Inhibition Of Hippocampus MiR-132Impairs Learning And Memory By Upregulation Of Mecp2

Part1Inhibition of Hippocampus miR-132impairs Learning and Meory by upregulation of Mecp2Background:miRNAs (microRNA) are short (approximately21-22nt),very conservative, non-coding RNA that regulat the translation of target messenger RNAs. miRNAs are processed from precursor molecules (pri-miRNAs), which are transcribed by RNA polymerase Ⅱ. Pri-miRNAs cutted by endonuclease Drosha/SGCR.8and transported to the cytoplasm by importin5. The produce of Drosha/SGCR8cleavage is an-70-nucleotide pre-miRNA. A single-stranded of this duplexes form reducing miRNA-induced silencing complexes (RISC) with Ago proteins, the other strand is usually degraded. Most miRNAs from animal binding with sequences in the3’-UTR (untranslated region) of target mRNAs by imperfectly base-pair, and inhibite protein synthesis by either repressing translation or promoting mRNA degradation. miRNAs play an important role in neuronal differentiation, migration, integration and even overall brain function by regulating the expression of some nerve-related proteins. miR-132enrich in nerve tissue, including the hippocampus and cortex, and specifically expressed in the neurons. The miR132level is downregulation in some neurodegeneration diseases including Huntington’s disease, schizophrenia and Alzheimer’s disease. More and more results show that miR-132have a significant effect on neuronal synapses occur, shape and number of dendritic spines as well as cognitive function of animal models related behaviors. However, the exact mechanism of miR-132function on learning and memory remains unclear.Objective:To explore the function miR132on learning and memory and its underlying mechanism through inhibition of hippocampus miR132function.Materials and methods:Experimental animals are adult (3month) and old mice(22month) male C57/BL mice. The level of miR132in old and adult mice were detected by real-time PCR and the synaptic related protein including PSD95, NR2B and GluR1were tested by Western blot. LNA-Inhibitor-miR132(LNA-miR132) were injected into DG (dentate gyrus) by syringe pump through Stereotactic injection technique to inhibit the function of endogeous miR132, LNA-Scramble as unrelated control and soldium as negative control.The Morris Water Maze, Step Down, Fear Conditioning were used to detect the function of learning and memory of the groups. The mRNA and protein level of synaptic related protein were tested by Western Blot and Real-Time PCR. The density of spines were tested by Laser scanning confocal microscope from LNA-miR132and LNA-Scramble group mice injected with AAV-GFP in DG region. The luciferase report system confirmed the direct binding between miR-132and the3’UTP of Mecp2. The induction and expression of LTP were recorded by Electrophysiological techniques in brain section from mice infused with LNA-miR132, LNA-Scramble and control group mice. The downstream protein interaction with Mecp2was confirmed by Co-IP. The expression of Mecp2was repressed specificly by siRNA in LNA-miR132and LAN-Scramble group mice. Learning and memory of the siRNA-mice were detected by Morris water maze and Fear conditioning test. Whole-cell patch-clamp recordings were made in brain sections from siRNA mice and LNA-miR132mice.Results:The expression of miR132in hippocampus and cortex of old mice significantly decreased compared with the adult mice. Accompanied with a reduction of synapse-associated protein PSD95, NR2B,GluR1. The results of Morris Water Maze, Step Down, Fear Conditioning show that mice injected by LNA-miR132have a serious impairment of learning and memory compared with mice injected by LNA-Scramble. In addition, the expression of protein including GluRl、GluR2、PSD95、NR2B and NR1in LNA-miR132group dramatically downregulation. Spine density of hippocampus neurons was significantly decreased in LNA-miR132mice. Long-term potentiation (LTP) of CA1neurons induced by four trains of high-frequency stimulation (HFS) on the Schaffer collaterals was observed in LNA-Scramble group but impaired in LNA-miR132group. The luciferase activity was significantly decreased in Mecp23’UTP group compared with Mecp23’UTP-mutation group after co-transfected with miR132expression vectors. The result of co-IP shows that there is a interaction between Mecp2and HDAC2. Finally, the impairment of learning and memory caused by inhibition of miR132can be rescued through repressed the expression of Mecp2by siRNA. LTP in siRNA-Mecp2mice was attenuated compared with the control mice.Conclusion:The inhibition of hippocampus miR132result in mecp2level upregulation. The interaction of Mecp2and HDAC2repressed the expression of synaptic related protein, decreased the spine density of hippocampus synapse, inhibited-the expression of LTP, impaired learning and momory. However, the impairment of learning and memory can be resuced by downregulation of the mecp2level Part2EPAC2inhibit the open probability of KATP Channel through interaction with SUR1ReceptorBackground:EPAC (Exchange Proteins Activated by cAMP) belongs to a novel class of cAMP receptors. There are two isoforms of EPAC proteins, namely EPAC1and EPAC2. EPAC proteins have multiple domains consisting of one (EPAC1) or two (EPAC2) cAMP regulatory binding motifs and a guanine nucleotide exchange factor (GEF). EPAC proteins almostly expressed in the brain, including in the hippocampus, striatum, and prefrontal cortex. We observed that the EPACl-/-(EPAC knock out) mice caused a reduction of the glutamate release from the presynapse terminals in the previously researches. The glutamate release required synaptic fusion with vesicle and Ca2+influx into the synapse terminals. We preciously show that there is the the ATP-sensitive potassium channel (KATP) composed by Kir6.1and type-1sulfonylurea receptor (SUR1) in presynaptic terminals of hippocampus. The KATP channel was sensitive to metabolic factors such as ATP/ADP ratios. In addition, the datas show that the interaction between EPAC and KATP channel control the secretion of insulin of pancreatic P-cells. However, whether EPAC interacts with KATP channels in the central neurons remains unknown.Objective:To explore the mechanism of potassium channel open probability regulated by EPAC in the central nervous system.Materials and methods:3-month-old mice C57/BL and C57/BL sources EPAC1knockout mice (EPAlC-/-), EPAC2(EPAC2-/-) knockout, EPAC1/2double knockout (EPAC-/-), SUR1knockout (SUR1-/-) and Kir6.1knockout (Kir6.1-/-) mice as the experimental animals. Hippocampus synaptosomes were extracted from wild-type and SUR1-/-mice and deteced the interaction between EPAC, SUR1and Kir6.1by immunoprecipitation method. A series of truncation mutants of SUR1and EPAC proteins were generation. Then, immunoprecipitation method was used to identify the key amino acids mediator the interaction of EPAC2and SUR1. we generated more phenotype transgenic mice through hybridization in those transgenic mice. The novel transgenic mice including EPAC positive SUR1knockout mice (EPAC-/-/SUR1+/+, EPAC Kir6.1double knockout mice (EPAC-/-TKir6.1-/-), EPAC positive Kir6.1knockout mice EPAC+/+/Kir6.1-/-and EPAC SUR1double knockout mice (EPAC-/-VSUR1-/-mice). The KATP open probability of novel mice were recording by single-channel recording technique. The rAAV-SUR1859-881were infused in hippocampus of EPAC-/-/SUR1+/+, EPAC-/-Kir6.1-/-and EPAC-/-/SUR1-/-mice. The KATP open probability was compaired between these mice expressed SUR1859-881. Results:The results of immunoprecipitation in wild-type mice show that there is the interaction between EPAC1, EPAC2and SUR1, Kir6.1and the precipitated signal in EPAC2with Kir6.1, SUR1stronger than that of EPAC1. But the interaction of EPAC and SUR1was destroyed in the synaptosomes from SUR1-/-mice. To validate this association, we generated a series of truncation mutants of SUR1and EPAC proteins. The immunoprecipitation between these mutant proteins confirmed that the interaction of EPAC2and SUR1occurring in the EPAC2351-458and SUR1859-881, and this association was inhibited by SUR159-881in vitro. Electrophysiological results show that the KATP open probability significantly decrased in wild-type mice (EPAC+/+/SUR1+/+) compaired with EPAC-/-/SUR1+/+, mice. However, the increased of open probability was absent in EPAC-/-/SUR1-/-and EPAC-/-/Kir6.1-/-mice. In addition, EPAC+/+/SUR1+/+mice expressed SUR1859-881protein hace a significant enhanced in KATP open probability comparied with other transgenic mice injected with rAAV-SUR1859-881.Conclusion:EPAC2regulates the open probability of potassium channel through direct inhibition of SUR1receptor.