Establishment of a dopaminergic neuron purification system in mice
Ontology highlight
ABSTRACT: Transcriptomic analysis of enriched of neuron population collected from in-vivo mouse brains has been a challenge in the neuroscience field due to its fragility in withstanding harsh condition during isolation and collection process. We established a fluorescent reporter mouse Ddc-hKO1 to facilitate the identification and collection of Ddc-expressing neurons (dopaminergic, serotonergic, cholinergic and adrenergic neurons) by the detection of red fluorescence signals using a FACs. We utilized an improved isolation protocol that ensure high yield and quality of viable neurons during collection that is suitable for RNA-sequencing. This is the first report of transcriptomic profiling of Ddc expressing (hKO1(+)) neurons. Successful collection of Ddc-expressing neurons were verified by gene markers of dopaminergic, serotonergic, cholinergic neurons in hKO1(+) populations while other neuron types in hKO1(-) population. Furthermore, GSEA analysis were performed on both hKO1(+) and hKO1(-) to further support the neuron types collected in respective population.
Project description:Performed RNA-seq analysis of animals with xbp-1s overexpression (ER stress response transcription factor) in specific neuron types: pan-neuronal, serotonergic neuron, dopaminergic neuron, and both serotonergic and dopaminergic neurons, all compared to a wild-type control. RNA-seq was performed on purified RNA extracted from ~1000 whole worms using a proprietary Genewiz protocol described briefly in the manuscript. 3 biological replicates are provided for each sample.
Project description:Dopaminergic neurons (expressing Pdat-1::mStrawberry) and serotonergic neurons (expressing Ptph-1::GFP) were purified from C. elegans embryos and larvae. We identified transcripts that were enriched in dopaminergic neurons compared to serotonin neurons.
Project description:Loss-of-function mutations in the parkin gene can cause early onset Parkinson’s disease, a movement disorder resulting from the selective degeneration of dopaminergic neurons in the basal ganglia. Analogously, movement deficits and loss of a subset of dopaminergic neurons are observed in Drosophila melanogaster homozygous for null mutations in parkin. Parkin is an E3 ubiquitin ligase that functions with the mitochondrial localized serine/threonine kinase PINK1 in a pathway required to maintain mitochondrial integrity. We previously established that the PINK1/Parkin pathway functions in Drosophila dopaminergic and cholinergic neurons to maintain mitochondrial membrane potential. However, the mechanisms through which the PINK/Parkin pathway selectively impacts dopaminergic neuron survival remains unclear, as do the mechanisms that lead to the selective vulnerability of dopaminergic neurons in Parkinson’s disease. Because the transcriptome of a cell determines its identity we hypothesized that knowledge of the transcriptional alterations that occur in dopaminergic neurons isolated from parkin null Drosophila would provide insight into the mystery of selective vulnerability in Parkinson's disease. Results: To test our hypothesis we measured the transcriptome of dopaminergic and cholinergic neurons isolated from isogenic heterozygous and homozygous parkin null mutants using a novel flow cytometry-based method we developed. Computational analysis and experimental confirmation demonstrate that our method allows for the successful expression analysis of defined neural subsets from the Drosophila brain. In addition, our dataset implicates iron handling and dopamine signaling as being significantly dysregulated in parkin null dopaminergic neurons. Conclusions: Our flow cytometry-based method allows for the isolation and microarray analysis of neuronal subsets from the adult Drosophila brain. Our microarray analyses implicate iron handling and dopamine metabolism as contributing factors in the etiology of parkin-associated early onset Parkinson’s disease. Here we provide a novel dataset that may serve as a foundation for subsequent functional analyses of the pathways underlying neuronal selective vulnerability in Parkinson's disease.
Project description:Microarray results revealed that EEA treatment induced variations in several genes associated with pro-inflammatory and dopaminergic, cholinergic, glutamatergic, and serotonergic synapses. DNA microarray analysis was conducted on isolated RNAs extracted from the limbic area of EEA-treated brains
Project description:SATB1 is a genetic master regulator in dopaminergic neurons. We try to identify the downstream regulated genes and pathways of SATB1 in human dopaminergic and CTX neurons. The RNA-Seq experiment was performed to investigate the role of the genetic master regulator SATB1 in human dopaminergic neurons in comparison to cortical neurons. We generated a human embryonic stem cell knockout clone for SATB1 and differentiated this clone into either dopaminergic or cortical neurons. Immature dopaminergic (day 30 of differentiation), mature dopaminergic (day 50 of differentiation) and mature cortical neurons (day 30 of differentiation) were subsequently subjected to RNA-Seq. We compared wild type and SATB1-KO neurons at the afore mentioned time points, to characterize the regulatory role of SATB1 in the different neuron subtypes.
Project description:Our microarray results showed there were up-regulated 28 genes and down-regulated 29 genes, which related depression and inflammatory response such as cytokine-cytokine receptor interaction, chemokine signaling pathway, dopaminergic synapse, glutamatergic synapse, GABAergic synapse, cholinergic synapse, and serotonergic synapse. Among these genes, especially, higher hippocampal mRNA expression of transthyretin (Ttr), Zinc finger protein of the cerebellum 1 (Zic1), and Ectonucleotide pyrophosphatase/phosphodiesterase 2 (Enpp2) was found in kososan-administered defeated mice than water-administered defeated mice
Project description:Despite the advances in our understanding of aging-associated behavioral decline, we know relatively little about how aging affect neural circuits that underlie specific behaviors. Specifically, we know little about how aging affect expression of genes in specific neural circuits. We have now addressed this problem by exploring a cholinergic neuron R15, an identified neuron of marine snail Aplysia. R15 is characterized by bursting action potentials and is implicated in reproduction, osmoregulation and locomotion. We examined changes in gene expression in R15 neurons during aging by microarray analyses of RNAs prepared from two different age groups, mature and old animals. Specifically we find that 1083 ESTs are differentially regulated in mature and old R15 neurons. Bioinformatics analyses of these genes have identified specific biological pathways and molecular processes that are up or down regulated in mature and old neurons. Comparison with human signaling networks using pathway analyses have identified three major networks that are altered in old R15 neurons. Furthermore, by single neuron qRTPCR we examined expression levels of candidate regulators involved in transcription (CREB1) and translation (S6 kinase) and find that aging is associated with a decrease in expression of these regulators. We next studied expression of CREB1 and S6 kinase in two different motor neurons (L7 and L11) and another cholinergic neuron R2 and find that these neurons have characteristic changes in gene expression during aging
Project description:Astrocytes are essential cells of the central nervous system, characterized by dynamic relationships with neurons that range from functional metabolic interactions and regulation of neuronal firing activities, to the release of neurotrophic and neuroprotective factors. In Parkinson’s disease (PD), dopaminergic neurons are a vulnerable population progressively lost during the course of the disease, but the effects of PD on astrocytes and astrocyte-to-neuron communication remains mostly unknown. This study focuses on the effects of the PD-related mutation LRRK2 G2019S in astrocytes, using patient-derived induced pluripotent stem cells. We report the alteration of extracellular vesicle (EV) biogenesis in astrocytes, and we identify the abnormal accumulation of key PD-related proteins within multi vesicular bodies (MVBs). We found that dopaminergic neurons internalize astrocyte-secreted EVs but LRRK2 G2019S EVs are abnormally enriched in the neurites and provide only marginal neurotrophic support to dopaminergic neurons. Thus, dysfunctional astrocyte-to-neuron communication via altered EV biological properties could participate in the progression of PD.
Project description:Personalized cell therapy is being explored as promising treatments for incurable diseases such as Parkinson’s disease (PD). Here, we directly reprogrammed human fibroblasts into induced dopaminergic neuronal progenitors (hiDPs) and assess their applicability for regenerative PD therapy. We found that hiDPs maintain progenitor characteristics over 20 passages and showed unaltered differentiation potential to midbrain dopaminergic neurons. By next-generation sequencing, we confirm that even long-term cultured hiDPs retain genomic stability, resulting in absence of tumorigenicity when transplanted, indicating preclinical safety. The hiDPs differentiate into highly pure dopaminergic neurons expressing markers associated with positive graft outcomes without unwanted serotonergic neurons known to cause graft-induced dyskinesia. The therapeutic effect and safety were confirmed by transplantation in rodent PD models. Because our optimized reprogramming conditions generate highly expandable hiDPs enabling scaled production of clonal cells and overcoming quality control issues, we propose that our hiDPs can become a preferred autologous therapy for aged and feeble PD patients vulnerable to immunosuppression regimens.
Project description:Personalized cell therapy is being explored as promising treatments for incurable diseases such as Parkinson’s disease (PD). Here, we directly reprogrammed human fibroblasts into induced dopaminergic neuronal progenitors (hiDPs) and assess their applicability for regenerative PD therapy. We found that hiDPs maintain progenitor characteristics over 20 passages and showed unaltered differentiation potential to midbrain dopaminergic neurons. By next-generation sequencing, we confirm that even long-term cultured hiDPs retain genomic stability, resulting in absence of tumorigenicity when transplanted, indicating preclinical safety. The hiDPs differentiate into highly pure dopaminergic neurons expressing markers associated with positive graft outcomes without unwanted serotonergic neurons known to cause graft-induced dyskinesia. The therapeutic effect and safety were confirmed by transplantation in rodent PD models. Because our optimized reprogramming conditions generate highly expandable hiDPs enabling scaled production of clonal cells and overcoming quality control issues, we propose that our hiDPs can become a preferred autologous therapy for aged and feeble PD patients vulnerable to immunosuppression regimens.