Dataset Information

Brain region differences in α1 and α5 subunit-containing GABAA receptor proteome revealed by affinity purification and Blue Native PAGE proteomics

ABSTRACT: GABAA receptors are the major inhibitory receptors in the brain. They are hetero-pentamers with a composition of predominantly two α, two β and one γ or δ subunit. From the six α subunit genes, the α5 subunit displays a limited spatial expression pattern and is known to mediate both phasic and tonic inhibition. In this study, using immunoaffinity-based proteomics we identified the α5 subunit containing receptor complexes in hippocampus and olfactory bulb. The α1-α5 interaction was identified in both brain regions albeit with significantly different stoichiometries. In line with this, reverse IPs using anti-α1 antibody showed the α5-α1 co-occurrence and validated the quantitative difference. In addition, we showed that the association of Neuroligin 2 with α1-containing receptors was much higher in olfactory bulb than hippocampus, which was confirmed using blue native gel electrophoresis and quantitative mass spectrometry. Finally, immunocytochemical staining revealed co-localization of α1 and α5 subunits in post-synaptic puncta in the hippocampus.

INSTRUMENT(S): timsTOF Pro 2

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Miao Chen

LAB HEAD: Kawan Li

PROVIDER: PXD047550 | Pride | 2024-01-26

REPOSITORIES: Pride

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Publications

Brain Region Differences in α1- and α5-Subunit-Containing GABA<sub>A</sub> Receptor Proteomes Revealed with Affinity Purification and Blue Native PAGE Proteomics.

Chen Miao M Koopmans Frank F Gonzalez-Lozano Miguel A MA Smit August B AB Li Ka Wan KW

Cells 20231220 1

GABA<sub>A</sub> receptors are the major inhibitory receptors in the brain. They are hetero-pentamers with a composition of predominantly two α, two β, and one γ or δ subunit. Of the six α subunit genes, the α5 subunit displays a limited spatial expression pattern and is known to mediate both phasic and tonic inhibition. In this study, using immunoaffinity-based proteomics, we identified the α5 subunit containing receptor complexes in the hippocampus and olfactory bulb. The α1-α5 interaction was ...[more]

PMID: 38201218

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Project description:Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic γ-aminobutyric acid type A receptors (GABAARs), which mediate phasic and tonic inhibition, respectively. These two GABAAR subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABAARs contain the α1-3 subunits whereas extrasynaptic GABAARs contain the α4-6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABAAR subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous α1- and α4-containing GABAARs from adult murine forebrains and examined their subunit composition and interacting proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantitative analysis. We found that the α1 and α4 subunits form separate populations of GABAARs and interact with distinct sets of binding proteins. We also discovered that the β3 subunit, which co-purifies with both the α1 and α4 subunits, has different levels of phosphorylation on serines 408 and 409 (S408/9) between the two receptor subtypes. To understand the role S408/9 plays in the assembly of α1- and α4-containing GABAARs, we examined the effects of S408/9A (alanine) knock-in mutation on the subunit composition of the two receptor subtypes using LC-MS/MS and quantitative analysis. We discovered that the S408/9A mutation results in the formation of novel α1α4-containing GABAARs. Moreover, in S408/9A mutants, the plasma membrane expression of the α4 subunit is increased whereas its retention in the endoplasmic reticulum is reduced. These findings suggest that S408/9 play a critical role in determining the subtype-specific assembly of GABAARs, and thus the efficacy of neuronal inhibition.

Project description:In this study, we demonstrate that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from hippocampus and olfactory bulb. Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampus and olfactory bulb-derived neural stem cells. Our analysis indicates that the balance between Wnt3, which triggers the expression of insulin via NeuroD1 transcription factor, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on the diabetic status. We also show that adult neural progenitors derived from diabetic animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of diabetic animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes. In this study, we demonstrate that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from hippocampus and olfactory bulb. Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampus and olfactory bulb-derived neural stem cells. Our analysis indicates that the balance between Wnt3, which triggers the expression of insulin via NeuroD1 transcription factor, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on the diabetic status. We also show that adult neural progenitors derived from diabetic animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of diabetic animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes. Total four different samples, gene expressions in hippocampal derived neural stem cells (HPC NSC), that in Olfactory bulb-derived neural stem cells (OB NSC), that in neurons derived from the HPC NSCs (HPC Neu) and that in neurons derived from the OB NSCs (OB Neu) were independently analyzed. Three independent experiments were performed to prepare each cell sample, and the extracted total RNAs from each cell source were mixed to apply following microarray analysis (Four independent RNA sample; HPC NSC, OB NSC, HPC Neu and OB Neu).

Dataset Information

Brain region differences in α1 and α5 subunit-containing GABAA receptor proteome revealed by affinity purification and Blue Native PAGE proteomics

Publications

Brain Region Differences in α1- and α5-Subunit-Containing GABA<sub>A</sub> Receptor Proteomes Revealed with Affinity Purification and Blue Native PAGE Proteomics.

Similar Datasets

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