RESUMO
Midbrain dopaminergic neurons (mDANs) control voluntary movement, cognition, and reward behavior under physiological conditions and are implicated in human diseases such as Parkinson's disease (PD). Many transcription factors (TFs) controlling human mDAN differentiation during development have been described, but much of the regulatory landscape remains undefined. Using a tyrosine hydroxylase (TH) human iPSC reporter line, we here generate time series transcriptomic and epigenomic profiles of purified mDANs during differentiation. Integrative analysis predicts novel regulators of mDAN differentiation and super-enhancers are used to identify key TFs. We find LBX1, NHLH1 and NR2F1/2 to promote mDAN differentiation and show that overexpression of either LBX1 or NHLH1 can also improve mDAN specification. A more detailed investigation of TF targets reveals that NHLH1 promotes the induction of neuronal miR-124, LBX1 regulates cholesterol biosynthesis, and NR2F1/2 controls neuronal activity.
Assuntos
Neurônios Dopaminérgicos , Células-Tronco Pluripotentes Induzidas , Humanos , Neurônios Dopaminérgicos/metabolismo , Multiômica , Mesencéfalo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genéticaRESUMO
Mitochondrial Rho GTPase 1 (Miro1) protein is a well-known adaptor for mitochondrial transport and also regulates mitochondrial quality control and function. Furthermore, Miro1 was associated with mitochondrial-endoplasmic reticulum (ER) contact sites (MERCs), which are key regulators of cellular calcium homeostasis and the initiation of autophagy. Impairments of these mechanisms were linked to neurodegeneration in Parkinson's disease (PD). We recently revealed that PD fibroblasts harboring Miro1 mutations displayed dysregulations in MERC organization and abundance, affecting mitochondrial homeostasis and clearance. We hypothesize that mutant Miro1 impairs the function of MERCs and mitochondrial dynamics, altering neuronal homeostasis and integrity in PD. PD skin fibroblasts harboring the Miro1-R272Q mutation were differentiated into patient-derived neurons. Live-cell imaging and immunocytochemistry were used to study mitophagy and the organization and function of MERCs. Markers of autophagy or mitochondrial function were assessed by western blotting. Quantification of organelle juxtapositions revealed an increased number of MERCs in patient-derived neurons. Live-cell imaging results showed alterations of mitochondrial dynamics and increased sensitivity to calcium stress, as well as reduced mitochondrial clearance. Finally, western blot analysis indicated a blockage of the autophagy flux in Miro1-mutant neurons. Miro1-mutant neurons display altered ER-mitochondrial tethering compared with control neurons. This alteration likely interferes with proper MERC function, contributing to a defective autophagic flux and cytosolic calcium handling capacity. Moreover, mutant Miro1 affects mitochondrial dynamics in neurons, which may result in disrupted mitochondrial turnover and altered mitochondrial movement.
Assuntos
Retículo Endoplasmático/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Doença de Parkinson/genética , Proteínas rho de Ligação ao GTP/genética , Cálcio/metabolismo , Diferenciação Celular/genética , Citosol/metabolismo , Homeostase/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Dinâmica Mitocondrial/genética , Mitofagia/genética , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologiaRESUMO
BACKGROUND: VPS35 is part of the retromer complex and is responsible for the trafficking and recycling of proteins implicated in autophagy and lysosomal degradation, but also takes part in the degradation of mitochondrial proteins via mitochondria-derived vesicles. The p.D620N mutation of VPS35 causes an autosomal-dominant form of Parkinson's disease (PD), clinically representing typical PD. OBJECTIVE: Most of the studies on p.D620N VPS35 were performed on human tumor cell lines, rodent models overexpressing mutant VPS35, or in patient-derived fibroblasts. Here, based on identified target proteins, we investigated the implication of mutant VPS35 in autophagy, lysosomal degradation, and mitochondrial function in induced pluripotent stem cell-derived neurons from a patient harboring the p.D620N mutation. METHODS: We reprogrammed fibroblasts from a PD patient carrying the p.D620N mutation in the VPS35 gene and from two healthy donors in induced pluripotent stem cells. These were subsequently differentiated into neuronal precursor cells to finally generate midbrain dopaminergic neurons. RESULTS: We observed a decreased autophagic flux and lysosomal mass associated with an accumulation of α-synuclein in patient-derived neurons compared to controls. Moreover, patient-derived neurons presented a mitochondrial dysfunction with decreased membrane potential, impaired mitochondrial respiration, and increased production of reactive oxygen species associated with a defect in mitochondrial quality control via mitophagy. CONCLUSION: We describe for the first time the impact of the p.D620N VPS35 mutation on autophago-lysosome pathway and mitochondrial function in stem cell-derived neurons from an affected p.D620N carrier and define neuronal phenotypes for future pharmacological interventions. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Doença de Parkinson , Proteínas de Transporte Vesicular , Neurônios Dopaminérgicos/metabolismo , Humanos , Mitocôndrias/metabolismo , Mutação/genética , Doença de Parkinson/metabolismo , Transporte Proteico , Proteínas de Transporte Vesicular/genética , alfa-Sinucleína/metabolismoRESUMO
We describe an induced pluripotent stem cell (iPSC) line that was derived from fibroblasts obtained from a Parkinson's disease (PD) patient carrying the p.G2019S mutation in the LRRK2 gene and the p.N409S mutation in the GBA1 gene. iPSCs were generated via Sendai virus transduction of Yamanaka factors. The presence of GBA1 p.N409S and LRRK2 p.G2019S was confirmed by Sanger sequencing. The iPSCs express pluripotency markers, are capable of in vitro differentiation into the three germ layers and have a normal karyotype. The newly generated line will be used for in vitro PD modeling by investigating the role of each mutation in iPSC-derived dopaminergic neurons.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Mutação/genética , Fibroblastos/metabolismo , Neurônios Dopaminérgicos/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genéticaRESUMO
We describe the generation of an isogenic control cell line DJ-1-delP GC13 from an induced pluripotent stem cell (iPSC) line DJ-1-delP LCSBi008-A that was derived from fibroblasts obtained from a Parkinson's disease (PD) patient. Using CRISPR/Cas9 technology, we corrected the disease causing c.471_473delGCC homozygous mutation in the PARK7 gene leading to p.158P deletion in the encoded protein DJ-1. The generated isogenic pair will be used for phenotypic analysis of PD-patient derived neurons and astrocytes.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Astrócitos/metabolismo , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismoRESUMO
Parkinson's disease (PD) is characterised by the degeneration of A9 dopaminergic neurons and the pathological accumulation of alpha-synuclein. The p.A30P SNCA mutation generates the pathogenic form of the alpha-synuclein protein causing an autosomal-dominant form of PD. There are limited studies assessing pathogenic SNCA mutations in patient-derived isogenic cell models. Here we provide a functional assessment of dopaminergic neurons derived from a patient harbouring the p.A30P SNCA mutation. Using two clonal gene-corrected isogenic cell lines we identified image-based phenotypes showing impaired neuritic processes. The pathological neurons displayed impaired neuronal activity, reduced mitochondrial respiration, an energy deficit, vulnerability to rotenone, and transcriptional alterations in lipid metabolism. Our data describes for the first time the mutation-only effect of the p.A30P SNCA mutation on neuronal function, supporting the use of isogenic cell lines in identifying image-based pathological phenotypes that can serve as an entry point for future disease-modifying compound screenings and drug discovery strategies.
Assuntos
Neurônios Dopaminérgicos/citologia , Mutação , Doença de Parkinson/patologia , alfa-Sinucleína/genética , Linhagem Celular , Neurônios Dopaminérgicos/metabolismo , Humanos , Mitocôndrias , Doença de Parkinson/genéticaRESUMO
Fibroblasts were obtained from a 76 year-old man diagnosed with Parkinson's disease (PD). The disease is caused by a heterozygous p.D620N mutation in VPS35. Induced pluripotent stem cells (iPSCs) were generated using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific). The presence of the c.1858G > A base exchange in exon 15 of VPS35 was confirmed by Sanger sequencing. The iPSCs are free of genomically integrated reprogramming genes, express pluripotency markers, display in vitro differentiation potential to the three germ layers and have karyotypic integrity. Our iPSC line will be useful for studying the impact of the p.D620N mutation in VPS35 in vitro.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Idoso , Diferenciação Celular , Heterozigoto , Humanos , Masculino , Mutação , Doença de Parkinson/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPSC clones into high-throughput 96-well microtiter plates. We used high-content screening (HCS) technology and generated an in-house developed algorithm to select the correctly edited isogenic clones for continued expansion and validation. In our model we have gene-corrected the iPSCs of a Parkinson's disease (PD) patient carrying the autosomal dominantly inherited heterozygous c.88G>C mutation in the SNCA gene, which leads to the pathogenic p.A30P form of the alpha-synuclein protein. Undertaking a PCR restriction-digest mediated clonal selection strategy prior to sequencing, we were able to post-sort validate each isogenic clone using a quadruple screening strategy prior to generating footprint-free isogenic iPSC lines, retaining a normal molecular karyotype, pluripotency and three germ-layer differentiation potential. Directed differentiation into midbrain dopaminergic neurons revealed that SNCA expression is reduced in the gene-corrected clones, which was validated by a reduction at the alpha-synuclein protein level. The generation of single-cell isogenic clones facilitates new insights in the role of alpha-synuclein in PD and furthermore is applicable across patient-derived disease models.
Assuntos
Células Clonais/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Doença de Parkinson/genética , alfa-Sinucleína/genética , Diferenciação Celular , Linhagem Celular , Humanos , Doença de Parkinson/patologiaRESUMO
Dermal fibroblasts from a patient carrying a heterozygous c.88G > C mutation in the SNCA gene that encodes alpha-synuclein were reprogrammed to pluripotency by retroviruses. This pathogenic mutation generates the p.A30P form of the alpha-synuclein protein leading to autosomal dominantly inherited Parkinson's disease (PD). Two clonal iPS cell lines were generated (A30P-3 and A30P-4) and characterised by validating the silencing of viral transgenes, the expression of endogenous pluripotency genes, directed differentiation into three germ layers in-vitro and a stable molecular genotype. These iPSC lines will serve as a valuable resource in determining the role of the p.A30P SNCA mutation in PD pathogenesis.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Linhagem Celular , Humanos , Mutação/genética , Doença de Parkinson/genética , alfa-Sinucleína/genéticaRESUMO
ACO2 is a mitochondrial protein, which is critically involved in the function of the tricarboxylic acid cycle (TCA), the maintenance of iron homeostasis, oxidative stress defense and the integrity of mitochondrial DNA (mtDNA). Mutations in the ACO2 gene were identified in patients suffering from a broad range of symptoms, including optic nerve atrophy, cortical atrophy, cerebellar atrophy, hypotonia, seizures and intellectual disabilities. In the present study, we identified a heterozygous 51 bp deletion (c.1699_1749del51) in ACO2 in a family with autosomal dominant inherited isolated optic atrophy. A complementation assay using aco1-deficient yeast revealed a growth defect for the mutant ACO2 variant substantiating a pathogenic effect of the deletion. We used patient-derived fibroblasts to characterize cellular phenotypes and found a decrease of ACO2 protein levels, while ACO2 enzyme activity was not affected compared to two age- and gender-matched control lines. Several parameters of mitochondrial function, including mitochondrial morphology, mitochondrial membrane potential or mitochondrial superoxide production, were not changed under baseline conditions. However, basal respiration, maximal respiration, and spare respiratory capacity were reduced in mutant cells. Furthermore, we observed a reduction of mtDNA copy number and reduced mtDNA transcription levels in ACO2-mutant fibroblasts. Inducing oxidative stress led to an increased susceptibility for cell death in ACO2-mutant fibroblasts compared to controls. Our study reveals that a monoallelic mutation in ACO2 is sufficient to promote mitochondrial dysfunction and increased vulnerability to oxidative stress as main drivers of cell death related to optic nerve atrophy.
Assuntos
Aconitato Hidratase/genética , Fibroblastos/metabolismo , Haploinsuficiência , Mitocôndrias/genética , Atrofia Óptica/genética , Nervo Óptico/patologia , Deleção de Sequência , Aconitato Hidratase/metabolismo , DNA Mitocondrial , Exoma , Feminino , Fibroblastos/patologia , Humanos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Atrofia Óptica/metabolismo , Atrofia Óptica/patologia , Nervo Óptico/metabolismoRESUMO
Parkinson's disease (PD) is a heterogeneous neurodegenerative disorder with monogenic forms representing prototypes of the underlying molecular pathology and reproducing to variable degrees the sporadic forms of the disease. Using a patient-based in vitro model of PARK7-linked PD, we identified a U1-dependent splicing defect causing a drastic reduction in DJ-1 protein and, consequently, mitochondrial dysfunction. Targeting defective exon skipping with genetically engineered U1-snRNA recovered DJ-1 protein expression in neuronal precursor cells and differentiated neurons. After prioritization of candidate drugs, we identified and validated a combinatorial treatment with the small-molecule compounds rectifier of aberrant splicing (RECTAS) and phenylbutyric acid, which restored DJ-1 protein and mitochondrial dysfunction in patient-derived fibroblasts as well as dopaminergic neuronal cell loss in mutant midbrain organoids. Our analysis of a large number of exomes revealed that U1 splice-site mutations were enriched in sporadic PD patients. Therefore, our study suggests an alternative strategy to restore cellular abnormalities in in vitro models of PD and provides a proof of concept for neuroprotection based on precision medicine strategies in PD.
Assuntos
Doença de Parkinson , Neurônios Dopaminérgicos , Éxons/genética , Humanos , Mutação/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Splicing de RNARESUMO
Aims: The outer mitochondrial membrane protein Miro1 is a crucial player in mitochondrial dynamics and calcium homeostasis. Recent evidence indicated that Miro1 mediates calcium-induced mitochondrial shape transition, which is a prerequisite for the initiation of mitophagy. Moreover, altered Miro1 protein levels have emerged as a shared feature of monogenic and sporadic Parkinson's disease (PD), but, so far, no disease-associated variants in RHOT1 have been identified. Here, we aim to explore the genetic and functional contribution of RHOT1 mutations to PD in patient-derived cellular models. Results: For the first time, we describe heterozygous RHOT1 mutations in two PD patients (het c.815G>A; het c.1348C>T) and identified mitochondrial phenotypes with reduced mitochondrial mass in patient fibroblasts. Both mutations led to decreased endoplasmic reticulum-mitochondrial contact sites and calcium dyshomeostasis. As a consequence, energy metabolism was impaired, which in turn caused increased mitophagy. Innovation and Conclusion: Our study provides functional evidence that ROTH1 is a genetic risk factor for PD, further implicating Miro1 in calcium homeostasis and mitochondrial quality control.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Homeostase , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Doença de Parkinson/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Idoso , Humanos , Pessoa de Meia-Idade , Proteínas Mitocondriais/genética , Mutação , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The infectivity of a recombinant murine norovirus (RecMNV) strain, previously isolated following in vitro coinfections, was evaluated in vivo in comparison with its parental strains (MNV-1-CW1 and WU20) in Balb/cByJ mice via measurement of weight loss and estimation of viral loads in faeces, tissues and organs 48 and 72h post-infection. The presence of infectious virus in all analysed tissues and organs suggests that, similarly to its parental viruses, RecMNV can disseminate beyond organs associated with the digestive tract. Our results also suggest that recombination occurring in vitro between two homologous murine norovirus strains can give rise to a chimeric strain which, despite slight differences, shows similar biological properties to its parental strains. This study provides the first report on in vivo replication of a recombinant norovirus strain isolated following in vitro coinfection. These results have great significance for norovirus genetic evolution and future vaccine development.