The Study On The Role And Mechanism Of MiR-9and EPAC In The Regulation Of Synaptic Plasticity

BackgroundMicroRNA (miRNA) is a class of small non-coding RNA transcripts, which is known to negatively regulate gene expression by inhibiting translation or promoting degradation of their target mRNAs. miR-9has been shown to be particularly abundant in the brain, it can not only promote the development and differentiation of neurons, but also participate in the regulation of neuritegrowth, branching and orientation. Abnormal expression of miR-9is closely related to cognitive dysfunction. Several studies have demonstrated the altered expression of miR-9in Alzheimer’s disease (AD) brains. It is conceivable that miR-9overexpression retards tau pathology in the early stages of AD by inhibition of SIRT1. Once amyloid pathology comes into play, the level of miR-9is decreased, resulting in the increased incorporation of tau and neurofilament H into NFTs. Our earlier study revealed that combined deletion of both the EPAC1and EPAC2genes (EPAC-’-) mice exhibit severe deficits in spatial learning and social behavior, miR-9was significantly downregulated in the forebrain of EPAC null alleles. If the learning and memory disorder of EPAC-/-mice is directly induced by inhibition of miR-9? Synaptic plasticity is an important basis of learning and memory, if downregulation of miR-9would also damage to synaptic plasticity? However, the effects of miR-9on synaptic plasticity and learning and memory are not clear. Foxp2is a member of the forkhead gene family, it is the first language-related genes found in humans and expressed throughout the whole brain. Foxp2protein plays an important regulatory function as a nuclear transcription factor, many of it’ target genes have played a key role for synaptic plasticity, such as growth of axons, dendritic remodeling and neurotransmission. Foxp2gene is predicted to be a target gene of miR-9. However, if miR-9involved in the regulation of synaptic plasticity and learning and memory through downregulation of Foxp2remains unknown.Objects 1. To identify the targeted regulatory relationship between miR-9and Foxp2.2. To explore underlying mechanisms of miR-9in the regulation of synaptic plasticity and learning and memory through downregulation of Foxp2.MethodsA construct of LNA-miR-9was used to inhibit the expression of miR-9in the brain. Following an intracerebroventricular injection of LNA-miR-9, we used Morris water maze to test spatial learning and memory of C57BL/6adult male mice. The emotional memory was tested by Fear conditioning test. Generation of FUGW2.1-miR-9was used to overexpress miR-9while LNA-miR-9was used to inhibit the expression of miR-9in HEK293cells. We used RT-PCR to test the levels of miR-9and Foxp2mRNA. The distribution of Foxp2protein in the whole brain was measured by immunohistochemistry. Synaptic morphologic plasticity was tested by Golgi staining. We used electrophysiological technique of patch clamp to test the plasticity of synaptic transmission. The expression level of Foxp2protein and synapse-associated proteins were measured by immunoblotting. Foxp2was knocked down by gene silencing to clarify the role of Foxp2protein in the synaptic plasticity and learning memory.Results1. Foxp2is regulated directly and negatively by miR-9To verify the direct nature of Foxp2inhibition by miR-9, we constructed recombinant plasmid FUGW2.1-miR-9, then transfected FUGW2.1vector and FUGW2.1-miR-9into HEK293cells. We also transfected LNA-miR-9(which achieves specific knockdown of endogenous miR-9) and LNA-scramble into HEK293cells. We used intracerebroventricular injection to deliver LNA-miR-9into the brain of C57BL/6adult mice. Then we use RT-PCR to test miR-9and Foxp2mRNA levels and western blots to show Foxp2protein levels in HEK293cells and mice respectively. We found that Foxp2protein level were lower in FUGW2.1-miR-9transfected cells and higher in LNA-miR-9transfected cells, compared to controls. In contrast, delivery of LNA-miR-9resulted in increased levels of Foxp2protein, compared to LNA-scrambled control. However, Foxp2mRNA levels were not altered in all groups. These results suggested that miR-9regulate Foxp2protein expression via a post-transcriptional mechanism.2. Downregulation of miR-9impairs spatial learning and memory and emotional memory of C57BL/6adult miceIn Morris water maze, miR-9downregulation mice displayed significantly increased escape latencies in the hidden platform paradigm, while spending less time in the target quadrant and the crossing times of original position of the platform had a significant decrease in a probe trial, but swimming ability were not affected compared to LNA-scramble injection mice. In tests of fear memory, miR-9downregulation mice exhibited a significant decrease in freezing behavior as evaluated by both contextual and tone-dependent fear conditioning paradigms. No differences were observed in the altered contextual fear conditioning test among the three groups. Together, these results show that miR-9downregulation impairs spatial learning and memory and fear memory formation in C57BL/6adult mice.3. Downregulation of miR-9damages structure and function of synaptic plasticity in C57BL/6miceWe next investigate the role of miR-9in synaptic plasticity. After behavior test, we used Golgi staining to test synaptic morphologic plasticity of C57BL/6adult mice. Golgi impregnation demonstrated that the dendritic spine density and the mushroom spines proportion in hippocampal CA1pyramidal neurons and cortex neurons are significantly decreased by inhibition of miR-9. Western blots showed that SNAP-25protein and NR1, NR2A, NR2B, GluRl subunit and PSD-95protein were all significantly decreased in hippocampus and cortex of miR-9downregulation mice. To extend analysis of synaptic transmission functions, we examined whether LTP (long-term potentiation) was altered in miR-9downregulation mice. We found inhibition of miR-9abrogated LTP of CA3-CA1. These results suggested that downregulation of miR-9regulated synapse formation and synaptic plasticity.4. Knockdown of Foxp2rescues the spatial learning and memory and emotional memory deficits induced by inhibition of miR-9Next, Foxp2was knocked down to clarify the role in the impairment of synaptic plasticity and learning and memory by inhibition of miR-9. Then we repeated the behavior test, the electrophysiological experiments, immunohistochemistry and Western blot. The results of Morris water maze and Fear conditioning test showed that knockdown of Foxp2rescued the spatial learning and memory impairment and fear memory deficits induced by inhibition of miR-9. The escape latencies, the target quadrant residence time and the crossing times of original position of the platform returned to normal. Moreover, injections of siFoxp2, but not siCon into miR-9downregulation mice markedly rescued contextual and tone-dependent fear memory formation. These data demonstrated that the spatial learning and memory and emotional memory deficits in miR-9downregulation mice can be reversed by Foxp2silencing.5. Knockdown of Foxp2rescues the damage of synaptic morphological and structural and functional plasticity induced by inhibition of miR-9After behavior test, we used Golgi staining to test synaptic morphological plasticity of trhree groups of mice. Golgi impregnation demonstrated that the reduction of dendritic spine density and ratio of mushroom in CA1pyramidal neurons and cortex neurons of miR-9downregulation mice were returned to normal by knockdown of Foxp2. We also found that Foxp2silencing restored LTP damage whereas the siControl containing a scrambled sequence failed to ameliorate the LTP defects in miR-9downregulation mice. Western blot showed that the decreased levels of SNAP-25protein and NR1, NR2A, NR2B, GluRl subunit and PSD-95protein by inhibition of miR-9were all increased.Together, our results suggested that miR-9is an important component in the positive regulation of synaptic plasticity and learning and memory by targeting Foxp2.ConclusionsTogether, our results identified the direct nature of Foxp2inhibition by miR-9from gene expression and function. Downregulation of miR-9reduced the dendritic spine density and t the mushroom spines proportion in hippocampal CA1pyramidal neurons and cortex neurons, decreased the levels of the SNAP-25protein and postsynaptic-associated protein in hippocampus and cortex, impaired LTP of CA3-CA1, then C57BL/6adult mice showed synaptic plasticity impairment and learning and memory and emotional memory disorder. The increased level of Foxp2protein induced by inhibition of miR-9is the key point. Knockdown of Foxp2rescued the synaptic plasticity damage and learning and memory deficit induced by inhibition of miR-9. Our research shows that the targeted regulatory relationship between miR-9and Foxp2plays an important role in learning and memory, so it maybe provides the study basis for the therapeutic intervention of learning and memory impairment. BackgroundEPAC (Exchange Proteins Activated by cAMP) belongs to a novel class of cAMP receptors. EPAC proteins have multiple domains consisting of one or two cAMP regulatory binding motifs and a guanine nucleotide exchange factor (GEF). When cAMP binds a regulatory motif, it causes a conformational change of EPAC proteins and hence activates a Ras-like small GTPase Rap1/2. In the cardiovascular system, EPAC1-Rap1signaling controls endothelial cell growth and vascular formation. In the pancreatic β3-cells, EPAC2regulates insulin secretion. Both EPAC1and EPAC2genes are expressed throughout the brain. But, their neurological functions are yet to be described.Our earlier study revealed that combined deletion of both the EPAC1and EPAC2genes (EPAC-/-) reduces glutamate release from the presynaptic terminals. Previously, we described ATP-sensitive potassium (KATP) channels consisting of Kir6.1subunit and type-1sulfonylurea receptor (SUR1) at the presynaptic terminals in the hippocampus. Compared to wild-type controls, mutant mice lacking the SUR1gene (SUR1-/-) or the Kir6.1gene (Kir6.1-/-) are vulnerable to epileptic seizures. However, whether EPAC interacts with KATP channels to regulate glutamate release and affect epileptic seizures in the central neurons is not known.Objects1. To identify the interaction between EPAC and KATP channels in the dentate granule cells.2. To study EPAC signaling pathway to control presynaptic neurotransmitter glutamate release at the granule cell terminals and epileptic seizures in adult mice.MethodsWe used gene targeting to develop EPAC mutant mice including EPAC1-/-, EPAC2-/-, EPAC-/-and SUR1-/-mutant mice. Then we used whole-cell patch clamp to record the spontaneous EPSCs. NMDA receptor-mediated EPSCs were evoked by paired-pulse stimulation of the mossy fiber tracks in the hilus of the dentate gyrus. Adult male mice were injected intraperitoneally with a single dose of kainic acid (25-40mg/kg in PBS). Mice were monitored continuously for3h. The severity of seizures was rated by the arbitrary scale. The averaged points for seizure severity were expressed as the seizure index.ResultsTo investigated whether an increase of KATP channel open probability in EPAC-/-mice alters glutamate transmitter release. We first analyzed the spontaneous EPSCs and found that their frequency were reduced in EPAC-/-mice compared to wild-type controls. We next recorded the paired-pulse facilitation of the evoked EPSCs at mossy fiber-CA3and found that EPAC-/-mice displayed greater paired-pulse facilitation than wild-type controls. The facilitation increased with an elevation of Ca2+. The similar reduction of glutamate release was achieved by interception of EPAC-SUR1association via expressing SUR1859-881peptide. Our earlier study revealed that, compared to wild-type controls, mutant mice lacking theSUR1gene (SUR1-/-) or the Kir6.1gene (Kir6.1-/-) are vulnerable to epileptic seizures. To hypothesize whether an increase of KATP channel open probability in EPAC-/-mice reduces seizure sensitivity, we examined adult mice for seizure activity after KA adminstration. We found that few EPAC-/-mice progressed in severity to the extent of EPAC+/+mice, the similar reduction of epileptic seizures was observed in wild-type mice expressing SUR1859-881. Therefore, EPAC protein confers seizure vulnerability through direct inhibition of the SUR1receptor. ConclusionsOur study suggested that EPAC physically and functionally interacts with KATP channels viadirect inhibition of the SUR1receptor in the dentate granule cells. We identified that the inhibition increases glutamate release and elevates seizure vulnerability in adult mice. So we supposed that EPAC signaling pathway could be a clinical therapeutic potential source of aberrant neuronal function in epilepsy.