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1.
Autophagy ; 16(2): 347-370, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30983487

RESUMO

Parkinson disease (PD) is an age-related neurodegenerative disorder associated with misfolded SNCA/α-synuclein accumulation in brain. Impaired catabolism of SNCA potentiates formation of its toxic oligomers. LRRK2 (leucine-rich repeat kinase-2) mutations predispose to familial and sporadic PD. Mutant LRRK2 perturbs chaperone-mediated-autophagy (CMA) to degrade SNCA. We showed greater age-dependent accumulation of oligomeric SNCA in striatum and cortex of aged LRRK2R1441G knockin (KI) mice, compared to age-matched wildtype (WT) by 53% and 31%, respectively. Lysosomal clustering and accumulation of CMA-specific LAMP2A and HSPA8/HSC70 proteins were observed in aged mutant striatum along with increased GAPDH (CMA substrate) by immunohistochemistry of dorsal striatum and flow cytometry of ventral midbrain cells. Using our new reporter protein clearance assay, mutant mouse embryonic fibroblasts (MEFs) expressing either SNCA or CMA recognition 'KFERQ'-like motif conjugated with photoactivated-PAmCherry showed slower cellular clearance compared to WT by 28% and 34%, respectively. However, such difference was not observed after the 'KFERQ'-motif was mutated. LRRK2 mutant MEFs exhibited lower lysosomal degradation than WT indicating lysosomal dysfunction. LAMP2A-knockdown reduced total lysosomal activity and clearance of 'KFERQ'-substrate in WT but not in mutant MEFs, indicating impaired CMA in the latter. A CMA-specific activator, AR7, induced neuronal LAMP2A transcription and lysosomal activity in MEFs. AR7 also attenuated the progressive accumulation of both intracellular and extracellular SNCA oligomers in prolonged cultures of mutant cortical neurons (DIV21), indicating that oligomer accumulation can be suppressed by CMA activation. Activation of autophagic pathways to reduce aged-related accumulation of pathogenic SNCA oligomers is a viable disease-modifying therapeutic strategy for PD.Abbreviations: 3-MA: 3-methyladenine; AR7: 7-chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine; CMA: chaperone-mediated autophagy; CQ: chloroquine; CSF: cerebrospinal fluid; DDM: n-dodecyl ß-D-maltoside; DIV: days in vitro; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GWAS: genome-wide association studies; HSPA8/HSC70: heat shock protein 8; KFERQ: CMA recognition pentapeptide; KI: knockin; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LDH: lactate dehydrogenase; LRRK2: leucine-rich repeat kinase 2; MEF: mouse embryonic fibroblast; NDUFS4: NADH:ubiquinone oxidoreductase core subunit S4; NE: novel epitope; PD: Parkinson disease; RARA/RARα: retinoic acid receptor, alpha; SNCA: synuclein, alpha; TUBB3/TUJ1: tubulin, beta 3 class III; WT: wild-type.

2.
Transl Neurodegener ; 8: 23, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428316

RESUMO

Background: Parkinson's disease (PD) is characterized by dopaminergic neuronal loss in the substantia nigra pars compacta and intracellular inclusions called Lewy bodies (LB). During the course of disease, misfolded α-synuclein, the major constituent of LB, spreads to different regions of the brain in a prion-like fashion, giving rise to successive non-motor and motor symptoms. Etiology is likely multifactorial, and involves interplay among aging, genetic susceptibility and environmental factors. Main body: The prevalence of PD rises exponentially with age, and aging is associated with impairment of cellular pathways which increases susceptibility of dopaminergic neurons to cell death. However, the majority of those over the age of 80 do not have PD, thus other factors in addition to aging are needed to cause disease. Discovery of neurotoxins which can result in parkinsonism led to efforts in identifying environmental factors which may influence PD risk. Nevertheless, the causality of most environmental factors is not conclusively established, and alternative explanations such as reverse causality and recall bias cannot be excluded. The lack of geographic clusters and conjugal cases also go against environmental toxins as a major cause of PD. Rare mutations as well as common variants in genes such as SNCA, LRRK2 and GBA are associated with risk of PD, but Mendelian causes collectively only account for 5% of PD and common polymorphisms are associated with small increase in PD risk. Heritability of PD has been estimated to be around 30%. Thus, aging, genetics and environmental factors each alone is rarely sufficient to cause PD for most patients. Conclusion: PD is a multifactorial disorder involving interplay of aging, genetics and environmental factors. This has implications on the development of appropriate animal models of PD which take all these factors into account. Common converging pathways likely include mitochondrial dysfunction, impaired autophagy, oxidative stress and neuroinflammation, which are associated with the accumulation and spread of misfolded α-synuclein and neurodegeneration. Understanding the mechanisms involved in the initiation and progression of PD may lead to potential therapeutic targets to prevent PD or modify its course.

3.
Transl Neurodegener ; 6: 27, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29046784

RESUMO

The clinical diagnosis of neurodegenerative disorders based on phenotype is difficult in heterogeneous conditions with overlapping symptoms. It does not take into account the disease etiology or the highly variable clinical course even amongst patients diagnosed with the same disorder. The advent of next generation sequencing (NGS) has allowed for a system-wide, unbiased approach to identify all gene variants in the genome simultaneously. With the plethora of new genes being identified, genetic rather than phenotype-based classification of Mendelian diseases such as spinocerebellar ataxia (SCA), hereditary spastic paraplegia (HSP) and Charcot-Marie-Tooth disease (CMT) has become widely accepted. It has also become clear that gene variants play a role in common and predominantly sporadic neurodegenerative diseases such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). The observation of pleiotropy has emerged, with mutations in the same gene giving rise to diverse phenotypes, which further increases the complexity of phenotype-genotype correlation. Possible mechanisms of pleiotropy include different downstream effects of different mutations in the same gene, presence of modifier genes, and oligogenic inheritance. Future directions include development of bioinformatics tools and establishment of more extensive public genotype/phenotype databases to better distinguish deleterious gene variants from benign polymorphisms, translation of genetic findings into pathogenic mechanisms through in-vitro and in-vivo studies, and ultimately finding disease-modifying therapies for neurodegenerative disorders.

4.
Neurobiol Aging ; 58: 238.e9-238.e15, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28709720

RESUMO

Genetic variants are implicated in the development of amyotrophic lateral sclerosis (ALS), but it is unclear whether the burden of rare variants in ALS genes has an effect on survival. We performed whole genome sequencing on 8 familial ALS (FALS) patients with superoxide dismutase 1 (SOD1) mutation and whole exome sequencing on 46 sporadic ALS (SALS) patients living in Hong Kong and found that 67% had at least 1 rare variant in the exons of 40 ALS genes; 22% had 2 or more. Patients with 2 or more rare variants had lower probability of survival than patients with 0 or 1 variant (p = 0.001). After adjusting for other factors, each additional rare variant increased the risk of respiratory failure or death by 60% (p = 0.0098). The presence of the rare variant was associated with the risk of ALS (Odds ratio 1.91, 95% confidence interval 1.03-3.61, p = 0.03), and ALS patients had higher rare variant burden than controls (MB, p = 0.004). Our findings support an oligogenic basis with the burden of rare variants affecting the development and survival of ALS.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/mortalidade , Estudos de Associação Genética , Variação Genética/genética , Adulto , Idoso , Esclerose Amiotrófica Lateral/complicações , Esclerose Amiotrófica Lateral/epidemiologia , D-Aminoácido Oxidase/genética , Proteínas de Ligação a DNA/genética , Feminino , Hong Kong/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Receptores Proteína Tirosina Quinases/genética , Insuficiência Respiratória/epidemiologia , Insuficiência Respiratória/etiologia , Risco , Superóxido Dismutase-1/genética , Sobrevida , Sequenciamento Completo do Genoma
5.
Sci Rep ; 7: 40887, 2017 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-28098219

RESUMO

Aging, genetics and environmental toxicity are important etiological factors in Parkinson's disease (PD). However, its pathogenesis remains unclear. A major obstacle is the lack of an appropriate experimental model which incorporates genetic susceptibility, aging and prolonged environmental toxicity. Here, we explored the interplay amongst these factors using mutant LRRK2R1441G (leucine-rich-repeat-kinase-2) knockin mice. We found that mutant primary cortical and mesencephalic dopaminergic neurons were more susceptible to rotenone-induced ATP deficiency and cell death. Compared with wild-type controls, striatal synaptosomes isolated from young mutant mice exhibited significantly lower dopamine uptake after rotenone toxicity, due to reduced striatal synaptosomal mitochondria and synaptic vesicular proton pump protein (V-ATPase H) levels. Mutant mice developed greater locomotor deficits in open-field tests than wild-type mice following low oral rotenone doses given twice weekly over 50 weeks (half their lifespan). The increased locomotor deficit was associated with specific reduction in striatal mitochondrial Complex-I (NDUFS4) in rotenone-treated mutant but not in similarly treated wild-type mice. Our unique experimental model which incorporates genetic effect, natural aging and prolonged oral environmental toxicity administered to mutant knockin LRRK2 mice over half their life span, with observable and measurable phenotype, is invaluable in further studies of the pathogenic process and therapeutics of PD.


Assuntos
Apoptose/efeitos dos fármacos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/patologia , Rotenona/farmacologia , Administração Oral , Envelhecimento , Animais , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Técnicas de Introdução de Genes , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Rotenona/uso terapêutico , Sinaptossomos/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
6.
Neuropsychologia ; 80: 102-114, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26529488

RESUMO

Previous studies have suggested that the deteriorated visuomotor control in patients with PD (Parkinson's disease) is due to deficits in various aspects of the sensory-motor processing rather than motor control itself. In the current study, by taking a control-theoretic approach, we systematically examined how PD and antiparkinsonian medication affect visuomotor control and the underlying sensory-motor system. We tested 20 PD patients in both ON and OFF medication states and 20 demographically matched healthy controls with a commonly used manual control task. Specifically, in each 95-s trial, participants were instructed to use a joystick to control a randomly moving target to keep it centered on a computer display. We found that although antiparkinsonian medication improved visuomotor control in PD patients, they still showed significantly decreased control precision (measured by RMS error) and response amplitude (gain) as well as increased response delay (phase lag) compared with healthy controls. Our model-driven analysis revealed that PD impairs the responsiveness and the predicting ability of the sensory-motor system as well as the stability of the neuromuscular system. Taking antiparkinsonian medication improves the responsiveness of the sensory-motor system. More importantly, it improves the ability of the sensory-motor system to make sensory predictions of the current control actions (see Wolpert et al., 1995) to anticipate the input error signals and generate control responses ahead of time up to the level of healthy controls. However, taking antiparkinsonian medication does not improve the stability of the neuromuscular system. These results support the claim that the effect of antiparkinsonian medication on visuomotor control is mainly through improving visual-stimulus-dependent sensory-motor processing. The present study provides the first quantitative examination of the effects of PD and antiparkinsonian medication on the visual-stimulus-dependent sensory-motor and visual-stimulus-independent neuromuscular systems underlying visuomotor control. The findings have practical implications for developing sensitive assessment tools to evaluate the efficacy of different therapies for PD and preliminary screening and training tools for fitness-to-drive in PD patients.


Assuntos
Doença de Parkinson/complicações , Transtornos Psicomotores/etiologia , Sensação/fisiologia , Idoso , Análise de Variância , Antiparkinsonianos/farmacologia , Antiparkinsonianos/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/tratamento farmacológico , Estimulação Luminosa , Transtornos Psicomotores/tratamento farmacológico , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Sensação/efeitos dos fármacos , Estatística como Assunto , Fatores de Tempo
7.
Brain Behav ; 5(4): e00321, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25798335

RESUMO

BACKGROUND: Familial spastic paraplegia (FSP) is a heterogeneous group of disorders characterized primarily by progressive lower limb spasticity and weakness. More than 50 disease loci have been described with different modes of inheritance. Recently, we described a novel missense mutation (c.803G>A, p.R268Q) in the plasma membrane calcium ATPase (PMCA4, or ATP2B4) gene in a Chinese family with autosomal dominant FSP. Further to this finding, here we describe the functional effect of this mutation. METHODS: As PMCA4 removes cytosolic calcium, we measured transient changes and the time-dependent decay of cytosolic calcium level as visualized by using fura-2 fluorescent dye with confocal microscopy in human SH-SY5Y neuroblastoma cells overexpressing either wild-type or R268Q mutant PMCA4. RESULTS: Overexpressing both wild-type and R268Q PMCA4 significantly reduced maximum calcium surge after KCl-induced depolarization as compared with vector control cells. However, cells overexpressing mutant PMCA4 protein demonstrated significantly higher level of calcium surge when compared with wild-type. Furthermore, the steady-state cytosolic calcium concentration in these mutant cells remained markedly higher than the wild-type after SERCA inhibition by thapsigargin. CONCLUSION: Our result showed that p.R268Q mutation in PMCA4 resulted in functional changes in calcium homeostasis in human neuronal cells. This suggests that calcium dysregulation may be associated with the pathogenesis of FSP.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Mutação de Sentido Incorreto , Paraplegia Espástica Hereditária/genética , Western Blotting , Cálcio/análise , Corantes Fluorescentes , Fura-2 , Humanos , Microscopia Confocal/métodos , ATPases Transportadoras de Cálcio da Membrana Plasmática , Reação em Cadeia da Polimerase em Tempo Real
8.
Ann Clin Transl Neurol ; 1(3): 199-208, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25356398

RESUMO

OBJECTIVE: Mutations in leucine-rich repeat kinase 2 (LRRK2) pose a significant genetic risk in familial and sporadic Parkinson's disease (PD). R1441 mutation (R1441G/C) in its GTPase domain is found in familial PD. How LRRK2 interacts with synaptic proteins, and its role in dopamine (DA) homeostasis and synaptic vesicle recycling remain unclear. METHODS: To explore the pathogenic effects of LRRK2(R1441G) mutation on nigrostriatal synaptic nerve terminals and locomotor activity, we generated C57BL/6N mice with homozygous LRRK2(R1441G) knockin (KI) mutation, and examined for early changes in nigrostriatal region, striatal synaptosomal [(3)H]-DA uptake and locomotor activity after reserpine-induced DA depletion. RESULTS: Under normal conditions, mutant mice showed no differences, (1) in amount and morphology of nigrostriatal DA neurons and neurites, (2) tyrosine hydroxylase (TH), DA uptake transporter (DAT), vesicular monoamine transporter-2 (VMAT2) expression in striatum, (3) COX IV, LC3B, Beclin-1 expression in midbrain, (4) LRRK2 expression in total cell lysate from whole brain, (5) α-synuclein, ubiquitin, and tau protein immunostaining in midbrain, (6) locomotor activity, compared to wild-type controls. However, after a single intraperitoneal reserpine dose, striatal synaptosomes from young 3-month-old mutant mice demonstrated significantly lower DA uptake with impaired locomotor activity and significantly slower recovery from the effects of reserpine. INTERPRETATION: Although no abnormal phenotype was observed in mutant LRRK2(R1441G) mice, the KI mutation increases vulnerability to reserpine-induced striatal DA depletion and perturbed DA homeostasis resulting in presynaptic dysfunction and locomotor deficits with impaired recovery from reserpine. This subtle nigrostriatal synaptic vulnerability may reflect one of the earliest pathogenic processes in LRRK2-associated PD.

9.
PLoS One ; 9(8): e104790, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25119969

RESUMO

Familial spastic paraplegia (FSP) is a heterogeneous group of disorders characterized primarily by progressive lower limb spasticity and weakness. More than 50 disease loci have been described with different modes of inheritance. In this study, we identified a novel missense mutation (c.803G>A, p.R268Q) in the plasma membrane calcium ATPase (PMCA4, or ATP2B4) gene in a Chinese family with autosomal dominant FSP using whole-exome sequencing and confirmed with Sanger sequencing. This mutation co-segregated with the phenotype in the six family members studied and is predicted to be pathogenic when multiple deleteriousness predictions were combined. This novel R268Q mutation was not present in over 7,000 subjects in public databases, and over 1,000 Han Chinese in our database. Prediction of potential functional consequence of R268Q mutation on PMCA4 by computational modeling revealed that this mutation is located in protein aggregation-prone segment susceptible to protein misfolding. Analysis for thermodynamic protein stability indicated that this mutation destabilizes the PMCA4 protein structure with higher folding free energy. As PMCA4 functions to maintain neuronal calcium homeostasis, our result showed that calcium dysregulation may be associated with the pathogenesis of FSP.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Paraplegia Espástica Hereditária/genética , Sequência de Bases , Exoma/genética , Genes Dominantes/genética , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Linhagem , Dobramento de Proteína , Análise de Sequência de DNA
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