MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

Abstract

JOURNAL/nrgr/04.03/01300535-202412000-00026/figure1/v/2024-04-08T165401Z/r/image-tiff Gamma-aminobutyric acid (GABA)ergic neurons, the most abundant inhibitory neurons in the human brain, have been found to be reduced in many neurological disorders, including Alzheimer's disease and Alzheimer's disease-related dementia. Our previous study identified the upregulation of microRNA-502-3p (miR-502-3p) and downregulation of GABA type A receptor subunit α-1 in Alzheimer's disease synapses. This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function. In vitro studies were performed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs. In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunit α-1 mRNA. Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunit α-1 gene and suppresses the luciferase activity. Furthermore, quantitative reverse transcription-polymerase chain reaction, miRNA in situ hybridization, immunoblotting, and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunit α-1 level, while suppression of miR-502-3p increased the level of GABA type A receptor subunit α-1 protein. Notably, as a result of the overexpression of miR-502-3p, cell viability was found to be reduced, and the population of necrotic cells was found to be increased. The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney (HEK) recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function, suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function. Additionally, the levels of proteins associated with Alzheimer's disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression. The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p. We propose that micro-RNA, in particular miR-502-3p, could be a potential therapeutic target to modulate GABAergic synapse function in neurological disorders, including Alzheimer's disease and Alzheimer's disease-related dementia.

Figure 1

In silico analysis of miR-502-3p binding at GABRα1 mRNA. (A) Hsa-miR-502-3p binding site at GABRα1 mRNA highlighted in red at position 29348. (B) Secondary structure of GABRα1-miR-502-3p hybrid showing the molecular interaction with nucleotides. The miR-502-3p binding sites at the GABRα1 mRNA are highlighted in red. (C) Entropy plot of GABRα1 3′-UTR with miR-502-3p where x-axis shows the position of the nucleotides and y-axis shows the entropy. (D) MiR-502-3p interaction sites at the GABRα1 mRNA. MiR-502-3p showed a 9-mer binding site, two 8-mer binding sites, six 7-mer binding sites, and twenty-eight 6-mer binding sites at the GABRα1 mRNA. GABRα1: Gamma-aminobutyric acid type A receptor subunit alpha1; miR: microRNA.

Figure 2

MiR-502-3p modulates GABRα1 levels. (A) Firefly/Renilla dual luciferase assay of hippocampal neurons (HT22) cells transfected with GABRα1 3’-UTR construct, control construct, miR-502-3p agomiRs and miR-502-3p antagomiRs. The luciferase activity was significantly reduced in the cells co-transfected with GABRα1 3’-UTR construct and miR-502-3p agomiRs relative to other groups. (B) qRT-PCR analysis of miR-502-3p in HT22 cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. MiR-502-3p expression was significantly increased in the miR-502-3p agomiRs transfected cells, while it was decreased in miR-502-3p antagomiRs transfected cells compared with scramble control. (C) qRT-PCR analysis of GABRα1 mRNA in HT22 cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. GABRα1 expression was decreased significantly in the miR-502-3p agomiRs transfected cells, while its levels were increased significantly in miR-502-3p antagomiRs transfected cells compared with scramble control. (D) Immunoblot analysis of GABRα1 protein in HT22 cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. GABRα1 protein levels were decreased in the miR-502-3p agomiRs transfected cells. (E) Densitometry analysis of GABRα1 blot shows a significantly reduced level of GABRα1 protein in miR-502-3p agomiRs transfected cells compared with scramble control, while the cells treated with miR-502-3p antagomiRs show an increased level of GABRα1 protein. (F) Enzyme-linked immunosorbent assay showed a significantly reduced level of GABRα1 protein in miR-502-3p agomiRs treated cells compared with scramble control and antagomiRs treated cells. For statistical analysis, one-way analysis of variance was used for analyzing the data between three groups of samples such as scramble control, agomiRs, and antagomiRs. Experiments were performed in triplicates. The P-values < 0.05 were considered statistically significant. GABRα1: Gamma-aminobutyric acid type A receptor subunit alpha1; miR: microRNA; qRT-PCR: quantitative reverse transcription-polymerase chain reaction.

Figure 3

Alterations in miR-502-3p levels modulate cell viability. (A, B) Cell viability analysis: Scatter plots showing the population of live cells, apoptotic cells, necrotic cells, and cell debris in the scramble control, miR-502-3p agomiRs and antagomiRs transfected hippocampal neurons (HT22) cells at 24 (A) and 48 hours (B) post-transfection. (C) Quantification of live cell population (%) in miR-502-3p agomiR and miR-502-3p antagomiR transfected HT22 cells at 24 and 48 hours. The cell population was significantly decreased in miR-502-3p agomiR transfected cells compared with scramble control at 24 and 48 hours. The percentage of viable cells was significantly increased in miR-502-3p antagomiR transfected cells relative to scramble control at 24 and 48 hours. (D) Quantification of necrotic cell population (%) in miR-502-3p agomiR and miR-502-3p antagomiR transfected HT22 cells at 24 and 48 hours. The necrotic cell population was significantly increased in miR-502-3p agomiR transfected cells compared with scramble control at 24 and 48 hours. The necrotic cell population was decreased in miR-502-3p antagomiR transfected cells relative to control and agomiR treated cells. (E) Cell proliferation analysis by cell counting kit-8 method showed the diminished cell survival in miR-502-3p agomiRs treated cells, while cell survival was gradually increased in miR-502-3p antagomiRs treated cells. For statistical analysis, one-way analysis of variance was used for analyzing the data between three groups of samples such as scramble control, agomiRs, and antagomiRs. Experiments were performed in quadruplicates. The P-values < 0.05 were considered statistically significant. miR: microRNA.

Figure 4

MiR-502-3p modulates GABRα1 protein level at cellular junctions. (A) MiRNA in situ hybridization analysis showed the increased signals of miR-502-3p probes in miR-502-3p agomiRs transfected hippocampal neurons (HT22) cells. The U6snRNA expression was consistent in all three groups of cells (magnification 20×; scale bars: 75 µm). (B) Immunofluorescence analysis of HT22 cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs (magnification 100×, scale bars: 10 µm). HT22 cells were stained with GABRα1 antibody detected by Alexa fluor (red) and nuclei were stained with DAPI (blue). MiR-502-3p antagomiR transfected cells showed high levels of GABRα1 protein at cell junctions as shown by the white arrows. (C) Fluorescence intensity quantification of GABRα1 protein in the cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. GABRα1 fluorescence intensity was significantly increased in miR-502-3p antagomiR transfected cells relative to control and agomiR treated cells. The P-values < 0.05 were considered statistically significant. DAPI: 4′,6-Diamidino-2-phenylindole; GABRα1: gamma-aminobutyric acid type A receptor subunit alpha1; miR: microRNA.

Figure 5

Modulation of GABRα1 protein by miR-502-3p at cellular junctions. (A) Confocal microscopy analysis of hippocampal neurons (HT22) cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. Cells were stained with GABRα1 antibody detected by Alexa fluor (red) and nuclei stained with DAPI (blue). MiR-502-3p antagomiR transfected cells showed high levels of GABRα1 protein at cell junctions as shown by the white arrow. Magnification 63×, scale bars: 20 µm. (B) Fluorescence intensity quantification of GABRα1 protein in the cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. GABRα1 fluorescence intensity was significantly increased in miR-502-3p antagomiR transfected cells compared with control and agomiR treated cells. DAPI: 4′,6-Diamidino-2-phenylindole; GABRα1: gamma-aminobutyric acid type A receptor subunit alpha1; miR: microRNA.

Figure 6

Patch-clamp analysis of cells treated with miR-502-3p. (A) Sweep plots showing the gamma-aminobutyric acid (GABA) current in the human-gamma-aminobutyric acid receptor A-α1/β2/γ2L (hGABAA-α1/β2/γ2L) human embryonic kidney (HEK) recombinant cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. The endogenous GABA channel was recorded at 300 μm GABA concentration for 4 seconds using the IonFlux system. (B) Quantification of GABA current in the parental, scramble control, miR-502-3p agomiRs, and antagomiR treated cells. The GABA current was reduced in agomiR transfected cells, while it was significantly increased in the miR-502-3p antagomiR transfected cells compared with scramble control. Experiments were performed a minimum 8 times in each group. The P values < 0.05 were considered statistically significant. miR: MicroRNA.

Figure 7

Modulation of AD proteins by miR-502-3p. (A) Immunoblot analysis of amyloid precursor protein (APP), tubulin associated unit (Tau), presenilin-1 (PSN1) and presenilin-2 (PSN2) and -actin protein in hippocampal neurons (HT22) cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. The levels of AD proteins were changed in miR-502-3p agomiR and antagomiR transfected cells. (B) Densitometry analysis of APP, Tau, PSN1 and PSN2 blot shows increased protein levels in miR-502-3p agomiR transfected cells compared with scramble control, while the cells treated with miR-502-3p antagomiR shows a reduced level of all AD proteins compared with agomiRs. For statistical analysis, Student’s t-test was used for analyzing the data between three groups of samples such as scramble control, agomiRs, and antagomiRs. Experiments were performed in triplicates. The P-values < 0.05 were considered statistically significant. AD: Alzheimer’s disease; miR: microRNA.