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Identification of differentially expressed proteins in human pluripotent stem cell derived into dopaminergic neurons

ABSTRACT: Dopaminergic neurons located in the ventral midbrain can be broadly subdivided into two distinct subpopulations. Substantia nigra (SN) dopaminergic neurons are highly sensitive to toxic insults and selectively degenerate in Parkinson’s disease, while ventral tegmental area (VTA) dopaminergic neurons are associated with other neurological disorders. Access to enriched cultures of SN and VTA dopaminergic neuronal subpopulations will facilitate disease modelling and give insight in the differential vulnerability, but it is unclear how the differentiation of human ES cells can be directed towards these distinct lineages. We found that overexpression of the lineage specifying transcription factors Sox6 and Otx2 can direct the differentiation of human ES cells into enriched populations of respectively SN or VTA neurons. Proteomic analysis of these cultures resulted in the identification of several differential expressed proteins and provided insight in pathways contributing to the selective vulnerability of SN.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Stem Cell, Cell Culture

DISEASE(S): Parkinson's Disease

SUBMITTER: Valerie LABAS

LAB HEAD: Lia Panman

PROVIDER: PXD028639 | Pride | 2021-11-03

REPOSITORIES: Pride

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Project description:Implications for neuroprotection in Parkinson's disease Parkinson’s disease and its characteristic symptoms are thought to arise from the progressive degeneration of specific midbrain dopamine (DA) neurons. In humans, DA neurons of the substantia nigra (SN) and their projections to the striatum show selective vulnerability, while neighboring DA neurons of the ventral tegmental area (VTA) are relatively spared from degeneration. This pattern of cell loss is mimicked in humans, primates, and certain rodents by the neurotoxin MPTP. In this study, we aimed to test the hypothesis that there are factors in the VTA that are potentially neuroprotective against MPTP and that these factors change over time. We have found a differential transcriptional response within the cells of the SN and VTA to sustained exposure to a low dose of MPTP. Specifically, the VTA has increased expression of 148 genes as an early response to MPTP and 113 genes as a late response to MPTP toxicity. This response encompasses many areas of cellular function, including protein regulation (Phf6) and ion/metal regulation (PANK2, Car4). Notably, these responses were largely absent from the cells of the SN. Our data show a clear dynamic response in maintaining the homeostasis and viability of the neurons in the VTA that is lacking in the SN after neurotoxin challenge. We used microarrays to analyze the differential response of the substantia nigra (SN) and ventral tegmental area (VTA) to a chronic low dose of the neurotoxin MPTP. Transgenic hTH-GFP mice were treated with MPTP (4mg/kg) for either 2 or 10 days. Control mice were given an equal volume of saline for 10 days. Dopamine neurons from the substantia nigra and ventral tegmental areas of control and MPTP treated animals were laser captured. The RNA was isolated and processed for microarray hybridization. Each group had three biological replicates, for a total of 18 samples. Three each in the following: Control SN, Control VTA, 2 day MPTP SN, 2 day MPTP VTA, 10 day MPTP SN, 10 day MPTP VTA. Samples were log2 transformed and RMA normalized using Agilent Genespring 10.0 GX.

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Identification of differentially expressed proteins in human pluripotent stem cell derived into dopaminergic neurons

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