Spatiotemporal Proteomic and Transcriptomic Landscape of DAT+ Dopaminergic Neurons Development and Function
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE285556 | GEO | 2025/04/16
REPOSITORIES: GEO
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Project description:Dopaminergic (DA) neurons marked by the dopamine transporter (DAT) have multiple physiological functions and are involved in the regulation of mental and neurological diseases, prompting in-depth studies into their development and functions. This research explores the spatiotemporal proteomic and transcriptomic changes in DAT+ DA neurons within key brain regions involved in DA signaling—the nucleus accumbens (NAc), substantia nigra (SNc), and ventral tegmental area (VTA). Utilizing cutting-edge multi-omics techniques, such as ultrasensitive trace sample proteomics and SMART-seq2 for transcriptomics, we examine the DA neuronal system at critical postnatal milestones: postnatal day 7 (P7), postnatal day 30 (P30), and postnatal day 60 (P60). The study reveals unique molecular profiles within DA neuron populations, showcasing their varied functional roles and developmental progression.
2024-09-03 | PXD055527 |
Project description:Groundbreaking research in the last decade has definitively established human pluripotent stem cells (hPSCs) as a powerful source for the unlimited generation of dopamine (DA) neurons used in cell-based therapy in Parkinson’s disease (PD). However, DA neurons constitute a heterogeneous group of cells with different molecular identities and, consequently, different functions and innervation targets. Moreover, the inaccessibility of the human brain has so far hindered our understanding of the highly complex human DA system and, therefore, the design of DA neuron subtype-specific differentiation protocols for more effective cell replacement therapies. Currently, DA neurons – located in human ventral midbrain – can only be reliably distinguished based on their projection patterns. Here, we used hPSCs to derive and then transplant DA neurons into a rat model of PD. After one year of transplantation, we performed single nucleus RNA sequencing (snRNAseq) of ~80,000 human nuclei to transcriptionally define cell type composition and obtained a comprehensive map of DA neuron molecular identities. Exploiting the fact that transplanted DA neurons innervated and integrated within the host circuitry, we then combined snRNAseq with axonal retrograde AAV barcoding to trace the projection patterns of molecularly distinct human DA neuron subtypes in the grafts. We thus defined human DA neuron transcriptional profiles based on their target-specific outgrowth in the host brain. The resulting datasets provide an unprecedented resource both for elucidating the molecular basis of human DA neuron diversity as well as for developing more targeted cell-based therapies, with major implications for outcomes of PD patients.
2024-11-03 | GSE233885 | GEO
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:Our goal was to develop a list of genes that could be used effectively to highlight the genomic profiling of human embryonic neural stem cells (hENSCs), and to identify genes involved in the process of their differentiation to dopaminergic (DA) neurons. Our results showed that Agilent's Whole Human Genome Oligonucleotide Microarray permits the monitoring of at least 41000 genes as cells differentiate. In this study, we identified 13525 genes to be differentially expressed between undifferentiated hENSC and DA cells isolated from human substansia nigra. Approximately 3737 genes were up-regulated in the embryonic NSC, and 4116 genes were up-regulated in DA cells. Careful analysis of the emerged data using the web-accessible program named Database for Annotation, Visualization and Integrated Discovery (DAVID) has permitted us to distinguish several genes and pathways that are involved in dopaminergic differentiation, and to identify the crucial signaling pathways that direct the process of differentiation. Our study elucidated that genes related to midbrain development, such as Nr4a2 (nuclear receptor subfamily 4, group A, member 2, Nurr1) and En1 (engrailed 1), were increased to 3.76- and 6.41-folds, respectively. In addition, the transcriptions of the genes for DA neuron phenotype, such as Ddc (doapmine decarboxylase, AADC), Slc6a3 (solute carrier family 6, member 3, DAT), and Th (tyrosine hydroxylase), were significantly increased to 8.08, 4.01, and 5.91, respectively. As data accumulate with different populations and different methods of differentiation, one will perhaps be able to identify the key regulators and biomarkers that may allow selective selection of limited number of genes or transcription factors to be used for direct reprogramming of NSC into DA cells, with an ultimate goal of obtaining different types of allogenic neurons including personalized DA neurons to be used in replacement therapy for neurodegenerative diseases such as Parkinson's disease (PD). Two-condition experiment: hENSC S vs. DA cells. Biological replicates: 2 hENSC, and 2 DA neuron samples from human substantia nigra cells.
2011-10-26 | E-GEOD-25931 | biostudies-arrayexpress
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:We studied human induced pluripotent stem cells (iPSCs)-derived dopaminergic (DA) neuron populations carrying CNVs of 16p11.2 duplication and 16p11.2 deletion.
Previously, healthy human iPSCs were edited using CRISPR-Cas9 method to produce isogenic lines with 16p11.2 deletion or 16p11.2 duplication. We differentiated these
isogenic iPSC lines into neural precursor cells and dopaminergic neurons and collected RNA samples for gene expression analyses with RNA sequencing. Our aim was to
identify differences in the expression of synaptic markers, neuronal differentiation markers, and neuron specific receptors that affect functionality of the neurons with 16p11.2
CNVs compared to isogenic control lines. We also studied physiological properties of these isogenic iPSC-derived DA neurons with 16p11.2 CNVs. In addition, we studied
expression and activation of a specific molecular pathway KCTD13-RHOA in the iPSC derived DA neuron populations with 16p11.2 CNVs.
| EGAS00001005137 | EGA
Project description:Elucidating the molecular mechanisms controlling dendrite development is key to understanding the pivotal role these structures play in influencing synaptic integration and neural function. Despite significant advances in this field, genetic pleiotropy remains a significant impediment to investigating such complex developmental processes. To circumvent this problem, we have applied class specific neuron transcriptional expression profiling coupled to an in vivo RNAi functional validation screen in order to dissect the molecular bases of Drosophila class IV dendritic arborization (da) neuron dendritogenesis. Microarray analyses reveal transcriptional regulation as one highly enriched biological and functional category with 420 transcription factors significantly expressed in class IV neurons. Among these, we identify roles for 268 genes in mediating a broad spectrum of functions including dendritic field coverage, branching, routing, and tiling. Collectively, our analyses provide a more comprehensive framework of the role complex transcriptional networks play in directing distinct aspects of class specific dendrite morphogenesis. Gene expression profiling of class IV da neurons performed at the third instar larval stage of development from two independent cell isolations from 40-50 age-matched third instar larvae expressing mCD8::GFP under the control of the GAL4ppk.1.9 driver
2014-04-01 | E-GEOD-28675 | biostudies-arrayexpress
Project description:While microRNAs (miRNAs) have recently emerged as critical post-transcriptional modulators of gene expression in neuronal development, very little is known regarding the roles of miRNA-mediated regulation in the specification of cell-type specific dendritic complexity. The dendritic arborization (da) sensory neurons of the Drosophila PNS offer an excellent model system for elucidating the molecular mechanisms governing class specific dendrite morphogenesis and for exploring miRNA-mediated control of this process. To facilitate functional analyses of miRNA regulation in da neurons, we have conducted whole-genome miRNA expression profiling as well as mRNA expression profiling of three distinct classes of da neurons, thereby generating a comprehensive molecular gene expression signature within these individual subclasses of da neurons. To further validate the role of these expressed miRNAs in directing dendritic architecture, we conducted a genome-wide UAS-miRNA phenotypic screen using live-image confocal microscopy followed by semi-automated neurometric quantification, to directly assess the effect of over/mis-expression of individual and clustered miRNAs on neurons of varying dendritic complexity. Through this approach, we have identified numerous miRNAs with previously unknown functions in dendritic development. Gain-of-function and loss-of-function analyses revealed an endogenous role miR-2b and miR-13b (members of the K-box family) and miR-12/283/304 in dendritic patterning in da neuron subclasses. Moreover, using an integrative bioinformatic analysis approach involving inverse correlation between miRNA and mRNA expression profiling data in combination with existing target prediction algorithms, we have identified putative target of these miRNAs in regulating da neuron dendritic development. Validation of these predicted miRNA-target relationships via phenotypic analyses as well as QPCR, revealed the regulatory effect of these molecules in restricting dendritic branching in da neurons.
2018-06-30 | GSE83609 | GEO
Project description:Genetic variation modulating risk of sporadic Parkinson’s disease (PD) has been primarily explored through genome wide association studies (GWAS). However, like many other common genetic diseases, the impacted genes remain largely unknown. Here, we used single-cell RNA-seq to characterize dopaminergic (DA) neuron populations in the mouse brain at embryonic and early postnatal timepoints. These data facilitated unbiased identification of DA neuron subpopulations through their unique transcriptional profiles, including a novel postnatal neuroblast population and substantia nigra (SN) DA neurons. We use these population-specific data to develop a scoring system to prioritize candidate genes in all 49 GWAS intervals implicated in PD risk, including known PD genes and many with extensive supporting literature. As proof of principle, we confirm that the nigrostriatal pathway is compromised in Cplx1 null mice. Ultimately, this systematic approach establishes biologically pertinent candidates and testable hypotheses for sporadic PD, informing a new era of PD genetic research.
2018-01-31 | GSE108020 | 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.
2011-11-07 | GSE32658 | GEO