Subcellular and regional localization of mRNA translation in midbrain dopamine neurons
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ABSTRACT:
PROVIDER: PRJNA750065 | ENA |
REPOSITORIES: ENA
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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:Maintenance of the drug-addicted state involves changes in gene expression in different neuronal cell types and neural circuits. Midbrain dopamine neurons in particular mediate numerous responses to drugs of abuse. Long noncoding RNAs (lncRNAs) regulate CNS gene expression through a variety of mechanisms, but very little is known about their role in drug abuse. The proportion of lncRNAs that are primate-specific provides a strong rationale for their study in human drug abusers. We examined the profile of lncRNA expression in postmortem human midbrain specimens from chronic cocaine abusers and well-matched control subjects (n=11 subject pairs) using a custom lncRNA microarray. Differential expression was validated by quantitative PCR, and cellular localization investigated by in situ hybridization histochemistry.A profile of lncRNAs significantly dysregulated in chronic cocaine abusers was determined. LncRNAs with dopamine cell-specific expression, differential subcellular distribution, covariance with protein-coding genes, and tissue-specific drug-responsiveness were identified. Dysregulation of midbrain lncRNA expression may reflect pathophysiological processes associated with chronic cocaine abuse. LncRNAs may be central mediators of cellular responses to drug abuse.
2015-07-04 | GSE67281 | GEO
Project description:The distribution of RNA in human embryonic stem cells (hESC) and the function of RNA localization in maintaining hESC pluripotency and differentiation are currently unknown. Here, by isolating five subcellular components of hESCs and differentiated cells, we uncovered the global subcellular RNA localization in hESC. For protein-coding mRNA, different transcripts of the same gene exhibit an “isoform switch” between subcellular components, which is regulated by localization cis-elements in their variable regions. For noncoding RNA, multiple sequence features such as polyA tail, length, and GC content jointly regulate their subcellular localization. In addition, we found that some developmental genes can be transcribed in advance and confined to chromatin in undifferentiated hESCs. Finally, we revealed significant changes in overall RNA distribution, mapped RNA dynamic localization atlas, and characterized different dynamic RNA localization patterns during hESC differentiation into mesoderm. The multiple RNA localization patterns we revealed will provide some new enlightenment for hESC stemness maintenance and differentiation.
2023-12-22 | GSE206328 | GEO
Project description:We report a novel midbrain dopamine neuron derivation protocol from human pluripotent stem cells (hPSC)s based on a two-step WNT signaling activation strategy. We perform genome-wide chromatin immunoprecipitation (ChIP-seq) for histone modifications including H3K4me3 and H3K27me3 with midbrain dopamine neuron differentiated cells from hPSC in Low-Chir and Boost-Chir (7.5 uM) conditions at day 11. Those results indicate that CHIR-boost promotes a more open chromatin structure at midbrain regions such as an EN1 locus based on increased H3K4me3 and decreased H3K27me3 levels. In contrast, analysis of the contaminating posterior (hindbrain) and anterior (diencephalic) lineage including NKX2-2 and PAX6 loci showed the opposite pattern with decreased H3K4me3 and increased H3K27me3 levels under CHIR-boost conditions. The protocol presented here is the basis for clinical grade dopamine neuron production and preclinical safety and efficacy studies.
2020-12-18 | GSE162882 | GEO
Project description:Maintenance of the drug-addicted state involves changes in gene expression in different neuronal cell types and neural circuits. Midbrain dopamine neurons in particular mediate numerous responses to drugs of abuse. Long noncoding RNAs (lncRNAs) regulate CNS gene expression through a variety of mechanisms, but very little is known about their role in drug abuse. The proportion of lncRNAs that are primate-specific provides a strong rationale for their study in human drug abusers. We examined the profile of lncRNA expression in postmortem human midbrain specimens from chronic cocaine abusers and well-matched control subjects (n=11 subject pairs) using a custom lncRNA microarray. Differential expression was validated by quantitative PCR, and cellular localization investigated by in situ hybridization histochemistry.A profile of lncRNAs significantly dysregulated in chronic cocaine abusers was determined. LncRNAs with dopamine cell-specific expression, differential subcellular distribution, covariance with protein-coding genes, and tissue-specific drug-responsiveness were identified. Dysregulation of midbrain lncRNA expression may reflect pathophysiological processes associated with chronic cocaine abuse. LncRNAs may be central mediators of cellular responses to drug abuse. cRNAs were generated from postmortem human midbrain specimens from chronic cocaine abusers and well-matched control subjects and hybridized to a custom Agilent 4 x 44,000-feature high-density oligonucleotide microarray platform using 60-mer probes. Cocaine control pairs were run together in a dye-flip two-color design, meaning each sample was run in quadruplicate, twice with each dye (Alexa-647 and Alexa-555; Invitrogen, Carlsbad, CA). Microarray slides were scanned with the default Agilent protocol and the intensity of fluorescence between dyes was normalized using a Loess correction. There are 7 probes per gene. Data across all cases and quadruplicates were quantile-normalized.
2015-07-04 | E-GEOD-67281 | biostudies-arrayexpress
Project description:We report a novel midbrain dopamine neuron derivation protocol from human pluripotent stem cells (hPSC)s based on a two-step WNT signaling activation strategy. By comparing next-generation sequencing (NGS) derived transcriptome profiling (RNA-seq) of dopamine neuron differentiated cells derived from hPSC with different concentration of Chir-boosting (day4 – day9) measured at day 11 including hPSC, we find that CHIR-boosting improves expression of midbrain markers such as EN1 while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. Furthermore, gene expression analysis from RNA-seq study confirmed the progressive differentiation and maturation of Boost CHIR treated precursors into mDA neurons by day 30 of differentiation. The protocol presented here is the basis for clinical grade dopamine neuron production and preclinical safety and efficacy studies.
2020-12-18 | GSE162883 | GEO
Project description:Subcellular RNA localization during Drosophila embryonic neurogenesis [fractionation-Seq]
| PRJNA415601 | ENA
Project description:Atlas of Subcellular RNA Localization Revealed by APEX-seq
| PRJNA476786 | ENA
Project description:Human-specific transcriptome of ventral and dorsal midbrain dopamine neurons
| PRJNA698999 | ENA
Project description:Objective: To assess the role of aldoketoreductases and other doxorubicin pharmacokinetic or pharmacogenomic genes in doxorubicin cytotoxicity, resistance, DNA binding activity, and subcellular localization, Methods: We conducted a whole genome microarray study to identify differences in between doxorubicin-sensitive MCF-7cc cells and doxorubicin-resistant MCF-7Dox2-12 cells in terms of their expression of genes related to doxorubicin pharmacokinetics or pharmacodynamics. Targets were then validated by pharmacologic inhibition in conjunction with drug metabolite profiling, drug localization, drug cytotoxicity, and drug DNA binding studies. Results: 2063 differentially expressed transcripts were identified, including 17% and 43% of genes or gene families associated with doxorubicin pharmacokinetics or pharmacodynamics (p values of significance of 0.05 and <0.0001, respectively). The largest changes in the expression of genes associated with doxorubicin pharmacokinetics and pharmacodynamics were chiefly among the aldo-keto reductases (AKRs) Akr1c2, Akr1c3 and Akr1b10 which convert doxorubicin to doxorubicinol. We observed that doxorubicinol exhibits dramatically reduced drug toxicity, reduced drug DNA-binding activity, and altered drug subcellular localization to lysosomes. Pharmacologic inhibition of these AKRs in MCF-7Dox2-12 cells restored drug cytotoxicity, and drug localization to the nucleus. Conclusion: These findings demonstrate the utility of using curated pharmacokinetic and pharmacodynamic knowledgebases to identify highly relevant genes associated with doxorubicin resistance. The products of one or more of these genes could effectively be shown to alter the drug’s properties, while inhibiting them restored drug DNA binding, cytotoxicity, and subcellular localization. Doxorubicin resistant cell lines of breast MCF-7 cells were generated for gene expression profilling. Two colour microarray of Agilent whole human genome nucleotide arrays was conducted with four labelling replicates of both forward and reverse labellings plus another set of 8 arrays with forward labelling. Sixteen arrays were used for this experiments. The co-cultured control cells MCF-7cc12 was generated by parallel selection process in the absence of drug.
2012-11-22 | E-GEOD-27254 | biostudies-arrayexpress