Overexpression Of Kir2.1in Excitatory Neurons Impairs Learning And Memory Via Aberrant Regulation Of MiR-9and FoxP2Translation

Background: Learning and memory are the dominant advanced function in the human brain and the cellular and molecular mechanisms of learning/memory are the most intersting topic in neuroscience research. It is known that brain is mainly constituted by neurons, which is highly correlated with synaptic structures and functions. Previous studies had suggested that the suppression of neuronal activity impaired synaptic plasticity resulted the learning and memory decline which is found in the many cognitive-deficits diseases, such as Alzheimer’s disease, Parkinson’s disease. Currently, plenty of epigenetic factors, such as aging or brain injury play important roles in the learning and memory deficits caused by neuronal activity suppression. However, the underlying mechanisms are remaining unknown. With the development of genomic sequencing and microarray technology, more and more epigenetic factors had been implicated in the regulation of learning and memory. There are more than hundred billions neurons expressed19,814protein-related genes and thousands of non-coding RNAs. The mutant and abnormally expression of ncRNAs is tightly correlated with pathogenesis of many diseases. It is known that miRNAs target many transcriptional factors and synaptic proteins in neurons and regulate local translation of those proteins to regulate the synaptogenesis and synaptis strength. However, the underlying mechanisms of miRNAs in neuronal-activity dependent learning and memory are not clear. Objective To unravel the molecular mechanisms of neuronal activity-dependent hippocampal neural circuit regulated by conditional transgenic mice model. Specifically, we expresed kir2.1channel in excitatory neurons and found the upregulation of miR-9, leads to the inhibition of Foxp2expression and the impairments of learning and memory. The current study will provide novel pharmaceutical targets for cognitive impaired diseases. Materials and methods We first crossed inducible overexpression Ai3-Flag-Kir2.1-2A-tdTomato mice (Tgl) with CamKII-a-CreERT2mice (Tg2) to get the homozygous mice that expressed Kir2.1in excitatory neurons upon the tamoxifen administering(Kir2.1(+) mice). The overexpression of Kir2.1protein in excitatory neurons were verified by immunohistochemistry,Western Blotting and qPCR. The induction and expression of LTP were recorded by electrophysiological techniques in brain section from mice.The Morris Water Maze, Fear Conditioning and T-maze tests were used to evaluate the function of learning and memory. The Open Field Test, Rotarod, Elevated Plus Maze and Forced Swimming Tests were used to detect the behaviors that not associated with learning and memory. RNA-seq screening uncover the neuronal activity dependent and learning and memory associated gene microRNA-9. The level of miR-9was double identified by q PCR. The luciferase report system confirmed the direct binding between miR-9and the3’UTP of FoxP2. LNA-miR-9were injected into DG by syringe pump through Stereotactic injection technique to inhibit the endogenous miR-9, by using LNA-Scramble as negativecontrol. The expression of FoxP2was repressed specifically by PUF Fox-P group mice. The mRNA and protein level of FoxP2were tested by qPCR andWestern Blotting. Results Our results show.(1) Successful generation of genetically modified Kir2.1Strains of Mice.(2) LTP but not normal synaptic transmission is impaired in Kir2.1(+) mice.(3) The Kir2.1(+) mice show the behavioral deficits of learning and memory.(4) miR-9and FoxP2are the newly identified learning and memory signaling pathway.(5) miR-9inhibits FoxP2protein translation.(6) Inhition of miR-9by specific inhibitor rescues the phenotypes of Kir2.1(+) mice.(7) Overexpression of miR-9results in the impairment of learning and memory in Kir2.1(-)mice.(8) Overexpression of FoxP2rescues the learning and memory in Kir2.1(+) mice. ConclusionTaken together, our results indicate that suppression of neuronal activity by overexpression of Kir2.1in excitatory neurons results in the upregulation of miR-9, which inhibits the protein translation of its target gene FoxP2and impairs synaptic plasticity in the hippocampal, as well as the dysfunction of learning and memory. Moreover, downregulation of miR-9, or upregulation of FoxP2can rescues those abnormalities, which could be a promisting therapeutic target for learning and memory deficit related disorders.