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1.
Ann Neurol ; 87(1): 139-153, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31658403

RESUMO

OBJECTIVE: The TMEM175/GAK/DGKQ locus is the 3rd strongest risk locus in genome-wide association studies of Parkinson disease (PD). We aimed to identify the specific disease-associated variants in this locus, and their potential implications. METHODS: Full sequencing of TMEM175/GAK/DGKQ followed by genotyping of specific associated variants was performed in PD (n = 1,575) and rapid eye movement sleep behavior disorder (RBD) patients (n = 533) and in controls (n = 1,583). Adjusted regression models and a meta-analysis were performed. Association between variants and glucocerebrosidase (GCase) activity was analyzed in 715 individuals with available data. Homology modeling, molecular dynamics simulations, and lysosomal localization experiments were performed on TMEM175 variants to determine their potential effects on structure and function. RESULTS: Two coding variants, TMEM175 p.M393T (odds ratio [OR] = 1.37, p = 0.0003) and p.Q65P (OR = 0.72, p = 0.005), were associated with PD, and p.M393T was also associated with RBD (OR = 1.59, p = 0.001). TMEM175 p.M393T was associated with reduced GCase activity. Homology modeling and normal mode analysis demonstrated that TMEM175 p.M393T creates a polar side-chain in the hydrophobic core of the transmembrane, which could destabilize the domain and thus impair either its assembly, maturation, or trafficking. Molecular dynamics simulations demonstrated that the p.Q65P variant may increase stability and ion conductance of the transmembrane protein, and lysosomal localization was not affected by these variants. INTERPRETATION: Coding variants in TMEM175 are likely to be responsible for the association in the TMEM175/GAK/DGKQ locus, which could be mediated by affecting GCase activity. ANN NEUROL 2020;87:139-153.


Assuntos
Canais de Potássio/genética , Sinucleinopatias/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença/genética , Genótipo , Glucosilceramidase/metabolismo , Humanos , Lisossomos/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Simulação de Dinâmica Molecular , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Polimorfismo de Nucleotídeo Único/genética , Canais de Potássio/fisiologia , Transtorno do Comportamento do Sono REM/genética , Transtorno do Comportamento do Sono REM/fisiopatologia , Sinucleinopatias/fisiopatologia
2.
Mov Disord ; 34(4): 526-535, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30788890

RESUMO

BACKGROUND: SMPD1 (acid-sphingomyelinase) variants have been associated with Parkinson's disease in recent studies. The objective of this study was to further investigate the role of SMPD1 mutations in PD. METHODS: SMPD1 was sequenced in 3 cohorts (Israel Ashkenazi Jewish cohort, Montreal/Montpellier, and New York), including 1592 PD patients and 975 controls. Additional data were available for 10,709 Ashkenazi Jewish controls. Acid-sphingomyelinase activity was measured by a mass spectrometry-based assay in the New York cohort. α-Synuclein levels were measured in vitro following CRISPR/Cas9-mediated knockout and siRNA knockdown of SMPD1 in HeLa and BE(2)-M17 cells. Lysosomal localization of acid-sphingomyelinase with different mutations was studied, and in silico analysis of their effect on acid-sphingomyelinase structure was performed. RESULTS: SMPD1 mutations were associated with PD in the Ashkenazi Jewish cohort, as 1.4% of PD patients carried the p.L302P or p.fsP330 mutation, compared with 0.37% in 10,709 Ashkenazi Jewish controls (OR, 3.7; 95%CI, 1.6-8.2; P = 0.0025). In the Montreal/Montpellier cohort, the p.A487V variant was nominally associated with PD (1.5% versus 0.14%; P = 0.0065, not significant after correction for multiple comparisons). Among PD patients, reduced acid-sphingomyelinase activity was associated with a 3.5- to 5.8-year earlier onset of PD in the lowest quartile versus the highest quartile of acid-sphingomyelinase activity (P = 0.01-0.001). We further demonstrated that SMPD1 knockout and knockdown resulted in increased α-synuclein levels in HeLa and BE(2)-M17 dopaminergic cells and that the p.L302P and p.fsP330 mutations impair the traffic of acid-sphingomyelinase to the lysosome. CONCLUSIONS: Our results support an association between SMPD1 variants, acid-sphingomyelinase activity, and PD. Furthermore, they suggest that reduced acid-sphingomyelinase activity may lead to α-synuclein accumulation. © 2019 International Parkinson and Movement Disorder Society.


Assuntos
Encéfalo/metabolismo , Predisposição Genética para Doença , Doença de Parkinson/genética , Esfingomielina Fosfodiesterase/genética , alfa-Sinucleína/metabolismo , Idoso , Encéfalo/patologia , Feminino , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Judeus/genética , Masculino , Pessoa de Meia-Idade , Mutação , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia
3.
Stroke ; 49(8): 1977-1980, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29986930

RESUMO

Background and Purpose- Absent or diminished α-galactosidase A (GLA) and acid α-glucosidase (GAA) enzyme activity are core features of Fabry and Pompe disease, respectively. Patients with Fabry or Pompe disease may have dilated intracranial arteries but whether lower GLA or GAA enzyme activity relates to brain arterial dilatation in other populations is unknown. Methods- Participants included Parkinson disease patients and nonblood-related controls, whose GLA and GAA enzymatic activities were measured in dried blood spots. Independent readers measured the axial arterial diameter of the ascending portion of the cavernous internal carotid arteries and the most proximal segment of the basilar artery in T2 black voids. Linear regression models were built to investigate the relationship between brain arterial diameters and lysosomal enzymatic activities. Results- The cohort included 107 participants (mean age, 66.5±10.3; 67% men). In an adjusted linear regression model, lower GLA activity was associated with larger brain arterial diameters (B=0.50±0.23, P=0.03). The strength of association was the greatest for the basilar artery diameter (B=0.80±0.33, P=0.02). Similarly, lower GAA activity was associated with an increased basilar arterial diameter (B=0.73±0.35, P=0.04). Conclusions- Lower GLA and GAA enzymatic activities were associated with larger brain arterial diameters, particularly the basilar artery diameter. Lower lysosomal enzymatic function in patients without Fabry or Pompe disease may play a role in brain arterial dilatation.


Assuntos
Artérias Cerebrais/diagnóstico por imagem , Artérias Cerebrais/enzimologia , Glucana 1,4-alfa-Glucosidase/metabolismo , Lisossomos/enzimologia , alfa-Galactosidase/metabolismo , Idoso , Encéfalo/irrigação sanguínea , Encéfalo/diagnóstico por imagem , Encéfalo/enzimologia , Estudos de Coortes , Dilatação Patológica/enzimologia , Ativação Enzimática/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Parkinsonianos/diagnóstico por imagem , Transtornos Parkinsonianos/enzimologia
4.
Mol Genet Metab ; 123(2): 135-139, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29100779

RESUMO

Deficiency of ß-Glucocerebrosidase (GBA) activity causes Gaucher Disease (GD). GD can be diagnosed by measuring GBA activity (Beutler and Kuhl, 1990). In this study, we assayed dried blood spots from a cohort (n=528) enriched for GBA mutation carriers (n=78) and GD patients (n=18) using both the tandem mass spectrometry (MS/MS) and fluorescence assays and their respective synthetic substrates. The MS/MS assay differentiated normal controls, which included GBA mutation carriers, from GD patients with no overlap. The fluorescence assay did not always differentiate normal controls including GBA mutation carriers from GD patients and false positives were observed. The MS/MS assay improved specificity compared to the fluorescence assay.


Assuntos
Biomarcadores/sangue , Teste em Amostras de Sangue Seco , Fluorescência , Doença de Gaucher/diagnóstico , Glucosilceramidase/sangue , Programas de Rastreamento , Espectrometria de Massas em Tandem/métodos , Bioensaio , Coleta de Amostras Sanguíneas , Estudos de Casos e Controles , Estudos de Coortes , Doença de Gaucher/metabolismo , Humanos
5.
J Biol Chem ; 290(23): 14361-80, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25878248

RESUMO

Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) Ca(2+) pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca(2+)-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca(2+) chelation. Interrogation of intracellular Ca(2+) handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca(2+) pools and in store-operated Ca(2+) uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca(2+) handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening.


Assuntos
Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Células-Tronco Neurais/patologia , Lipofuscinoses Ceroides Neuronais/patologia , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Chaperonas Moleculares/genética , Mutação , Células-Tronco Neurais/metabolismo , Lipofuscinoses Ceroides Neuronais/tratamento farmacológico , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
Brain ; 138(Pt 9): 2648-58, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26117366

RESUMO

Glucocerebrosidase (GBA) mutations have been associated with Parkinson's disease in numerous studies. However, it is unknown whether the increased risk of Parkinson's disease in GBA carriers is due to a loss of glucocerebrosidase enzymatic activity. We measured glucocerebrosidase enzymatic activity in dried blood spots in patients with Parkinson's disease (n = 517) and controls (n = 252) with and without GBA mutations. Participants were recruited from Columbia University, New York, and fully sequenced for GBA mutations and genotyped for the LRRK2 G2019S mutation, the most common autosomal dominant mutation in the Ashkenazi Jewish population. Glucocerebrosidase enzymatic activity in dried blood spots was measured by a mass spectrometry-based assay and compared among participants categorized by GBA mutation status and Parkinson's disease diagnosis. Parkinson's disease patients were more likely than controls to carry the LRRK2 G2019S mutation (n = 39, 7.5% versus n = 2, 0.8%, P < 0.001) and GBA mutations or variants (seven homozygotes and compound heterozygotes and 81 heterozygotes, 17.0% versus 17 heterozygotes, 6.7%, P < 0.001). GBA homozygotes/compound heterozygotes had lower enzymatic activity than GBA heterozygotes (0.85 µmol/l/h versus 7.88 µmol/l/h, P < 0.001), and GBA heterozygotes had lower enzymatic activity than GBA and LRRK2 non-carriers (7.88 µmol/l/h versus 11.93 µmol/l/h, P < 0.001). Glucocerebrosidase activity was reduced in heterozygotes compared to non-carriers when each mutation was compared independently (N370S, P < 0.001; L444P, P < 0.001; 84GG, P = 0.003; R496H, P = 0.018) and also reduced in GBA variants associated with Parkinson's risk but not with Gaucher disease (E326K, P = 0.009; T369M, P < 0.001). When all patients with Parkinson's disease were considered, they had lower mean glucocerebrosidase enzymatic activity than controls (11.14 µmol/l/h versus 11.85 µmol/l/h, P = 0.011). Difference compared to controls persisted in patients with idiopathic Parkinson's disease (after exclusion of all GBA and LRRK2 carriers; 11.53 µmol/l/h, versus 12.11 µmol/l/h, P = 0.036) and after adjustment for age and gender (P = 0.012). Interestingly, LRRK2 G2019S carriers (n = 36), most of whom had Parkinson's disease, had higher enzymatic activity than non-carriers (13.69 µmol/l/h versus 11.93 µmol/l/h, P = 0.002). In patients with idiopathic Parkinson's, higher glucocerebrosidase enzymatic activity was associated with longer disease duration (P = 0.002) in adjusted models, suggesting a milder disease course. We conclude that lower glucocerebrosidase enzymatic activity is strongly associated with GBA mutations, and modestly with idiopathic Parkinson's disease. The association of lower glucocerebrosidase activity in both GBA mutation carriers and Parkinson's patients without GBA mutations suggests that loss of glucocerebrosidase function contributes to the pathogenesis of Parkinson's disease. High glucocerebrosidase enzymatic activity in LRRK2 G2019S carriers may reflect a distinct pathogenic mechanism. Taken together, these data suggest that glucocerebrosidase enzymatic activity could be a modifiable therapeutic target.


Assuntos
Regulação Enzimológica da Expressão Gênica/genética , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Mutação/genética , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Idoso , Estudos de Coortes , Feminino , Genótipo , Humanos , Judeus/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/genética , Índice de Gravidade de Doença
7.
Sci Rep ; 10(1): 2479, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051502

RESUMO

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Better understanding of the underlying disease mechanism(s) is an urgent need for the development of disease-modifying therapeutics. Limited studies have been performed in large patient cohorts to identify protein alterations in cerebrospinal fluid (CSF), a proximal site to pathology. We set out to identify disease-relevant protein changes in CSF to gain insights into the etiology of Parkinson's disease and potentially assist in disease biomarker identification. In this study, we used liquid chromatography-tandem mass spectrometry in data-independent acquisition (DIA) mode to identify Parkinson's-relevant biomarkers in cerebrospinal fluid. We quantified 341 protein groups in two independent cohorts (n = 196) and a longitudinal cohort (n = 105 samples, representing 40 patients) consisting of Parkinson's disease and healthy control samples from three different sources. A first cohort of 53 Parkinson's disease and 72 control samples was analyzed, identifying 53 proteins with significant changes (p < 0.05) in Parkinson's disease relative to healthy control. We established a biomarker signature and multiple protein ratios that differentiate Parkinson's disease from healthy controls and validated these results in an independent cohort. The second cohort included 28 Parkinson's disease and 43 control samples. Independent analysis of these samples identified 41 proteins with significant changes. Evaluation of the overlapping changes between the two cohorts identified 13 proteins with consistent and significant changes (p < 0.05). Importantly, we found the extended granin family proteins as reduced in disease, suggesting a potential common mechanism for the biological reduction in monoamine neurotransmission in Parkinson's patients. Our study identifies several novel protein changes in Parkinson's disease cerebrospinal fluid that may be exploited for understanding etiology of disease and for biomarker development.


Assuntos
Cromograninas/líquido cefalorraquidiano , Doença de Parkinson/líquido cefalorraquidiano , Idoso , Biomarcadores/líquido cefalorraquidiano , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica , Espectrometria de Massas em Tandem
8.
Ann Clin Transl Neurol ; 7(10): 1816-1830, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32888397

RESUMO

OBJECTIVE: Reduction in glucocerebrosidase (GCase; encoded by GBA) enzymatic activity has been linked to Parkinson's disease (PD). Here, we correlated GCase activity and PD phenotype in the Parkinson's Progression Markers Initiative (PPMI) cohort. METHODS: We measured GCase activity in dried blood spots from 1559 samples of participants in the inception PPMI cohort, collected in four annual visits (from baseline visit to Year-3). Participants (PD, n = 392; controls, n = 175) were fully sequenced for GBA variants by means of genome-wide genotyping arrays, whole-exome sequencing, whole-genome sequencing, Sanger sequencing, and RNA-sequencing. RESULTS: Fifty-two PD participants (13.4%) and 13 (7.4%) controls carried a GBA variant. GBA status was strongly associated with GCase activity. Among noncarriers, GCase activity was similar between PD and controls. Among GBA p.E326K carriers (PD, n = 20; controls, n = 5), activity was significantly lower in PD carriers than control carriers (9.53 µmol/L/h vs. 11.68 µmol/L/h, P = 0.035). Glucocerebrosidase activity was moderately (r = 0.45) associated with white blood cell (WBC) count. Next, we divided the noncarriers with PD to tertiles based on WBC count-corrected enzymatic activity. Members of the lower tertile had higher MDS-Unified Parkinson's Disease Rating Scale motor score in the "off" medication examination at year-III exam. Longitudinal analyses demonstrated slight reduction of activity in samples collected earlier on in the study, likely because of longer storage time. INTERPRETATION: GCase activity is associated with GBA genotype, WBC count, and among p.E326K variant carriers, with PD status. Reduced activity may also be associated with worse phenotype but longer follow up is required to confirm this observation.


Assuntos
Demência/fisiopatologia , Glucosilceramidase/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Adulto , Demência/complicações , Demência/patologia , Progressão da Doença , Feminino , Genótipo , Glucosilceramidase/genética , Heterozigoto , Humanos , Masculino , Testes de Estado Mental e Demência , Pessoa de Meia-Idade , Mutação/genética , Doença de Parkinson/complicações , Fenótipo
9.
Artigo em Inglês | MEDLINE | ID: mdl-27110593

RESUMO

Mutations in glucocerebrosidase (GBA) are a common risk factor for Parkinson's disease (PD). The scavenger receptor class B member 2 (SCARB2) gene encodes a receptor responsible for the transport of glucocerebrosidase (GCase) to the lysosome. Two common SNPs in linkage disequilibrium with SCARB2, rs6812193 and rs6825004, have been associated with PD and Lewy Body Disease in genome wide association studies. Whether these SNPs are associated with altered glucocerebrosidase enzymatic activity is unknown. Our objective was to determine whether SCARB2 SNPs are associated with PD and with reduced GCase activity. The GBA gene was fully sequenced, and the LRRK2 G2019S and SCARB2 rs6812193 and rs6825004 SNPs were genotyped in 548 PD patients and 272 controls. GCase activity in dried blood spots was measured by tandem mass spectrometry. We tested the association between SCARB2 genotypes and PD risk in regression models adjusted for gender, age, and LRRK2 G2019S and GBA mutation status. We compared GCase activity between participants with different genotypes at rs6812193 and rs6825004. Genotype at rs6812193 was associated with PD status. PD cases were less likely to carry the T allele than the C allele (OR=0.71; p=0.004), but GCase enzymatic activity was similar across rs6812193 genotypes (C/C: 11.88 µmol/l/h; C/T: 11.80 µmol/l/h; T/T: 12.02 µmol/l/h; p=0.867). Genotype at rs6825004 was not associated with either PD status or GCase activity. In conclusion, our results support an association between SCARB2 genotype at rs6812193 and PD, but suggest that the increased risk is not mediated by GCase activity.

10.
Mol Genet Metab Rep ; 3: 55-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26937397

RESUMO

Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann-Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann-Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls.

11.
BMC Neurosci ; 5: 57, 2004 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-15588329

RESUMO

BACKGROUND: JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a approximately 1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Deltaex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology. RESULTS: To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Deltaex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced. CONCLUSIONS: These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival.


Assuntos
Linhagem Celular , Cerebelo/citologia , Glicoproteínas de Membrana/genética , Mitocôndrias/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Chaperonas Moleculares/genética , Lipofuscinoses Ceroides Neuronais/genética , Animais , Transporte Biológico , Catepsina D/metabolismo , Cerebelo/metabolismo , Cerebelo/ultraestrutura , Modelos Animais de Doenças , Endocitose , Homozigoto , Lisossomos/ultraestrutura , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Chaperonas Moleculares/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco/metabolismo , Células-Tronco/ultraestrutura
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