HnRNP M Deficiency Leads To Cognitive Deficits Via Disrupting Synaptic Plasticity

RNA binding proteins(RBPs)play a key role in the transcriptome's maintenance by controlling RNA processing and transport.They are a particular type of protein that have been connected to multiple functions.RBPs are proteins that bind RNA by one or more globular RNA-binding domains(RBDs)and alter the fate or function of the RNAs they bind.RBPs bind RNA molecules at unique sequences or secondary structures in the nucleus and cytoplasm to assist with many stages in RNA processing.RNA binding proteins act by integrating a variety of diverse and closely regulated RNA metabolism processes,including alternative splicing,transcription,intracellular delivery,translation,and degradation or decay.In terms of brain functions,especially cognition,the roles of RNA binding proteins in these processes are largely unknown.The function of hnRNP M,an RBP member of the heterogeneous nuclear ribonucleoproteins,was investigated in this study.We found that the level of hnRNP M was depleted in the aging mouse brain,especially in the hippocampus,which is responsible for the encoding of declarative(episodic)memories,context-dependent spatial learning,and emotional(anxiety)regulation.This indicates that hnRNP M plays a crucial role in hippocampus-related cognitive functions.As a result,we chose to examine the physiological importance of hnRNP M in cognition by depleting hnRNP M levels in the hippocampus of young wild-type mice and then studying the phenotype.By sterotaxically injecting the CA1 area of the hippocampus with an AAV-mediated shRNA,the level of hnRNP M was significantly decreased.The mRNA and protein levels of hnRNP M were determined using qPCR and western blot respectively after four weeks of injection.Following confirmation of hnRNP M knockdown,control and hnRNP M hippocampus knocked down mice were subjected to series of behavioral experiments to see if hnRNP M depletion affected hippocampus function.The hnRNP M knocked down mice displayed substantial cognitive deficits compared to the control group,including failure to discern a novel object from a familiar one and problems with spatial learning abilities.Anxiety behavioral features,on the other hand,tend to be negligible in both the hnRNP M knockdown and control group mice.As a result,we posit that the depletion of hnRNP M is related to the deterioration of cognitive functions.We performed a nucleotide blast for a reported target sequence for hnRNP M(GGUUGGUU)and studied the Gene Ontology enrichment since hnRNP M RNA recognition/binding domains recognize and bind poly U/G nucleotide combinations avidly.The GO term enrichment analysis reveals that hnRNP M target mRNAs play a significant role in the regulation of synaptic plasticity,as well as other neuronal functions.We then examined the expression of target mRNAs typical with the enriched terms in N2a cells under hnRNP M knocked down and overexpressed conditions.Only synaptophysin and PSD-95 showed significant and corresponding response to changes in hnRNP M levels,out of all the mRNAs assessed.We further compared the levels of synaptophysin and PSD-95 mRNA and protein in the hnRNP M knocked down mice hippocampus to those in the control group.The levels of these synaptic proteins were shown to be substantially lowered in the hippocampus of hnRNP M deficient mice.Also,the PSD-95 and synaptophysin immunochemistry stains showed significant loss of these two proteins in the hnRNP M knocked down mice hippocampus as compared to the control.Since hnRNP M influences the levels of synaptophysin and PSD-95 synaptic proteins,we therefore examined the effect of hnRNP M deficiency on dendritic spine morphology.The knockdown of hnRNP M in the CA1 region of the hippocampus in young wild-type mice results in a substantial reduction in dendritic spine density,which affects cognitive functions in the hnRNP M knocked down mice.We provided an in vitro justification for our in vivo findings by repressing and enhancing the level of hnRNP M in primary cultured neuron and checking the levels of these synaptic proteins.Similar to our in vivo findings,the levels of synaptophysin and PSD-95 were significantly decreased in hnRNP M knockdown primary cultured neuron and increased significantly in hnRNP M overexpressed primary cultured neurons.Since hnRNPs are a broad family of proteins with known interactions,we tested the expression levels of other hnRNPs in hnRNP M knocked down primary cultured neurons to see whether the cognitive relevance of hnRNP M was exclusively due to hnRNP M or was possibly affected by other hnRNP family proteins.Our findings revealed that other hnRNPs have no effect on the cognitive deficits induced by hnRNP M knockdown as the levels of other hnRNPs in hnRNP M knocked down primary neurons remained unchanged.Since RNA binding proteins regulate mRNA decay,which is a key control point in gene expression,the mRNA stability of PSD-95 and synaptophysin under the influence of hnRNP M was investigated at different time points.Our results indicate that hnRNP M-deficient N2a cells have shorter mRNA half-lives for both PSD-95 and synaptophysin,while hnRNP M-administered N2a cells have longer mRNA half-lives for both PSD-95 and synaptophysin.Subsequently,we validated the association between PSD-95/synaptophysin and hnRNP M through RNA immunoprecipitation.Our results showed significant enrichment of PSD-95 and synaptophysin in total hnRNP M pulled down RNA.We next determined where the interactions between hnRNP M and these synaptic proteins precisely occur,since hnRNP M has both nuclear and cytoplasmic distributions.In hnRNP M enhanced N2a cells,we fractionated the nuclear and cytoplasmic lysates,and performed the immunoprecipitation of each lysate.Our result showed that significant interactions between the synaptic proteins and hnRNP M occur in the cytoplasmic compartment.Recently,the 3'UTR of PSD-95 and synaptophysin mRNA were predicted to harbor two hnRNP M putative binding sites each,with one in the last exon(exon 6)of synaptophysin.We determined the binding of hnRNP M on these predicted sites on PSD-95 and synaptophysin mRNA through luciferase activities.Our results indicate that hnRNP M binds to PSD-95 and synaptophysin directly,primarily through the 3'UTR binding motifs.As a result,we report that hnRNP M binds to PSD-95 and synaptophysin mRNA directly and then modulates their stability and expression.This study therefore reports that hnRNP M is necessary for brain function,and that its alteration affects synaptic plasticity by influencing synaptophysin and PSD-95 levels,disrupting dendritic spine density,and eventually triggering learning and memory deficits.