Fragile X and Synaptic Plasticity

描述（由申请人提供）：努力确定脆性X综合征（FXS）中记忆和认知缺陷的原因，导致在该疾病的敲除小鼠模型（Fmr1-KO）的皮层中发现突触可塑性损伤。申请人通过证明由学习相关的传入活动模式诱导的海马长期增强（LTP）在Fmr1-KO小鼠中未能稳定，扩展了这些结果。对持续LTP所需的细胞骨架变化的分析指出，在LTP诱导后的最初几分钟内，一个关键的fxs缺陷涉及新肌动蛋白丝的稳定失败。初步结果显示，敲除（KOs）中几种肌动蛋白相关蛋白的异常表达支持了这一观点。拟议研究的目的是：(1)确定Fmr1-KO小鼠丝状(F)肌动蛋白稳定和LTP失败的原因，(2)开发使细胞骨架变化正常化和稳定LTP的治疗方法。前期研究表明，使用mGluR5拮抗剂MPEP或阳性AMPA受体调节剂（ampakine）治疗可以恢复Fmr1-KO海马的稳定LTP。进一步的结果表明，这两种药物也逆转了ko中异常脊柱形态的测量。拟议的研究将以这些发现为基础，有四个具体目标。目的1将验证MPEP可以使成年Fmr1-KO小鼠海马切片中脊柱f -肌动蛋白和LTP的稳定正常化的假设。进一步的研究将测试LTP损伤是否被翻译抑制剂所抵消，并与整合素相关酪氨酸激酶的异常信号传导有关。目的2将测试Fmr1-KO树突棘中肌动蛋白调节蛋白的基础水平异常是否会导致TBS诱导的肌动蛋白细胞骨架信号的畸变。研究将采用反褶积免疫荧光技术来测试θ波突发传入刺激对KO和WT小鼠脊柱靶蛋白水平的影响。Aim 3将使用急性切片来测试MPEP和ampakine治疗是否在Fmr1-KO小鼠海马LTP的拯救中具有附加或协同作用（3A）。随后的急性切片实验将测试挽救LTP的治疗是否也使（3B）锥体细胞脊柱测量和（3C）水平正常化，以及海马区CA1脊柱肌动蛋白调控蛋白的活动诱导变化。Aim 4的研究补充了Aim 3的研究，以测试拯救海马LTP的药物是否也能恢复Fmr1-KO小鼠体感觉新皮层切片中的稳定增强（4A）和脊柱测量（4B）。然后，Aim 4C将测试使用ampakine、MPEP或两者同时使用的体内治疗是否能使体感觉皮层和海马区CA1的脊柱测量正常化。目的3和4将使用分散锥体细胞中组成性表达黄色荧光蛋白（YFP）的Fmr1-KO和WT小鼠来提供树突棘的明亮标记。这些研究有望为脆性X基因突变干扰脊柱可塑性和结构的原因提供一个具体的解释，并为纠正这些缺陷提供潜在的治疗方法。公共卫生相关性：努力确定与脆性X综合征相关的智力迟钝的原因，导致在该疾病的小鼠模型中发现突触可塑性损伤。目前的研究将验证一种假设，即损伤是由于肌动蛋白调节蛋白的异常水平造成的，而肌动蛋白调节蛋白对学习过程中突触功能的变化至关重要。研究还将测试纠正这些突触缺陷的潜在治疗方法，这些缺陷可能会改善这种综合征和其他自闭症谱系障碍的学习能力。

DESCRIPTION (provided by applicant): Efforts to identify causes of memory and cognition deficits in Fragile X Syndrome (FXS) led to the discovery of synaptic plasticity impairments in cortex of a knock-out mouse model (Fmr1-KO) of the disorder. The applicants have extended these results by showing that hippocampal long-term potentiation (LTP), induced by learning-related patterns of afferent activity, fails to stabilize in Fmr1-KO mice. Analysis of cytoskeletal changes required for lasting LTP pointed to the hypothesis that a critical FXS-defect involves failed stabilization of new actin filaments during the first few minutes after LTP induction. Preliminary results showing abnormal expression of several actin-associated proteins in the knockouts (KOs) support this argument. Objectives of the proposed studies are to (1) identify causes for the failure in filamentous (F) actin stabilization and LTP in Fmr1-KO mice, and (2) develop treatments for normalizing cytoskeletal changes and stable LTP. Pilot studies have shown that treatment with the mGluR5 antagonist MPEP, or with a positive AMPA receptor modulator (ampakine), can restore stable LTP to Fmr1-KO hippocampus. Further results indicate that both drugs also reverse measures of aberrant spine morphology in the KOs. The proposed research will build on these findings in 4 specific aims. Aim 1 will test the hypothesis that MPEP can normalize stabilization of spine F-actin and LTP in hippocampal slices from adult Fmr1-KO mice. Further studies will test if LTP impairments are offset by translation inhibitors and linked to aberrant signaling by integrin-associated tyrosine kinases. Aim 2 will test if abnormal basal levels of actin regulatory proteins in Fmr1-KO dendritic spines lead to aberrations in TBS- induced signaling to the actin cytoskeleton. Studies will employ deconvolution immunofluorescent techniques to test effects of theta burst afferent stimulation on levels of target proteins in spines of KO and WT mice. Aim 3 will use acute slices to test if MPEP and ampakine treatments have additive or synergistic effects in the rescue of hippocampal LTP in Fmr1-KO mice (3A). Follow on acute slice experiments will test if the treatments that rescue LTP also normalize (3B) pyramidal cell spine measures and (3C) levels and activity-induced changes in spine actin-regulatory proteins in hippocampal field CA1. Studies in Aim 4 complement those in Aim 3 to test if drugs that rescue hippocampal LTP also restore stable potentiation (4A) and spine measures (4B) in slices from somatosensory neocortex of Fmr1-KO mice. Aim 4C will then test if in vivo treatments with an ampakine, MPEP, or both, normalize spine measures in somatosensory cortex and hippocampal field CA1. Aims 3 and 4 will use Fmr1-KO and WT mice that constitutively express yellow fluorescent protein (YFP) in scattered pyramidal cells to provide a bright label of dendritic spines. These studies are expected to produce a specific explanation for why spine plasticity and structure are disturbed by the Fragile X mutation, and to generate potential therapies for correcting the defects. PUBLIC HEALTH RELEVANCE: Efforts to identify causes of mental retardation associated with Fragile X Syndrome led to the discovery of synaptic plasticity impairments in a mouse model of the disorder. The present studies will test the hypothesis that impairments are due to abnormal levels of actin regulatory proteins, which are critical for changes in synaptic function during learning. Studies will also test potential therapeutics for correcting these synaptic defects that might improve learning in this syndrome and other autism spectrum disorders.