Single nucleus RNAseq of midbrain dopaminergic neuronal nuclei isolated by FACS
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE222558 | GEO | 2023/04/26
REPOSITORIES: GEO
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Project description:Midbrain dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) project widely throughout the central nervous system, playing critical roles in voluntary movements, reward processing, and working memory. Many of these neurons are highly sensitive to neurodegeneration in Parkinson’s Disease (PD), and their loss correlates strongly with the pathognomonic symptoms. To characterize these populations molecularly, we developed a protocol to enrich and transcriptionally profile DA neuron nuclei from postmortem human SNpc of both PD patients and matched controls. We identified a total of ten distinct populations, including one that was primate-specific. A single subtype, marked by the gene AGTR1, was highly susceptible to degeneration, and was enriched for expression of genes associated with PD in genetic studies, suggesting many risk loci act within this subtype to influence its neurodegeneration. The AGTR1 subtype also showed the strongest upregulation of TP53 and its downstream targets, nominating a potential pathway of degeneration in vivo. The transcriptional characterization of differentially disease-vulnerable DA neurons in the SNpc will inform the development of laboratory models, enable the nomination of novel disease biomarkers, and guide further studies of pathogenic disease mechanisms.
2021-06-16 | GSE178265 | GEO
Project description:Midbrain dopaminergic (DA) neurons are involved in diverse neurological functions, including control of movements, emotions or reward. In return, their dysfunctions cause severe clinical manifestations in humans, such as the appearance of motor and cognitive symptoms in Parkinson’s Disease. The physiology and pathophysiology of these neurons are widely studied, mostly with respect to molecular mechanisms implicating protein-coding genes. In contrast, the contribution of non-coding elements of the genome to DA neurons function is poorly investigated. In this study, we isolated DA neurons from E14.5 ventral mesencephalons, and used RNA-seq and ATAC-seq to establish and describe the repertoires of long non-coding RNAs (lncRNAs) and putative DNA regulatory regions, such as enhancers, specific to this neuronal population. We identified 990 novel lncRNAs out of 1,363 expressed in DA neurons, and most of them were not found in hindbrain serotonergic (5-HT) neurons, indicating a high degree of cell-specificity. This feature was also observed regarding open chromatin regions, as 39% of the ATAC-seq peaks from the DA repertoire were not detected in the 5-HT neurons. Our work provides for the first time DA-specific catalogues of non-coding elements of the genome that will undoubtedly participate in deepening our knowledge regarding DA neuronal development and dysfunctions.
2019-01-09 | GSE108917 | GEO
Project description:Here, we show that midbrain dopamine (DA) neurons derived from human pluripotent stem cells (hPSCs) are selectively permissive to SARS-CoV-2 infection both in vitro and upon transplantation in vivo, and that SARS-CoV-2 infection triggers a DA neuron inflammatory and cellular senescence response. A high-throughput screen in hPSC-derived DA neurons identified several FDA approved drugs, including riluzole, metformin, and imatinib, that can rescue the cellular senescence phenotype and prevent SARS-CoV-2 infection. RNA-seq analysis of human ventral midbrain tissue from COVID-19 patients, using formalin-fixed paraffin-embedded autopsy samples, confirmed the induction of an inflammatory and cellular senescence signature and identified low levels of SARS-CoV-2 transcripts. Our findings demonstrate that hPSC-derived DA neurons can serve as a disease model to study neuronal susceptibility to SARS-CoV-2 and to identify candidate neuroprotective drugs for COVID-19 patients. The susceptibility of hPSC-derived DA neurons to SARS-CoV-2 and the observed inflammatory and senescence transcriptional responses suggest the need for careful, long-term monitoring of neurological problems in COVID-19 patients.
2021-10-20 | GSE174745 | GEO
Project description:Ventral midbrain (VM) dopaminergic progenitor cells derived from human pluripotent stem cells have the potential to replace endogenously lost dopamine neurons and are currently in preclinical and clinical development for treatment of Parkinson’s Disease (PD). However, one main challenge in the quality control of the cells is that rostral and caudal VM progenitors are extremely similar transcriptionally though only the caudal VM cells give rise to dopaminergic neurons with functionality in PD. Therefore, it is critical to develop assays which can rapidly and reliably discriminate rostral from caudal VM cells during clinical manufacturing. Here, we applied shotgun proteomics to search for novel secreted biomarkers specific for caudal VM progenitors compared to rostral VM progenitors and validated key hits by ELISA. From this, we identified novel secreted markers (CPE, LGI1 and PDGFC) significantly enriched in caudal versus rostral VM progenitor cultures, whereas the markers CNTN2 and CORIN were significantly enriched in rostral VM cultures. With this data, we suggest and test in clinical grade samples a panel of coupled ELISA assays that can be applied as a quality control tool for assessing the correct patterning of cells during clinical manufacturing.
2023-12-13 | PXD039510 | Pride
Project description:Dopaminergic (DA) neurons are the predominant cell type in the midbrain that synthesize dopamine, a neurotransmitter implicated in various behavioural processes, including motor function, the reward pathway, and satiety. In diseases affecting these neurons, such as in Parkinson’s disease (PD), there is growing evidence that the gut-brain axis and selective vulnerability of DA neurons plays a crucial role in disease. Most investigations relating to DA neurons in the gut rely on immunoreactivity to tyrosine hydroxylase (TH) - a rate-limiting enzyme in the production of dopamine. However, the reliability of TH staining as a marker of DA neurons has been questioned in recent years. Our aim is to perform a comprehensive characterization of DA neurons in the gut using a well-accepted reporter mouse line, expressing a fluorescent protein under the dopamine transporter promoter (DAT). Our findings confirm a unique localization of DA neurons in the gut, and unveil that there are discrete subtypes of DA neurons in the gut, which we characterized using both immunofluorescence and single-cell transcriptomics. We observed distinct subtypes of DAT neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, and a smaller population likely releasing nitric oxide; while others were positive for a slew of canonical DA markers (Vmat2, Girk2, Foxa2). Given the clear heterogeneity of DA gut neurons, further investigation is warranted to define their functional signatures and discover their inherent biological differences that predispose these cells to neurodegeneration.
2023-07-16 | GSE237170 | GEO
Project description:Human pluripotent stem cells (hPSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of hPSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However the effective use of hPSCs for cell therapy has lagged far behind. While mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease, DA neurons derived from human PSCs generally display poor in vivo performance. There are also considerable safety concerns for hPSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor plate-based strategy for the derivation of human DA neurons that efficiently engraft, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor plate precursors are derived from hPSCs in days following exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signaling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive in vitro molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of hPSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in PD animal models in three host species. Long-term engraftment in 6-OHDA-lesioned mouse and rats demonstrates robust survival of midbrain DA neurons, complete restoration of amphetamine-induced rotation behavior and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into Parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models tested indicate considerable promise for the development of cell based therapies in PD. Differentiated hESC with three conditions (LSB, LSB/S/F8, LSB/S/F8/CHIR) were subjected to RNA extraction in specific timepoint (day 0, 1, 3, 5, 7, 11, 13, 25) and hybridization on Illumina microarrays. Each sample has 3 or 4 biological repeats. Based on previous study* of dual SMAD inhibition neural induction, we developed new midbrain dopamine neuron protocol. It depends on time specific treatment of below factors (LSB/S/F8/CHIR): L (LDN193189 (BMP inhibitor) , day 0-11), SB (SB431542 (TGF-b signal inhibitor), day 0-5), S (SHH + Purmorphamine (Smo agonist), day 1-7), F8 (FGF8, day 1-7) and CHIR (CHIR99021 (GSK3b inhibitor), day 3-13) LSB and LSB/S/F8 are limited control conditions of dual SMAD only (LSB) or traditional patterning with Sonic and FGF (LSB/S/F8) *Chambers,S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275-280 (2009).
2011-11-07 | E-GEOD-32658 | biostudies-arrayexpress
Project description:COVID-19 patients commonly present with neurological signs of central nervous system (CNS) and/or peripheral nervous system dysfunction. However, which neural cells are permissive to infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been controversial. Here, we show that midbrain dopamine (DA) neurons derived from human pluripotent stem cells (hPSCs) are selectively susceptible and permissive to SARS-CoV-2 infection, and that SARS-CoV-2 infection triggers a DA neuron inflammatory and cellular senescence response. A high-throughput screen in hPSC-derived DA neurons identified several FDA approved drugs, including riluzole, metformin, and imatinib, that can rescue the cellular senescence phenotype by preventing SARS-CoV-2 infection. RNA-seq analysis of human ventral midbrain tissue from COVID-19 patients, using formalin-fixed paraffin-embedded autopsy samples, confirmed the induction of an inflammatory and cellular senescence signature and identified low levels of SARS-CoV-2 transcripts. Our findings demonstrate that hPSC-derived DA neurons can serve as a disease model to study neuronal susceptibility to SARS-CoV-2 and to identify candidate neuroprotective drugs for COVID-19 patients. The susceptibility of hPSC-derived DA neurons to SARS-CoV-2 and the observed inflammatory and senescence transcriptional responses suggest the need for careful, long-term monitoring of neurological problems in COVID-19 patients.
2023-12-04 | GSE248989 | GEO
Project description:Local translation within excitatory and inhibitory neurons is known to be involved in neuronal development and synaptic plasticity. Despite the extensive dendritic and axonal arborizations of monoaminergic neurons, the subcellular localization of protein synthesis has not been well-characterized in these populations. Here, we investigated mRNA localization in midbrain dopaminergic (mDA) neurons, cells with enormous axonal and dendritic projections, both of which can release dopamine (DA). Using highly-sensitive sequencing and imaging approaches in mDA axons, we found no evidence for axonal mRNA localization or translation. In contrast, we found that mDA neuronal dendritic projections into the substantia nigra reticulata (SNr) contain ribosomes and mRNAs encoding the DA synthesis, release, and reuptake machinery. Surprisingly, we found dendritic localization of mRNAs encoding synaptic vesicular release proteins in mDA neurons. Our results are consistent with a role for local translation in the regulation of DA transmission from dendrites, but not striatal axons. Finally, we defined a molecular signature of sparse mDA neurons in the SNr, including enrichment of an ER calcium pump previously undescribed in mDA neurons.
2021-07-27 | GSE180913 | GEO
Project description:The adult offspring of MHV68 infected dams had a significantly decreased expression of genes linked to dopamine neurons (Th, lmx1b, and foxa1). Deconvolution analysis revealed that the proportion of dopamine neurons in the midbrain was reduced. Pathway analyses indicated mitochondrial dysfunction, with reductions in genes associated with ATP synthesis, mitochondrial respiratory chain, and mitochondrial translation in the offspring of dams infected with MHV68. TIGAR (a negative regulator of mitophagy) and SDHA (mitochondrial complex II subunit) protein levels were increased, and the levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum were increased in these offspring. No such changes were observed in the offspring of dams infected with MCMV.
2022-04-14 | GSE162566 | 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