RESUMO
Mutations in CHCHD2 are linked to a familial, autosomal dominant form of Parkinson's disease (PD). The gene product may regulate mitochondrial respiratory function. However, whether mitochondrial dysfunction induced by CHCHD2 mutations further yields α-synuclein pathology is unclear. Here, we provide compelling genetic evidence that mitochondrial dysfunction induced by PD-linked CHCHD2 T61I mutation promotes α-synuclein aggregation using brain autopsy, induced pluripotent stem cells (iPSCs) and Drosophila genetics. An autopsy of an individual with CHCHD2 T61I revealed widespread Lewy pathology with both amyloid plaques and neurofibrillary tangles that appeared in the brain stem, limbic regions and neocortex. A prominent accumulation of sarkosyl-insoluble α-synuclein aggregates, the extent of which was comparable to that of a case with α-synuclein (SNCA) duplication, was observed in CHCHD2 T61I brain tissue. The prion-like activity and morphology of α-synuclein fibrils from the CHCHD2 T61I brain tissue were similar to those of fibrils from SNCA duplication and sporadic PD brain tissues. α-Synuclein insolubilization was reproduced in dopaminergic neuron cultures from CHCHD2 T61I iPSCs and Drosophila lacking the CHCHD2 ortholog or expressing the human CHCHD2 T61I. Moreover, the combination of ectopic α-synuclein expression and CHCHD2 null or T61I enhanced the toxicity in Drosophila dopaminergic neurons, altering the proteolysis pathways. Furthermore, CHCHD2 T61I lost its mitochondrial localization by α-synuclein in Drosophila. The mislocalization of CHCHD2 T61I was also observed in the patient brain. Our study suggests that CHCHD2 is a significant mitochondrial factor that determines α-synuclein stability in the etiology of PD.
Assuntos
Proteínas de Ligação a DNA/genética , Mutação com Perda de Função , Doença de Parkinson/genética , Fatores de Transcrição/genética , alfa-Sinucleína/química , Idoso , Animais , Autopsia , Encéfalo/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Drosophila , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Neurônios/citologia , Doença de Parkinson/metabolismo , Linhagem , Agregados Proteicos , Estabilidade Proteica , Fatores de Transcrição/metabolismoRESUMO
Recently, the genetic variability in lysosomal storage disorders has been implicated in the pathogenesis of Parkinson's disease. Here, we found that variants in prosaposin (PSAP), a rare causative gene of various types of lysosomal storage disorders, are linked to Parkinson's disease. Genetic mutation screening revealed three pathogenic mutations in the saposin D domain of PSAP from three families with autosomal dominant Parkinson's disease. Whole-exome sequencing revealed no other variants in previously identified Parkinson's disease-causing or lysosomal storage disorder-causing genes. A case-control association study found two variants in the intronic regions of the PSAP saposin D domain (rs4747203 and rs885828) in sporadic Parkinson's disease had significantly higher allele frequencies in a combined cohort of Japan and Taiwan. We found the abnormal accumulation of autophagic vacuoles, impaired autophagic flux, altered intracellular localization of prosaposin, and an aggregation of α-synuclein in patient-derived skin fibroblasts or induced pluripotent stem cell-derived dopaminergic neurons. In mice, a Psap saposin D mutation caused progressive motor decline and dopaminergic neurodegeneration. Our data provide novel genetic evidence for the involvement of the PSAP saposin D domain in Parkinson's disease.
Assuntos
Predisposição Genética para Doença/genética , Doença de Parkinson/genética , Saposinas/genética , Idoso , Animais , Estudos de Casos e Controles , Neurônios Dopaminérgicos/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Mutantes , Pessoa de Meia-Idade , Degeneração Neural/genética , Degeneração Neural/patologia , Doença de Parkinson/patologiaRESUMO
Parkinson's disease (PD) is characterized as a chronic and progressive neurodegenerative disorder, and the deposition of specific protein aggregates of α-synuclein, termed Lewy bodies, is evident in multiple brain regions of PD patients. Although there are several available medications to treat PD symptoms, these medications do not prevent the progression of the disease. Soluble epoxide hydrolase (sEH) plays a key role in inflammation associated with the pathogenesis of PD. Here we found that MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced neurotoxicity in the mouse striatum was attenuated by subsequent repeated administration of TPPU, a potent sEH inhibitor. Furthermore, deletion of the sEH gene protected against MPTP-induced neurotoxicity, while overexpression of sEH in the striatum significantly enhanced MPTP-induced neurotoxicity. Moreover, the expression of the sEH protein in the striatum from MPTP-treated mice or postmortem brain samples from patients with dementia of Lewy bodies (DLB) was significantly higher compared with control groups. Interestingly, there was a positive correlation between sEH expression and phosphorylation of α-synuclein in the striatum. Oxylipin analysis showed decreased levels of 8,9-epoxy-5Z,11Z,14Z-eicosatrienoic acid in the striatum of MPTP-treated mice, suggesting increased activity of sEH in this region. Interestingly, the expression of sEH mRNA in human PARK2 iPSC-derived neurons was higher than that of healthy control. Treatment with TPPU protected against apoptosis in human PARK2 iPSC-derived dopaminergic neurons. These findings suggest that increased activity of sEH in the striatum plays a key role in the pathogenesis of neurodegenerative disorders such as PD and DLB. Therefore, sEH may represent a promising therapeutic target for α-synuclein-related neurodegenerative disorders.
Assuntos
Epóxido Hidrolases/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Linhagem Celular , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Células HEK293 , Humanos , Corpos de Lewy/efeitos dos fármacos , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/metabolismo , Degeneração Neural/patologia , RNA Mensageiro/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Bilirubin, the end product of heme redox metabolism, has cytoprotective properties and is an essential metabolite associated with cardiovascular disease, inflammatory bowel disease, type 2 diabetes, and neurodegenerative diseases including Parkinson's disease (PD). PD is characterized by progressive degeneration of nigral dopaminergic neurons and is associated with elevated oxidative stress due to mitochondrial dysfunction. In this study, using a ratiometric bilirubin probe, we revealed that the mitochondrial inhibitor, rotenone, which is widely used to create a PD model, significantly decreased intracellular bilirubin levels in HepG2 cells. Chemical screening showed that BRUP-1 was a top hit that restored cellular bilirubin levels that were lowered by rotenone. We found that BRUP-1 up-regulated the expression level of heme oxygenase-1 (HO-1), one of the rate-limiting enzyme of bilirubin production via nuclear factor erythroid 2-related factor 2 (Nrf2) activation. In addition, we demonstrated that this Nrf2 activation was due to a direct inhibition of the interaction between Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) by BRUP-1. Both HO-1 up-regulation and bilirubin restoration by BRUP-1 treatment were significantly abrogated by Nrf2 silencing. In neuronal PC12D cells, BRUP-1 also activated the Nrf2-HO-1 axis and increased bilirubin production, resulted in the suppression of neurotoxin-induced cell death, reactive oxygen species production, and protein aggregation, which are hallmarks of PD. Furthermore, BRUP-1 showed neuroprotective activity against rotenone-treated neurons derived from induced pluripotent stem cells. These findings provide a new member of Keap1-Nrf2 direct inhibitors and suggest that chemical modulation of heme metabolism using BRUP-1 may be beneficial for PD treatment.
Assuntos
Bilirrubina/metabolismo , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson Secundária/prevenção & controle , Animais , Inativação Gênica , Heme Oxigenase-1/metabolismo , Células Hep G2 , Humanos , Células-Tronco Pluripotentes Induzidas , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Neurotoxinas/toxicidade , Células PC12 , Doença de Parkinson Secundária/induzido quimicamente , RNA Interferente Pequeno/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Rotenona/toxicidade , Desacopladores/toxicidadeRESUMO
OBJECTIVE: The objective of this study was to determine comprehensive metabolic changes of caffeine in the serum of patients with parkinsonian disorders including Parkinson's disease (PD), progressive supranuclear palsy (PSP), and multiple system atrophy (MSA) and to compare this with healthy control serum. METHODS: Serum levels of caffeine and its 11 downstream metabolites from independent double cohorts consisting of PD (n = 111, 160), PSP (n = 30, 19), MSA (n = 23, 17), and healthy controls (n = 43, 31) were examined by liquid chromatography-mass spectrometry. The association of each metabolite with clinical parameters and medication was investigated. Mutations in caffeine-associated genes were investigated by direct sequencing. RESULTS: A total of 9 metabolites detected in more than 50% of participants in both cohorts were decreased in 3 parkinsonian disorders compared with healthy controls without any significant association with age at sampling, sex, or disease severity (Hoehn and Yahr stage and Unified Parkinson's Disease Rating Scale motor section) in PD, and levodopa dose or levodopa equivalent dose in PSP and MSA. Of the 9 detected metabolites, 8 in PD, 5 in PSP, and 3 in MSA were significantly decreased in both cohorts even after normalizing to daily caffeine consumption. No significant genetic variations in CYP1A2 or CYP2E1 were detected when compared with controls. CONCLUSION: Serum caffeine metabolic profiles in 3 parkinsonian diseases show a high level of overlap, indicative of a common potential mechanism such as caffeine malabsorption from the small intestine, hypermetabolism, increased clearance of caffeine, and/or reduced caffeine consumption. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Atrofia de Múltiplos Sistemas , Transtornos Parkinsonianos , Paralisia Supranuclear Progressiva , Cafeína , Humanos , Metaboloma , Atrofia de Múltiplos Sistemas/tratamento farmacológico , Transtornos Parkinsonianos/tratamento farmacológicoRESUMO
The ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase Parkin, two gene products associated with young-onset Parkinson's disease (PD), participate in mitochondrial quality control. The phosphorylation of mitochondrial polyUb by PINK1, which is activated in a mitochondrial membrane potential (ΔΨm)-dependent manner, facilitates the mitochondrial translocation and concomitant enzymatic activation of Parkin, leading to the clearance of phospho-polyUb-tagged mitochondria via mitophagy. Thus, Ub phosphorylation is a key event in PINK1-Parkin-mediated mitophagy. Here, we examined the role of phospho-Ub signaling in the pathogenesis of PD using fly PD models, human brain tissue and dopaminergic neurons derived from induced pluripotent stem cells (iPSCs) containing Parkin or PINK1 mutations, as well as normal controls. We report that phospho-Ub signaling is highly conserved between humans and Drosophila, and that phospho-Ub signaling and the relocation of axonal mitochondria upon ΔΨm reduction are indeed compromised in human dopaminergic neurons containing Parkin or PINK1 mutations. Moreover, phospho-Ub signaling is prominent in tyrosine hydroxylase-positive neurons compared with tyrosine hydroxylase-negative neurons, suggesting that PINK1-Parkin signaling is more required for dopaminergic neurons. These results shed light on the particular vulnerability of dopaminergic neurons to mitochondrial stress.
Assuntos
Doença de Parkinson/genética , Proteínas Quinases/genética , Ubiquitina/metabolismo , Animais , Encéfalo/metabolismo , Neurônios Dopaminérgicos/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Doença de Parkinson/etiologia , Fosforilação , Proteínas Quinases/metabolismo , Transporte Proteico , Transdução de Sinais , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
INTRODUCTION: Chemical intervention of autophagy has been investigated in clinical trials for various age-related conditions such as sarcopenia and neurodegeneration. However, at present, no autophagy inducer has been established as a disease-modifying agent against neurodegenerative diseases. METHODS: We screened a library consisting of 796 medicines clinically approved (in Japan) for autophagy enhancers as potential neurodegeneration therapeutics using HeLa cells stably expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) followed by an analysis of the molecular mechanisms using various neuronal models. RESULTS: The primary screening identified 152 hits in a static cellular state. A widely available Alzheimer's disease drug, memantine, which antagonizes N-Methyl-d-aspartate receptor (NMDAR), was one of the hits. Memantine increased the levels of LC3-II in a dose-dependent and time-dependent manner, and upregulated autophagic flux. In addition, the pharmacological effects of memantine on autophagy were independent of mTORC1 activity and NMDAR activation. Furthermore, a VPS34 inhibitor suppressed the memantine-induced LC3-II upregulation, suggesting that memantine may affect VPS34 complex activity. Notably, intracellular Huntington's disease-specific aggregates of elongated huntingtin, a well-established autophagy substrate, were significantly decreased by memantine. In addition, memantine enhanced elimination of degraded mitochondrial in neurons derived from induced pluripotent stem cells of PARK2 or PARK6 patients, who exhibited defective PINK1/parkin-mediated mitophagy, suggests that memantine accelerated the clearance of damaged mitochondria. CONCLUSION: These findings indicate that memantine may be beneficial for the treatment of neurodegeneration characterized by the abnormal accumulation of autophagy or mitophagy substrates.
Assuntos
Autofagia/efeitos dos fármacos , Memantina/farmacologia , Fármacos Neuroprotetores/farmacologia , Actinas/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Fatores de Tempo , Regulação para Cima/efeitos dos fármacosRESUMO
Patient-specific induced pluripotent stem cells (iPSCs) show promise for use as tools for in vitro modeling of Parkinson's disease. We sought to improve the efficiency of dopaminergic (DA) neuron induction from iPSCs by the using surface markers expressed in DA progenitors to increase the significance of the phenotypic analysis. By sorting for a CD184high/CD44- fraction during neural differentiation, we obtained a population of cells that were enriched in DA neuron precursor cells and achieved higher differentiation efficiencies than those obtained through the same protocol without sorting. This high efficiency method of DA neuronal induction enabled reliable detection of reactive oxygen species (ROS) accumulation and vulnerable phenotypes in PARK2 iPSCs-derived DA neurons. We additionally established a quantitative system using the mt-mKeima reporter system to monitor mitophagy in which mitochondria fuse with lysosomes and, by combining this system with the method of DA neuronal induction described above, determined that mitophagy is impaired in PARK2 neurons. These findings suggest that the efficiency of DA neuron induction is important for the precise detection of cellular phenotypes in modeling Parkinson's disease.
Assuntos
Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Apoptose , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Receptores de Hialuronatos/metabolismo , Mitofagia/fisiologia , Modelos Neurológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Ethambutol is a common medicine used for the treatment of tuberculosis, which can have serious side effects, such as retinal and liver dysfunction. Although ethambutol has been reported to impair autophagic flux in rat retinal cells, the precise molecular mechanism remains unclear. Using various mammalian cell lines, we showed that ethambutol accumulated in autophagosomes and vacuolated lysosomes, with marked Zn(2+) accumulation. The enlarged lysosomes were neutralized and were infiltrated with Zn(2+) accumulations in the lysosomes, with simultaneous loss of acidification. These results suggest that EB neutralizes lysosomes leading to insufficient autophagy, implying that some of the adverse effects associated with EB in various organs may be of this mechanism.
Assuntos
Antituberculosos/administração & dosagem , Etambutol/administração & dosagem , Lisossomos/fisiologia , Fagossomos/fisiologia , Zinco/farmacocinética , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Taxa de Depuração Metabólica/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Fagossomos/ultraestrutura , RatosRESUMO
BACKGROUND: The benefits of escalating the dose of donepezil in patients who are already receiving long-term treatment with it have not been well evaluated. Therefore, an exploratory study to assess the effects of donepezil dose escalation in patients with Parkinson's disease with dementia, and specifically on patients receiving long-term treatment with donepezil, was performed. METHODS: Patients treated with 5-mg/day donepezil for at least 3 months and having a Mini-Mental State Examination (MMSE) score between 10 and 26 were included in this study. Donepezil dosage was then increased to 10 mg/day for 12 weeks. The outcome measures were a modified form of the Neuropsychiatric Inventory (NPI) with an extra domain for additional evaluation of fluctuation in cognitive functions (NPI-11) and the MMSE. RESULTS: Of the nine patients enrolled, two withdrew because of nausea and inability to be assessed on the predetermined date; this left seven participants (four men and three women) with a mean age of 74.6 ± 6.9 years, a mean period of Parkinson's disease of 11.7 ± 7.5 years, and median donepezil use of 7 months (range: 3-56 months). At baseline, the mean total NPI-11 and mean MMSE scores were 18.3 ± 5.6 points and 21.3 ± 5.3 points, respectively. At week 12, they improved by 8.3 points (P < 0.01) and 3.0 points (P = 0.08), respectively, from the baseline. The NPI symptom domains that improved by 1 or more points were hallucination (1.3 points), depression (1.0 points), anxiety (1.6 points), and aberrant motor behaviour (1.7 points). None of the patients withdrew because of worsening of parkinsonism. CONCLUSIONS: The present results suggest that treatment with dose escalation of donepezil from 5 mg/day to 10 mg/day may be therapeutically useful for patients with Parkinson's disease with dementia who have taken donepezil 5 mg/day in the long term.
Assuntos
Inibidores da Colinesterase/administração & dosagem , Demência/tratamento farmacológico , Indanos/administração & dosagem , Doença de Parkinson/tratamento farmacológico , Piperidinas/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Inibidores da Colinesterase/efeitos adversos , Cognição/efeitos dos fármacos , Donepezila , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Humanos , Indanos/efeitos adversos , Assistência de Longa Duração , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/psicologia , Piperidinas/efeitos adversos , Estudos Prospectivos , Tomografia Computadorizada de Emissão de Fóton Único , Resultado do TratamentoRESUMO
The CMT1A variant accounts for over 60% of cases of Charcot-Marie-Tooth disease (CMT), one of the most common human neuropathies. The cause of CMT1A has been identified as the duplication of PMP22, a myelin protein expressed in Schwann cells. Yet, the pathological mechanisms have not been elucidated, and no treatment is currently available. In our study, we established an iPS cell line from a CMT1A patient with PMP22 duplication. The generated iPSCs maintain pluripotency and in vitro differentiation potency.
Assuntos
Doença de Charcot-Marie-Tooth , Células-Tronco Pluripotentes Induzidas , Proteínas da Mielina , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Linhagem Celular , Diferenciação Celular , Duplicação Gênica , MasculinoRESUMO
Appropriate control induced pluripotent stem cells (iPSCs) are essential for studying iPSCs derived from patients with Parkinson's disease (PD). In this study, we established an iPSC line from a healthy male donor. The iPSCs showed pluripotency, capacity to differentiate into three germ layers, and normal karyotypes. Additionally, we confirmed that the iPSC line did not exhibit any PD-related gene abnormalities. This iPSC line will be useful for PD research.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Humanos , Masculino , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Linhagem CelularRESUMO
The appropriate control of induced pluripotent stem cells (iPSCs) is essential for studying iPSCs derived from patients with Parkinson's disease (PD). Here, we established an iPSC line from a healthy female donor. The iPSCs were pluripotent, could differentiate into three germ layers, and had normal karyotypes. We also confirmed that the iPSC line exhibited no PD-related gene abnormalities. This iPSC line will be useful for PD research.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Humanos , Feminino , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Linhagem Celular , Camadas Germinativas/metabolismoRESUMO
PARK2 is the most common autosomal recessive form of Parkinson's disease and is caused by mutations in parkin that result in early-onset loss of dopaminergic neurons in the substantia nigra. In this study, we established an induced pluripotent stem cell (iPSC) line from a patient harboring a homozygous exon 3 deletion in PARK2. The established iPSCs showed pluripotency, the capacity to differentiate into the three germ layers, and normal karyotypes.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Transtornos Parkinsonianos , Humanos , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Transtornos Parkinsonianos/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Parkinson's disease is the second most common neurodegenerative disorder and is pathologically characterized by synuclein-rich aggregations (Lewy bodies) in neurons. Multiplication of the synuclein gene (SNCA) increases the mRNA and protein levels of synuclein, resulting in autosomal dominant hereditary Parkinson's disease. In the present study, we established three isogenic induced pluripotent stem cells (iPSCs) from a patient harboring SNCA duplication, which showed pluripotency, three-germ layer differentiation capacity, and normal karyotypes.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Humanos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Clonais/metabolismo , Diferenciação CelularRESUMO
PURPOSE: Parkinson disease (PD) is a common progressive neurodegenerative disorder in our ageing society. Early-stage PD biomarkers are desired for timely clinical intervention and understanding of pathophysiology. Since one of the characteristics of PD is the progressive loss of dopaminergic neurons in the substantia nigra pars compacta, we propose a feature extraction method for analysing the differences in the substantia nigra between PD and non-PD patients. METHOD: We propose a feature-extraction method for volumetric images based on a rank-1 tensor decomposition. Furthermore, we apply a feature selection method that excludes common features between PD and non-PD. We collect neuromelanin images of 263 patients: 124 PD and 139 non-PD patients and divide them into training and testing datasets for experiments. We then experimentally evaluate the classification accuracy of the substantia nigra between PD and non-PD patients using the proposed feature extraction method and linear discriminant analysis. RESULTS: The proposed method achieves a sensitivity of 0.72 and a specificity of 0.64 for our testing dataset of 66 non-PD and 42 PD patients. Furthermore, we visualise the important patterns in the substantia nigra by a linear combination of rank-1 tensors with selected features. The visualised patterns include the ventrolateral tier, where the severe loss of neurons can be observed in PD. CONCLUSIONS: We develop a new feature-extraction method for the analysis of the substantia nigra towards PD diagnosis. In the experiments, even though the classification accuracy with the proposed feature extraction method and linear discriminant analysis is lower than that of expert physicians, the results suggest the potential of tensorial feature extraction.
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Cadherin 23 (CDH23) is one of the most common genes responsible for hereditary hearing loss; a mutation of CDH23 can cause a wide range of symptoms depending on the variant. In this study, an iPSC line was generated from a patient with late-onset, progressive high frequency hearing loss caused by c.[719C > T];[6085C > T]:p.[P240L];[R2029W] compound heterozygous variants of CDH23. The cells were confirmed to have a normal karyotype, express markers of pluripotency, and have tri-embryonic differentiation potential. This disease-specific iPSC line will further the construction of disease models and the elucidation of the pathophysiology of CDH23 mutations.
Assuntos
Caderinas , Perda Auditiva , Células-Tronco Pluripotentes Induzidas , Mutação , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Caderinas/genética , Caderinas/metabolismo , Perda Auditiva/genética , Perda Auditiva/patologia , Linhagem Celular , Diferenciação Celular , Masculino , Proteínas Relacionadas a CaderinasRESUMO
Endoplasmic reticulum-mitochondrial contact sites (ERMCS) play an important role in mitochondrial dynamics, calcium signaling, and autophagy. Disruption of the ERMCS has been linked to several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). However, the etiological role of ERMCS in these diseases remains unclear. We previously established tyrosine hydroxylase reporter (TH-GFP) iPSC lines from a PD patient with a PRKN mutation to perform correlative light-electron microscopy (CLEM) analysis and live cell imaging in GFP-expressing dopaminergic neurons. Here, we analyzed ERMCS in GFP-expressing PRKN-mutant dopaminergic neurons from patients using CLEM and a proximity ligation assay (PLA). The PLA showed that the ERMCS were significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control under normal conditions. The reduction of the ERMCS in PRKN-mutant patient dopaminergic neurons was further enhanced by treatment with a mitochondrial uncoupler. In addition, mitochondrial calcium imaging showed that mitochondrial Ca2+ flux was significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control. These results suggest a defect in calcium flux from ER to mitochondria is due to the decreased ERMCS in PRKN-mutant patient dopaminergic neurons. Our study of ERMCS using TH-GFP iPSC lines would contribute to further understanding of the mechanisms of dopaminergic neuron degeneration in patients with PRKN mutations.
RESUMO
Background: Parkinson's disease (PD) is a progressive neurodegenerative condition that primarily affects motor functions; it is caused by the loss of midbrain dopaminergic (mDA) neurons. The therapeutic effects of transplanting human-induced pluripotent stem cell (iPSC)-derived mDA neural progenitor cells in animal PD models are known and are being evaluated in an ongoing clinical trial. However, However, improvements in the safety and efficiency of differentiation-inducing methods are crucial for providing a larger scale of cell therapy studies. This study aimed to investigate the usefulness of dopaminergic progenitor cells derived from human iPSCs by our previously reported method, which promotes differentiation and neuronal maturation by treating iPSCs with three inhibitors at the start of induction. Methods: Healthy subject-derived iPS cells were induced into mDA progenitor cells by the CTraS-mediated method we previously reported, and their proprieties and dopaminergic differentiation efficiency were examined in vitro. Then, the induced mDA progenitors were transplanted into 6-hydroxydopamine-lesioned PD model mice, and their efficacy in improving motor function, cell viability, and differentiation ability in vivo was evaluated for 16 weeks. Results: Approximately ≥80% of cells induced by this method without sorting expressed mDA progenitor markers and differentiated primarily into A9 dopaminergic neurons in vitro. After transplantation in 6-hydroxydopamine-lesioned PD model mice, more than 90% of the engrafted cells differentiated into the lineage of mDA neurons, and approximately 15% developed into mature mDA neurons without tumour formation. The grafted PD model mice also demonstrated significantly improved motor functions. Conclusion: This study suggests that the differentiation protocol for the preparation of mDA progenitors is a promising option for cell therapy in patients with PD.
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Parkinson's disease (PD) is a common neurodegenerative disorder that results from the loss of dopaminergic neurons. Mutations in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) gene cause a familial form of PD with α-Synuclein aggregation, and we here identified the pathogenesis of the T61I mutation, the most common disease-causing mutation of CHCHD2. In Neuro2a cells, CHCHD2 is in mitochondria, whereas the T61I mutant (CHCHD2T61I ) is mislocalized in the cytosol. CHCHD2T61l then recruits casein kinase 1 epsilon/delta (Csnk1e/d), which phosphorylates neurofilament and α-Synuclein, forming cytosolic aggresomes. In vivo, both Chchd2T61I knock-in and transgenic mice display neurodegenerative phenotypes and aggresomes containing Chchd2T61I , Csnk1e/d, phospho-α-Synuclein, and phospho-neurofilament in their dopaminergic neurons. Similar aggresomes were observed in a postmortem PD patient brain and dopaminergic neurons generated from patient-derived iPS cells. Importantly, a Csnk1e/d inhibitor substantially suppressed the phosphorylation of neurofilament and α-Synuclein. The Csnk1e/d inhibitor also suppressed the cellular damage in CHCHD2T61I -expressing Neuro2a cells and dopaminergic neurons generated from patient-derived iPS cells and improved the neurodegenerative phenotypes of Chchd2T61I mutant mice. These results indicate that Csnk1e/d is involved in the pathogenesis of PD caused by the CHCHD2T61I mutation.