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Global proteome of Atg5flox:Slc32a1-Cre:tdTomato FACS sorted WT and KO neurons

ABSTRACT: The experiment was performed to identify autophagy targets in wildtype and autophagy-deficient forebrain inhibitory neurons. Therefore, neurons were isolated from the cortex, hippocampus and striatum of 2-3 weeks old Atg5flox/flox:Slc32a1-Cretg/wt:tdTomato+ (KO) and Atg5wt/wt:Slc332a1-Cretg/wt:tdTomato+ (WT) mice. Neurons in suspension were FACS sorted and inhibitory forebrain neurons expressing tdTomato were forwarded to global proteome analysis assessed by LC-MS/MS.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Hippocampus

SUBMITTER: Prerana Wagle

LAB HEAD: N.L. Kononenko

PROVIDER: PXD031535 | Pride | 2022-11-15

REPOSITORIES: Pride

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			Action	DRS
	20200822001950_E2_FAIMS_Col_PF001_30cm_2222_01.raw	Raw
	20200822020916_E2_FAIMS_Col_PF001_30cm_2222_02.raw	Raw
	20200822035846_E2_FAIMS_Col_PF001_30cm_2222_03.raw	Raw
	20200822054820_E2_FAIMS_Col_PF001_30cm_2222_04.raw	Raw
	20200822073755_E2_FAIMS_Col_PF001_30cm_2222_05.raw	Raw

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Project description:Ischemic stroke is a severe medical condition that leads to neurological impairments, including the loss of sensory, motor, and cognitive functions. Focal cerebral ischemia (FCI) occurs when blood supply to a specific brain region is interrupted, resulting in cell death. Cells expressing neural-glial antigen 2 (NG2) include glial cells, primarily oligodendrocyte precursors, and perivascular cells. In both cell types, the canonical Wnt signaling pathway is active, with its activity increasing after ischemic injury, influencing the tissue’s response to damage. Following ischemia, NG2 glia rapidly proliferate, migrate to the injury site, contribute to glial scar formation alongside astrocytes, and play a role in brain tissue regeneration. Their potential to differentiate into cell types beyond oligodendrocytes has been documented. Perivascular cells, which regulate vascular contraction, serve as an informational bridge between vascular endothelial cells and glial cells, particularly astrocytes. The plasticity of pericytes following brain injury has also been suggested. Previously, we analyzed NG2-expressing cells in Rosa26-tdTomato/Cspg4-CreERT2 mice three days after middle cerebral artery occlusion (MCAO) or sham surgery (CTRL) without Wnt pathway modulation (ArrayExpress accession number: E-MTAB-11967). In this study, we extended our analysis by inhibiting the Wnt signaling pathway, which is crucial for stem cell maintenance, proliferation, and neuronal differentiation. To investigate these processes, we isolated NG2 cells and their derivatives labeled with the red fluorescent protein tdTomato from the cortex of Rosa26-tdTomato/Cspg4-CreERT2 mice three days after MCAO. Mice subjected to sham surgery served as healthy controls. Wnt pathway inhibition was achieved using an allele that enables inducible expression of the Wnt inhibitor Dickkopf1 (Dkk1). Our aim was to determine how the Wnt pathway inhibition affects the distribution of NG2 glial and perivascular cell subtypes, identify their derivatives in the cortex of healthy and ischemic mice, and characterize their abundance and gene expression profiles.

Project description:Ischemic stroke is a severe medical condition that leads to neurological impairments, including the loss of sensory, motor, and cognitive functions. Focal cerebral ischemia (FCI) occurs when blood supply to a specific brain region is interrupted, resulting in cell death. Cells expressing neural-glial antigen 2 (NG2) include glial cells, primarily oligodendrocyte precursors, and perivascular cells. In both cell types, the canonical Wnt signaling pathway is active, with its activity increasing after ischemic injury, influencing the tissue’s response to damage. Following ischemia, NG2 glia rapidly proliferate, migrate to the injury site, contribute to glial scar formation alongside astrocytes, and play a role in brain tissue regeneration. Their potential to differentiate into cell types beyond oligodendrocytes has been documented. Perivascular cells, which regulate vascular contraction, serve as an informational bridge between vascular endothelial cells and glial cells, particularly astrocytes. The plasticity of pericytes following brain injury has also been suggested. Previously, we analyzed NG2-expressing cells in Rosa26-tdTomato/Cspg4-CreERT2 mice three days after middle cerebral artery occlusion (MCAO) or sham surgery (CTRL) without Wnt pathway modulation (ArrayExpress accession number: E-MTAB-11967). In this study, we extended our analysis by constitutively activating the Wnt/β-catenin signaling pathway, which is crucial for stem cell maintenance, proliferation, and neuronal differentiation. To investigate these processes, we isolated NG2 cells and their derivatives labeled with the red fluorescent protein tdTomato from the cortex of Rosa26-tdTomato/Cspg4-CreERT2 mice three days after MCAO. Mice subjected to sham surgery served as healthy controls. Wnt pathway activation was achieved using an allele that enables constitutive stabilization of β-catenin (Ctnnb1 delEx3). Our aim was to determine how the Wnt pathway overactivation affects the distribution of NG2 glial and perivascular cell subtypes, identify their derivatives in the cortex of healthy and ischemic mice, and characterize their abundance and gene expression profiles.

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Global proteome of Atg5flox:Slc32a1-Cre:tdTomato FACS sorted WT and KO neurons

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