Novel microRNAs targeting NMDA receptor subunits in animal models of schizophrenia

Abstract

N-methyl-D-aspartate receptors (NMDAR) are downregulated in schizophrenia possibly through microRNAs (miRNAs) that are differentially expressed in this condition. We screened the miRNAs that are altered in schizophrenia against the targets, Grin2A and Grin2B subunits of NMDAR using bioinformatic tools. Among the predicted miRNAs some interacted with the 3`UTR sequences of Grin2A (miR-296, miR-148b, miR-129-2, miR-137) and Grin2B (miR-296, miR-148b, miR-129-2, miR-223) in dual luciferase assays. This was supported by downregulation of the GluN2B protein in primary hippocampal neurons upon overexpressing Grin2B targeting miRNAs. In two models of schizophrenia- pharmacological MK-801 model and neurodevelopmental methylazoxymethanol acetate (MAM) model which showed cognitive deficits - protein levels of GluN2A and GluN2B were downregulated but their transcript levels were upregulated. MiR-296-3p, miR-148b-5p and miR-137 levels showed upregulation in both models which could have interacted with Grin2A/Grin2B transcripts resulting in translational arrest. In MAM model, reciprocal changes in the expression of the 3p and 5p forms of miR-148b and miR-137 were observed. Expression of neuregulin 1 (NRG1), BDNF and CaMKIIα, genes implicated in schizophrenia, were also altered in these models. This is the first report of downregulation of GluN2A and GluN2B by miR-296, miR-148b and miR-129-2. Mining miRNAs regulating NMDA receptors might give insights into the pathophysiology of this disorder, providing avenues in therapeutics.

miR-146a and miR-200b alter cognition by targeting NMDA receptor subunits

“…The amplified pre-miRNAs were cloned into the expression vector, pRIPM that has Ds Red as marker.. The 3’UTRs of Grin2A and Grin2B in the vector backbone psiCHECK2 described before (Gunasekaran et al, 2021) were also used for performing the dual luciferase assay. All the clones were confirmed by restriction digestion and sequencing.…”

“…The procedure for dual luciferase assay was performed using Human Embryonic kidney-293 (HEK-293) cells as described before (Gunasekaran et al, 2021). In brief, The psiCHECK2 and pRIPM vector constructs carrying the 3’UTRs and the pre-miRNAs respectively were used for the assay.…”

“…Pregnant female dams of E-18-19 were sacrificed and the hippocampus was dissected, trypsinised and the dissociated cells were cultured in coated dishes. The primary neurons were maintained up to 20 days in vitro (DIV 20) (Gunasekaran et al, 2021;Chandran et al, 2021; Salazar et al, 2017).…”

“…Weaning of the pups was performed on postnatal day 30 (P30). Detailed procedure was described before (Gunasekaran et al, 2021).…”

“…miR-19a and miR-539 can also target and regulate NMDAR (Corbel et al, 2015). In our previous study, we found miR-129-2, miR-148a and miR-296 to be targeting NMDAR in vitro and inanimal models in which NMDAR is dysregulated (Gunasekaran et al, 2021).…”