Single cell RNA sequencing analysis of midbrain organoids with SNCA gene triplication modeling key features of synuclienopathy
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE186780 | GEO | 2021/11/02
REPOSITORIES: GEO
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Project description:Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with progressive loss of dopaminergic neurons in the substantia nigra, and accumulation of aggregates of α-synuclein. However, the sequence of molecular events that proceed from the SNCA mutation during development, to its end stage pathology is unknown. Utilising human induced pluripotent stem cells (hiPSCs) with SNCA mutations, we resolved the temporal sequence of pathophysiological events that occur during neuronal differentiation in order to discover the early, and likely causative, events in synucleinopathies. We adapted a small molecule-based protocol that generates highly enriched midbrain dopaminergic (mDA) neurons (>80%). We characterised their molecular identity using single-cell RNA sequencing and their functional identity through the synthesis and secretion of dopamine, the ability to generate action potentials, and form functional synapses and networks. RNA velocity analyses confirmed the developmental transcriptomic trajectory of midbrain neural precursors into different mDA neuronal clusters. To characterise the synucleinopathy, we adopted super-resolution methods to determine the number, size, and structure of aggregates in SNCA-mutant mDA neurons. By day 27 of differentiation, prior to maturation to mDA neurons of molecular and functional identity, we demonstrate the formation of small aggregates; specifically, β-sheet rich oligomeric aggregates, in SNCA-mutant midbrain immature neurons. The aggregation progresses over time to accumulate phosphorylated and fibrillar aggregates. When the midbrain neurons were functional, we observed impaired intracellular calcium signalling, evidenced with an increased basal calcium level and impairments in both cytosolic and mitochondrial calcium rearrangements. Once midbrain identity fully developed, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling as well as an upregulation of mitophagy and autophagy. In addition, SNCA-mutant neurons displayed pathophysiological excitability, revealed as a depolarised resting membrane potential, an increased input resistance, and impaired firing properties. Ultimately these multiple cellular stresses lead to an increase in cell death by day 62 post-differentiation. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD, and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.
2023-03-11 | PXD035500 | Pride
Project description:Since the discovery of radial glia as the source of neurons, their heterogeneity in regard to neurogenesis has been described by clonal and time-lapse analysis in vitro. However, the molecular determinants specifying neurogenic radial glia differently from radial glia that mostly self-renew remain ill-defined. Here, we isolated two radial glial subsets that co-exist at mid-neurogenesis in the developing cerebral cortex and their immediate progeny. While one subset generates neurons directly, the other is largely non-neurogenic but also gives rise to Tbr2-positive basal precursors, thereby contributing indirectly to neurogenesis. Isolation of ; these distinct radial glia subtypes allowed determining interesting differences in their transcriptome. These transcriptomes were also strikingly different from the transcriptome of radial glia isolated at the end of neurogenesis. This analysis therefore identifies, for the first time, the lineage origin of basal progenitors and the molecular differences of this lineage in comparison to directly neurogenic and gliogenic radial glia. Experiment Overall Design: Comparison of radial glial subtypes
2008-06-18 | E-GEOD-8034 | biostudies-arrayexpress
Project description:Alpha synuclein (SNCA) has been linked to neurodegenerative diseases (synucleinopathies) that include Parkinson’s disease (PD). Although the primary neurodegeneration in PD involves nigrostriatal dopaminergic neurons, more extensive yet regionally selective neurodegeneration is observed in other synucleinopathies. Furthermore, SNCA is ubiquitously expressed in neurons and numerous neuronal systems are dysfunctional in PD. Therefore it is of interest to understand how overexpression of SNCA affects neuronal function in regions not directly targeted for neurodegeneration in PD. To gain a better understanding of the consequences of excessive SNCA expression on basal ganglia function, we performed transcriptome analysis of striatal tissue from male Thy1-aSyn-mice and wt littermates. The present study investigated the consequences of SNCA overexpression on cellular processes and functions in the striatum of mice overexpressing wild-type, human SNCA under the Thy1 promoter (Thy1-aSyn mice) by transcriptome analysis. The analysis revealed alterations in multiple biological processes in the striatum of Thy1-aSyn mice, including synaptic plasticity, signaling, transcription, apoptosis, and neurogenesis. The present study investigated the consequences of SNCA overexpression on cellular processes and functions in the striatum of mice overexpressing wild-type, human SNCA under the Thy1 promoter (Thy1-aSyn mice) by transcriptome analysis. The analysis revealed alterations in multiple biological processes in the striatum of Thy1-aSyn mice, including synaptic plasticity, signaling, transcription, apoptosis, and neurogenesis.
2012-02-11 | GSE35723 | GEO
Project description:A major barrier to research on Parkinson’s disease (PD) is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells (iPSCs) from patients with PD and differentiate them into neurons affected by disease. We created an iPSC model of PD caused by triplication of SNCA encoding ?-synuclein. ?-Synuclein dysfunction is common to all forms of PD, and SNCA triplication leads to fully penetrant familial PD with accelerated pathogenesis. After differentiation of iPSCs into neurons enriched for midbrain dopaminergic subtypes, those from the patient contain double ?-synuclein protein compared to those from an unaffected relative, precisely recapitulating the cause of PD in these individuals. A measurable biomarker makes this model ideal for drug screening for compounds that reduce levels of ?-synuclein, and for mechanistic experiments to study PD pathogenesis. This SNP microarray study was carried out to confirm presence of SNCA triplication in the affected subject and the derived cell lines. 11 samples were analysed: genomic DNA from the two subjects in the study, the two parent fibroblast lines (AST denoting alpha-synuclein triplication and NAS denoting normal alpha-synuclein), two iPSC lines from each parent fibroblast line (four in total), a human embryonic stem cell line (SHEF4) and two neuronal samples one each from AST and NAS iPSCs).
2011-09-14 | E-GEOD-28366 | biostudies-arrayexpress
Project description:Midbrain organoid derived from human induced pluriopotent stem cells a new model of Parkinson's Disease. Organ specific organoids can be used to model many diseases, however little is know about these models. We created a flow cytometry workflow to identify cell types in midbrain organoids. We then selected four populations and sorted these populations: Neurons1(CD24++), Neurons2(CD56++), Astrocytes and Radial Glia, using FACS and the antibody intensity gates defined by our workflow. We then performed scRNAseq on the sorted population and identified cell types within the sorted populations.
2023-03-15 | GSE226890 | GEO
Project description:Parkinson’s disease (PD) is a prevalent neurodegenerative disorder that is characterized by the selective loss of midbrain dopamine (DA)-producing neurons and the formation of α-synuclein (α-syn)-containing inclusions named Lewy bodies (LBs). Here, we report that the loss of glucocerebrosidase (GCase), coupled with α-syn overexpression, result in substantial accumulation of detergent-resistant α-syn aggregates and Lewy body-like inclusions (LBLIs) in human midbrain-like organoids (hMLOs). These LBLIs exhibit a highly similar structure to PD-associated LBs, by displaying a spherically symmetric morphology with an eosinophilic core, and containing α-syn and ubiquitin. Importantly, hMLOs generated from PD patient-derived inducible pluripotent stem cells (iPSCs) harboring SNCA triplication also exhibit subsequent degeneration of DA neurons and LBLI formation upon chronic GCase inhibitor treatment. Taken together, our hMLOs harbouring two major PD risk factors (GCase deficiency and overproduced α-syn) successfully recapitulate major pathophysiological signatures of the disease, and highlight the broad utility of brain organoid technology in modeling human neurodegenerative diseases.
2021-08-13 | E-MTAB-7302 | biostudies-arrayexpress
Project description:A major barrier to research on Parkinson’s disease (PD) is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells (iPSCs) from patients with PD and differentiate them into neurons affected by disease. We created an iPSC model of PD caused by triplication of SNCA encoding α-synuclein. α-Synuclein dysfunction is common to all forms of PD, and SNCA triplication leads to fully penetrant familial PD with accelerated pathogenesis. After differentiation of iPSCs into neurons enriched for midbrain dopaminergic subtypes, those from the patient contain double α-synuclein protein compared to those from an unaffected relative, precisely recapitulating the cause of PD in these individuals. A measurable biomarker makes this model ideal for drug screening for compounds that reduce levels of α-synuclein, and for mechanistic experiments to study PD pathogenesis. This SNP microarray study was carried out to confirm presence of SNCA triplication in the affected subject and the derived cell lines.
2011-09-15 | GSE28366 | GEO
Project description:A major barrier to research on Parkinson’s disease (PD) is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells (iPSCs) from patients with PD and differentiate them into neurons affected by disease. We created an iPSC model of PD caused by triplication of SNCA encoding α-synuclein. α-Synuclein dysfunction is common to all forms of PD, and SNCA triplication leads to fully penetrant familial PD with accelerated pathogenesis. After differentiation of iPSCs into neurons enriched for midbrain dopaminergic subtypes, those from the patient contain double α-synuclein protein compared to those from an unaffected relative, precisely recapitulating the cause of PD in these individuals. A measurable biomarker makes this model ideal for drug screening for compounds that reduce levels of α-synuclein, and for mechanistic experiments to study PD pathogenesis. This gene expression microarray study was carried out as part of the validation process for demonstrating that the generated iPSC lines are pluripotent. 5 samples were analysed: two clonal iPSC lines from each of two genotypes (four in total; AST denoting alpha-synuclein triplication and NAS denoting normal alpha-synuclein), a human embryonic stem cell line (SHEF4). All cultured in self-renewal conditions, mTeSR1
2011-09-14 | E-GEOD-30792 | biostudies-arrayexpress
Project description:Abnormal neuronal aggregation of a-synuclein is implicated in the development of Parkinson’s disease. Glial cells also show extensive a-synuclein pathology and are thought to contribute to disease progression. However, the mechanism that produces the glial a-synuclein pathology and the interaction between neurons and glia in the disease-inflicted microenvironment remain unknown. Here, we show that in neuronal cells misfolded a-synuclein proteins are selectively translocated into vesicles, leading to exocytosis of the aggregated forms. More importantly, our data demonstrate that astrocytes take up the neuron-derived a-synuclein aggregates and produce a-synuclein inclusions similar to the ones found in human brains. This uptake is paralleled by changes in the gene expression profile reflecting an inflammatory response. These results suggest that astroglial a-synuclein pathology is produced by cell-to-cell transmission of neuronal a-synuclein aggregates. This transmission step is thus an important mediator of pathogenic glial responses and could qualify as a new therapeutic target. To determine how astrocytes respond to extracellular a-synuclein, gene expression profiles were established and analyzed for nearly 22,000 rat genes using the Illumina RatRef-12 Expression BeadChip. Total RNA was isolated from astrocytes exposed for 6h or 24h to conditioned medium from cultures of a-synuclein or lacZ expressing cells.
2010-01-11 | E-GEOD-11574 | biostudies-arrayexpress
Project description:A major barrier to research on Parkinson’s disease (PD) is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells (iPSCs) from patients with PD and differentiate them into neurons affected by disease. We created an iPSC model of PD caused by triplication of SNCA encoding α-synuclein. α-Synuclein dysfunction is common to all forms of PD, and SNCA triplication leads to fully penetrant familial PD with accelerated pathogenesis. After differentiation of iPSCs into neurons enriched for midbrain dopaminergic subtypes, those from the patient contain double α-synuclein protein compared to those from an unaffected relative, precisely recapitulating the cause of PD in these individuals. A measurable biomarker makes this model ideal for drug screening for compounds that reduce levels of α-synuclein, and for mechanistic experiments to study PD pathogenesis. This gene expression microarray study was carried out as part of the validation process for demonstrating that the generated iPSC lines are pluripotent. 15 samples were analysed: the two parent fibroblast lines (AST denoting alpha-synuclein triplication and NAS denoting normal alpha-synuclein), two iPSC lines from each parent fibroblast line (four in total), a human embryonic stem cell line (SHEF4) and eight neuronal samples (each iPSC line differentiated into a neuronal population enriched for dopaminergic neurons, at two different time points).
2011-09-14 | E-GEOD-28365 | biostudies-arrayexpress