pattern of lysosomal dysfunction might explain the pathophysiological link between these disorders.


Assuntos
Atrofia de Múltiplos Sistemas/genética , Doença de Niemann-Pick Tipo C/complicações , Paralisia Supranuclear Progressiva/genética , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Doença de Niemann-Pick Tipo C/genética
6.
Neurosci Lett ; 706: 43-50, 2019 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-31067492

RESUMO

Niemann-Pick disease type C (NPC) is a fatal neurodegenerative condition with no FDA-approved therapy. Previous studies demonstrated that neuroinflammation is an early pathologic event and a disease modifier of NPC, affecting symptomatic onset and overall lifespan. Therefore, NPC-specific anti-inflammatory therapy may result in clinical benefit. However, to date, the initial trigger of the inflammatory onset and the mechanism driving the sustained chronic neuroinflammation remain unknown. In this study, we utilized a genome-wide transcriptome analysis to identify the key pathways involved in early NPC. Our results showed that an atypical pattern of interferon downstream signaling that involves both IFN-γ- and IFN-α-responsive genes is activated in pre-symptomatic Npc1-/- cerebella. Functional analysis of the differentially expressed genes highlighted microglial activation, anti-viral response, and T-lymphocyte activation and chemotaxis pathways. Multiplex protein analysis confirmed that a potent IFN-γ-responsive cytokine, IP-10/CXCL10 was significantly upregulated in the pre-symptomatic stage and further exacerbated in the terminal stage. In addition, several IFN-γ-responsive cytokines were elevated in the terminal stage Npc1-/- cerebella, including MIG/CXCL9, MCP-1/CCL2, MIP-1α/CCL3, MIP-1ß/CCL4, RANTES/CCL5, M-CSF, and IL-1α. Together, our results describe a novel activation pattern of interferon downstream signaling in pre-symptomatic NPC, as well as key inflammatory mediators that could serve as potential targets for NPC-specific anti-inflammatory therapy.


Assuntos
Cerebelo/metabolismo , Interferons/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Transdução de Sinais/genética , Animais , Citocinas/metabolismo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Interferons/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Knockout , Doença de Niemann-Pick Tipo C/genética , Sintomas Prodrômicos
7.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(5): 480-483, 2019 May 10.
Artigo em Chinês | MEDLINE | ID: mdl-31030438

RESUMO

OBJECTIVE: To delineate the clinical and genetic features of a Chinese boy suspected for Niemann-Pick disease type C. METHODS: The patient underwent clinical examination and was subjected to next generation sequencing. Suspected mutations were validated by Sanger sequencing. Potential impact of the novel mutation was predicted by SIFT, PolyPhen-2 and MutationTaster software. RESULTS: The child has featured hepatosplenomegaly, increased direct bilirubin, jaundiced skin and liver damage. DNA sequencing showed that he has carried compound heterozygous mutations of NPC1 gene, namely c.2728GG (p.P90R), which were inherited from his mother and father, respectively. The c.2728G>A (p.G910S) mutation was previously reported, while the c.269C>G (p.P90R) was a novel mutation. CONCLUSION: The child has suffered from Niemann-Pick disease type C due to mutations of NPC1 gene. Above finding has enriched the spectrum of NPC1 mutations and provided a basis for genetic counseling and prenatal diagnosis.


Assuntos
Proteínas de Transporte/genética , Glicoproteínas de Membrana/genética , Doença de Niemann-Pick Tipo C , Grupo com Ancestrais do Continente Asiático , Bilirrubina , Criança , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Doença de Niemann-Pick Tipo C/genética
8.
Mol Neurobiol ; 56(9): 6426-6435, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30820861

RESUMO

Niemann-Pick type C (NP-C) is a rare autosomal recessive disorder characterized by storage of unesterified glycolipids and cholesterol in lysosome and/or late endosome due to mutations in either NPC1 or NPC2 gene. This study aims to identify the spectrum of sequence alterations associated to NP-C in individuals with clinical suspicion of this disease. The entire coding region and flanking sequences of both genes associated to NP-C were evaluated in a total of 265 individuals that were referred to our laboratory. Clinical and/or biochemical suspicion of NP-C was confirmed by molecular analysis in 54 subjects. In this cohort, 33 different sequence alterations were identified in NPC1 and one in NPC2. Among those, 5 novel alterations in NPC1 gene were identified as follows: one deletion (p.Lys38_Tyr40del), one frameshift (p.Asn195Lysfs*2), and three missense mutations (p.Cys238Arg, p.Ser365Pro and, p.Val694Met) that are likely to be pathogenic through different approaches, including in silico tools as well as multiple sequence alignment throughout different species. We have also reported main clinical symptoms of patients with novel alterations and distribution of frequent symptoms in the cohort. Findings reported here contribute to the knowledge of mutation spectrum of NP-C, defining frequent mutations as well as novel sequence alterations associated to the disease.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação/genética , Doença de Niemann-Pick Tipo C/genética , Adolescente , Adulto , Alelos , Sequência de Aminoácidos , Sequência de Bases , Pré-Escolar , Feminino , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular/química , Masculino , Estrutura Secundária de Proteína
9.
J Appl Genet ; 60(2): 175-178, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30815842

RESUMO

We previously reported the altered pulmonary function and pathology found in the mouse model of infantile Niemann-Pick C1 disease, the Npc1-/- mouse. Despite its salutary properties on brain and liver parameters, we did not find efficacious effects of hydroxypropyl-ß-cyclodextrin (HPBCD) on pulmonary pathology. Since we had previously shown the beneficial effects of probucol on the somatic phenotype in the Npc1-/- mice, we have now studied the effects of combined therapy with HPBCD and probucol on the lung with mostly negative results. Body weight and lung weight for body weight were increased in parallel while inspiratory capacity for body weight was markedly decreased. Other physical, biochemical, and pulmonary function parameters were not much changed. There were trends towards improved lung elastance (p = 0.09) and compliance (p = 0.07).


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/administração & dosagem , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Probucol/administração & dosagem , Proteínas/genética , Animais , Colesterol/genética , Modelos Animais de Doenças , Combinação de Medicamentos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Knockout , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia
10.
Mol Genet Metab ; 125(4): 345-350, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30392741

RESUMO

Niemann-Pick disease, type C1 (NPC1) is an inborn error of metabolism that results in endolysosomal accumulation of unesterified cholesterol. Clinically, NPC1 manifests as cholestatic liver disease in the newborn or as a progressive neurogenerative condition characterized by cerebellar ataxia and cognitive decline. Currently there are no FDA approved therapies for NPC1. Thus, understanding the pathological processes that contribute to neurodegeneration will be important in both developing and testing potential therapeutic interventions. Neuroinflammation and necroptosis contribute to the NPC1 pathological cascade. Receptor Interacting Protein Kinase 1 and 3 (RIPK1 and RIPK3), are protein kinases that play a central role in mediating neuronal necroptosis. Our prior work suggested that pharmacological inhibition of RIPK1 had a significant but modest beneficial effect; however, the inhibitors used in that study had suboptimal pharmacokinetic properties. In this work we evaluated both pharmacological and genetic inhibition of RIPK1 kinase activity. Lifespan in both Npc1-/- mice treated with GSK'547, a RIPK1 inhibitor with better pharmacokinetic properties, and Npc1-/-:Ripk1kd/kd double mutant mice was significantly increased. In both cases the increase in lifespan was modest, suggesting that the therapeutic potential of RIPK1 inhibition, as a monotherapy, is limited. We thus investigated the potential of combining RIPK1 inhibition with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) therapy HPßCD has been shown to slow neurological disease progression in NPC1 mice, cats and patients. HPßCD appeared to have an additive positive effect on the pathology and survival of Npc1-/-:Ripk1kd/kd mice. RIPK1 and RIPK3 are both critical components of the necrosome, thus we were surprised to observe no increase survival in Npc1-/-;Ripk3-/- mice compared to Npc1-/- mice. These data suggest that although necroptosis is occurring in NPC1, the observed effects of RIPK1 inhibition may be related to its RIPK3-independent role in neuroinflammation and cytokine production.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Apoptose , Necrose , Doença de Niemann-Pick Tipo C/terapia , Proteínas/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Animais , Terapia Combinada , Modelos Animais de Doenças , Excipientes/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores
11.
J Appl Genet ; 59(4): 441-447, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30209687

RESUMO

Early onset Niemann-Pick C diseases are extremely rare, especially Niemann-Pick C2. Perhaps unusually for autosomal recessive diseases, heterozygotes for mutations in NPC1 manifest many biological variations. NPC2 deficiency has large effects on fertility. These features of NPC1 and NPC2 are reviewed in regard to possible negative selection for heterozygotes carrying null and hypomorphic alleles.


Assuntos
Proteínas de Transporte/genética , Estudo de Associação Genômica Ampla , Glicoproteínas/genética , Heterozigoto , Glicoproteínas de Membrana/genética , Doença de Niemann-Pick Tipo C/genética , Alelos , Animais , Fertilidade/genética , Humanos , Mutação , Seleção Genética
12.
Adv Biol Regul ; 70: 82-88, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30205942

RESUMO

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder resulting from mutations in either the NPC1 (95%) or NPC2 (5%) genes. NPC typically presents in childhood with visceral lipid accumulation and complex progressive neurodegeneration characterized by cerebellar ataxia, dysphagia, and dementia, resulting in a shortened lifespan. While cholesterol is widely acknowledged as the principal storage lipid in NPC, multiple species of sphingolipids accumulate as well. This accumulation of sphingolipids led to the initial assumption that NPC disease was caused by a deficiency in a sphingolipid catabolism enzyme, similar to sphingomyelinase deficiencies with which it shares a family name. It took about half a century to determine that NPC was in fact caused by a cholesterol trafficking defect, and still as we approach a century after the initial identification of the disease, the mechanisms by which sphingolipids accumulate remain poorly understood. Here we focus on the defects of sphingolipid catabolism in the endolysosomal compartment and how they contribute to the biology and pathology observed in NPC disease. This review highlights the need for further work on understanding and possibly developing treatments to correct the accumulation of sphingolipids in addition to cholesterol in this currently untreatable disease.


Assuntos
Doença de Niemann-Pick Tipo C/metabolismo , Animais , Humanos , Lisossomos/metabolismo , Proteína C1 de Niemann-Pick/genética , Proteína C1 de Niemann-Pick/metabolismo , Doença de Niemann-Pick Tipo C/genética , Esfingolipidoses/genética , Esfingolipidoses/metabolismo , Esfingolipídeos/metabolismo
13.
Proteins ; 86(11): 1165-1175, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30183109

RESUMO

The Niemann-Pick type C1 (NPC1) protein is a large transmembrane protein located in lysosomes/endosomes. NPC1 binds cholesterol (CLR) and transports it to cellular membrane and endoplasmic reticulum. Mutations in NPC1 cause Niemann-Pick type C (NPC) disease, a rare autosomal disorder characterized by intracellular accumulations of CLR and subsequent neurodegeneration leading to premature death. Among known disease-causing mutations in NPC1, Q92R is the one that is located in the N-terminal cholesterol-binding domain [NTD]. Here we study the effect of the mutation on the ability of NPC1 (NTD) to bind and retain CLR in the binding pocket using structural analysis. We compare characteristics of the Q92R and Q92S mutant type (MT) protein, which is predicted to be benign. We provide detailed investigation of the CLR-NPC1 (NTD) binding process; and propose the mechanism, by which Q92R mutation causes NPC disease. We show that although Q92 residue neither directly participates in catalytic activity of the NPC1 (NTD), nor defines its CLR-binding specificity - it is important for the overall protein structure as well as for providing favorable electrostatic environment for CLR transfer. Our results suggest that a negative electrostatic potential of the CLR binding site (the S-opening) might promote NPC2 interaction with NPC1 (NTD) and/or proper CLR orientation and its enforced transfer. We show that in contrast to the benign Q92S mutation, Q92R significantly reduces electrostatic potential around S-opening, and thus likely affects NPC1 (NTD)-NPC2 interaction and/or CLR transfer from NPC2 to NPC1.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutação Puntual , Sítios de Ligação , Proteínas de Transporte/química , Humanos , Glicoproteínas de Membrana/química , Simulação de Dinâmica Molecular , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , Ligação Proteica , Domínios Proteicos , Eletricidade Estática
14.
BMC Neurol ; 18(1): 117, 2018 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-30119649

RESUMO

BACKGROUND: Niemann-Pick disease type C (NPC) is a lysosomal storage disorder with severe prognosis. Disease-specific therapy is crucial to prevent disease progression; however, diagnosing NPC is quite difficult because of remarkably variable clinical presentations. The NPC Suspicion Index (NPC-SI) was developed to overcome this problem. Identifying preclinical cases is important for prevention and therapy. Here, we report three newly diagnosed NPC cases, one typical juvenile-onset case and the cases of two sisters with symptoms neurologically/psychiatrically indistinguishable from dystonia and schizophrenia, respectively. CASE PRESENTATION: In Case 1, a 25-year-old man presented with a 14-year history of intellectual disability, clumsiness, spastic ataxia, dysphagia, and frequent falls. Neurological examination revealed vertical supranuclear gaze palsy and involuntary movements. Ultrasonography revealed mild splenomegaly, and filipin staining of skin fibroblasts was positive with a variant staining pattern. NPC1 gene analysis showed compound heterozygous mutations, including c.1421C > T (p.P474L), a known causative mutation, and c.3722 T > C (p.L1241S), a new mutation. In Case 2, a 28-year-old woman, the proband, who had marked splenomegaly in her childhood, survived well, contrary to the expected severe prognosis of infantile NPC. She had minor neuropsychiatric symptoms including auditory hallucinations, nocturnal urination, and sleep paralysis. At the age of 28 years, she presented with a 1-year history of orofacial and oromandibular painful dystonia. The patient's 35-year-old sister (Case 3) was diagnosed with schizophrenia. In both cases, filipin staining of skin fibroblasts was positive with variant staining patterns, as well as elevated levels of urinary bile acids. NPC1 gene analysis showed compound heterozygous mutations including c.3011C > T (p.S1004 L), a known causative mutation, and c.160_161insG (p.D54GfsX4), a new mutation. Their mother, who was under therapy with modafinil for narcolepsy, shared the latter mutation. CONCLUSIONS: Marked clinical variability was observed in our three cases. NPC could masquerade as a pure neuropsychiatric disorder such as dystonia or schizophrenia. Abdominal ultrasonography, history evaluation, and neurological examination were quite important in the diagnostic process.


Assuntos
Proteínas de Transporte/genética , Glicoproteínas de Membrana/genética , Doença de Niemann-Pick Tipo C , Esquizofrenia , Adulto , Variação Biológica da População , Feminino , Humanos , Masculino , Doença de Niemann-Pick Tipo C/complicações , Doença de Niemann-Pick Tipo C/genética , Esquizofrenia/complicações , Esquizofrenia/genética
15.
Artigo em Inglês | MEDLINE | ID: mdl-29971198

RESUMO

Background: Adult-onset Niemann-Pick Type C is a rare neurogenetic lysosomal disorder, whose diagnosis is often delayed and missed because of its heterogeneous clinical presentations and rarity as well as the lack of awareness of characteristic eye findings among neurologists. Phenomenology Shown: Impaired smooth pursuits, saccades, and optokinetic nystagmus in the vertical direction, with relatively normal eye movements in the horizontal direction, and ataxia features on finger chase testing, tandem walking, and gait ataxia. Educational Value: Impairment of vertical eye movements in combination with ataxia, cognitive impairment, and/or psychiatric symptoms in an adult patient should always raise clinical suspicion of Niemann-Pick Type C.


Assuntos
Doença de Niemann-Pick Tipo C/complicações , Doença de Niemann-Pick Tipo C/diagnóstico , Transtornos da Motilidade Ocular/etiologia , Adulto , Encéfalo/diagnóstico por imagem , Proteínas de Transporte/genética , Humanos , Imagem por Ressonância Magnética , Masculino , Glicoproteínas de Membrana/genética , Mutação/genética , Doença de Niemann-Pick Tipo C/diagnóstico por imagem , Doença de Niemann-Pick Tipo C/genética , Transtornos da Motilidade Ocular/diagnóstico por imagem
16.
Hum Mol Genet ; 27(17): 3079-3098, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29878115

RESUMO

Niemann-Pick type C disease (NP-C) is a fatal neurodegenerative lysosomal storage disorder. It is caused in 95% of cases by a mutation in the NPC1 gene that encodes NPC1, an integral transmembrane protein localized to the limiting membrane of the lysosome. There is no cure for NP-C but there is a disease-modifying drug (miglustat) that slows disease progression but with associated side effects. Here, we demonstrate in a well-characterized mouse model of NP-C that a single administration of AAV-mediated gene therapy to the brain can significantly extend lifespan, improve quality of life, prevent or ameliorate neurodegeneration, reduce biochemical pathology and normalize or improve various indices of motor function. Over-expression of human NPC1 does not cause adverse effects in the brain and correctly localizes to late endosomal/lysosomal compartments. Furthermore, we directly compare gene therapy to licensed miglustat. Even at a low dose, gene therapy has all the benefits of miglustat but without adverse effects. On the basis of these findings and on-going ascendency of the field, we propose intracerebroventricular gene therapy as a potential therapeutic option for clinical use in NP-C.


Assuntos
Adenoviridae/genética , Proteínas de Transporte/administração & dosagem , Modelos Animais de Doenças , Transtornos Neurológicos da Marcha/prevenção & controle , Terapia Genética , Longevidade/genética , Glicoproteínas de Membrana/administração & dosagem , Doença de Niemann-Pick Tipo C/prevenção & controle , Animais , Proteínas de Transporte/fisiologia , Transtornos Neurológicos da Marcha/genética , Transtornos Neurológicos da Marcha/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Inflamação/prevenção & controle , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Mutação , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia
17.
Biol Chem ; 399(8): 903-910, 2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-29897878

RESUMO

Niemann-Pick type C disease (NPC) is a rare human disease, with limited effective treatment options. Most cases of NPC disease are associated with inactivating mutations of the NPC1 gene. However, cellular and molecular mechanisms responsible for the NPC1 pathogenesis remain poorly defined. This is partly due to the lack of a suitable animal model to monitor the disease progression. In this study, we used CRISPR to construct an NPC1-/- zebrafish model, which faithfully reproduced the cardinal pathological features of this disease. In contrast to the wild type (WT), the deletion of NPC1 alone caused significant hepatosplenomegaly, ataxia, Purkinje cell death, increased lipid storage, infertility and reduced body length and life span. Most of the NPC1-/- zebrafish died within the first month post fertilization, while the remaining specimens developed slower than the WT and died before reaching 8 months of age. Filipin-stained hepatocytes of the NPC1-/- zebrafish were clear, indicating abnormal accumulation of unesterified cholesterol. Lipid profiling showed a significant difference between NPC1-/- and WT zebrafish. An obvious accumulation of seven sphingolipids was detected in livers of NPC1-/- zebrafish. In summary, our results provide a valuable model system that could identify promising therapeutic targets and treatments for the NPC disease.


Assuntos
Modelos Animais de Doenças , Doença de Niemann-Pick Tipo C , Peixe-Zebra , Animais , Tamanho Corporal , Metabolismo dos Lipídeos , Fígado/química , Fígado/metabolismo , Atividade Motora , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
18.
Hum Mol Genet ; 27(12): 2101-2112, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29659804

RESUMO

Niemann-Pick type C (NPC) disease is a rare lysosomal storage disease caused primarily by mutations in NPC1. NPC1 encodes the lysosomal cholesterol transport protein NPC1. The most common NPC1 mutation is a missense mutation (NPC1I1061T) that causes misfolding and rapid degradation of mutant protein in the endoplasmic reticulum. Cholesterol accumulates in enlarged lysosomes as a result of decreased levels of lysosomal NPC1I1061T protein in patient cells. There is currently no cure or FDA-approved treatment for patients. We sought to identify novel compounds that decrease lysosomal cholesterol storage in NPC1I1061T/I1061T patient fibroblasts using a high-content screen with the cholesterol dye, filipin and the lysosomal marker, LAMP1. A total of 3532 compounds were screened, including 2013 FDA-approved drugs, 327 kinase inhibitors and 760 serum metabolites. Twenty-three hits were identified that decreased both filipin and LAMP1 signals. The majority of hits (16/21) were histone deacetylase (HDAC) inhibitors, a previously described class of modifiers of NPC cholesterol storage. Of the remaining hits, the antimicrobial compound, alexidine dihydrochloride had the most potent lysosomal cholesterol-reducing activity. Subsequent analyses showed that alexidine specifically increased levels of NPC1 transcript and mature protein in both control and NPC patient cells. Although unsuitable for systemic therapy, alexidine represents a unique tool compound for further NPC studies and as a potent inducer of NPC1. Together, these findings confirm the utility of high-content image-based compound screens of NPC1 patient cells and support extending the approach into larger compound collections.


Assuntos
Proteínas de Transporte/genética , Colesterol/genética , Inibidores de Histona Desacetilases/administração & dosagem , Glicoproteínas de Membrana/genética , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Biguanidas/administração & dosagem , Colesterol/metabolismo , Avaliação Pré-Clínica de Medicamentos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Fibroblastos/efeitos dos fármacos , Filipina/metabolismo , Inibidores de Histona Desacetilases/isolamento & purificação , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/genética , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Metaboloma/efeitos dos fármacos , Mutação de Sentido Incorreto , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia
19.
Hum Mol Genet ; 27(12): 2076-2089, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29617956

RESUMO

Niemann-Pick disease, type C1 (NPC1) is a neurodegenerative disorder with limited treatment options. NPC1 is associated with neuroinflammation; however, attempts to therapeutically target neuroinflammation in NPC1 have had mixed success. We show here that NPC1 neuroinflammation is characterized by an atypical microglia activation phenotype. Specifically, Npc1-/- microglia demonstrated altered morphology, reduced levels of lineage markers and a shift toward glycolytic metabolism. Treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD), a drug currently being studied in a phase 2b/3 clinical trial, reversed all microglia-associated defects in Npc1-/- animals. In addition, impairing microglia mediated neuroinflammation by genetic deletion of IRF8 led to decreased symptoms and increased lifespan. We identified CD22 as a marker of dysregulated microglia in Npc1 mutant mice and subsequently demonstrated that elevated cerebrospinal fluid levels of CD22 in NPC1 patients responds to HPßCD administration. Collectively, these data provide the first in-depth analysis of microglia function in NPC1 and suggest possible new therapeutic approaches.


Assuntos
Inflamação/tratamento farmacológico , Proteína C1 de Niemann-Pick/genética , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , 2-Hidroxipropil-beta-Ciclodextrina/administração & dosagem , Adolescente , Adulto , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/patologia , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Humanos , Lactente , Inflamação/genética , Inflamação/patologia , Fatores Reguladores de Interferon , Masculino , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Doença de Niemann-Pick Tipo C/líquido cefalorraquidiano , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/líquido cefalorraquidiano
20.
J Neurol Sci ; 386: 56-63, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29406968

RESUMO

Niemann-Pick Type C (NPC) disease is a rare neurovisceral disorder caused by mutations of either NPC1 or NPC2 gene and characterized by defective intracellular transport of cholesterol and glycosphingolipids, leading to neuron loss and myelin aberration in the central nervous system. In this study, by comparing protein expression in the cortical white matter tracts from mice at different postnatal days, we identified that in the NPC1 mutant (NPC1-/-) mice, the onset of myelination is delayed and the amount of the major myelin protein MBP and PLP, and oligodendrocyte regulatory factor Olig1 and Olig2, but not NG2 and Sox10, decreased significantly, suggesting a disruption of oligodendrocyte differentiation. Furthermore, in in vitro oligodendrocyte cultivation, NPC1-/- oligodendrocytes showed less response to the stimulation of neuron-conditioned medium (CdM), indicating a defect of oligodendrocyte per se. Interestingly, lovastatin restores the number of mature myelin-forming oligodendrocytes by increasing Olig1 and Olig2 expressions. Our data suggest a potential strategy for improving myelination using lovastatin in NPC disease.


Assuntos
Anticolesterolemiantes/uso terapêutico , Lovastatina/uso terapêutico , Mutação/genética , Bainha de Mielina/fisiologia , Proteína C1 de Niemann-Pick/genética , Doença de Niemann-Pick Tipo C , Oligodendroglia/patologia , Animais , Animais Recém-Nascidos , Antivirais/uso terapêutico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Filipina/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Proteína C1 de Niemann-Pick/metabolismo , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo
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