Fragile X and Synaptic Plasticity

描述（由申请人提供）：鉴定脆性X综合征（FXS）中记忆和认知缺陷的原因的努力导致在该疾病的敲除小鼠模型（Fmr 1-KO）的皮质中发现突触可塑性损伤。申请人通过显示由学习相关的传入活动模式诱导的海马长时程增强（LTP）在Fmr 1-KO小鼠中不能稳定来扩展这些结果。对持续LTP所需的细胞骨架变化的分析指出了一个假设，即一个关键的FXS缺陷涉及在LTP诱导后的最初几分钟内新的肌动蛋白丝的稳定性失败。初步结果表明，在敲除（科斯）的几个肌动蛋白相关蛋白的异常表达支持这一论点。本研究的目的是（1）确定Fmr 1-KO小鼠中丝状（F）肌动蛋白稳定化和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。初步研究表明，用mGluR 5拮抗剂MPEP或阳性AMPA受体调节剂（ampakine）治疗可以恢复Fmr 1-KO海马的稳定LTP。进一步的结果表明，这两种药物也逆转了科斯中异常脊柱形态的测量。拟议的研究将在4个具体目标的基础上建立这些发现。目的1将验证MPEP可以使成年Fmr 1-KO小鼠海马脑片中脊髓F-actin和LTP的稳定性正常化的假设。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将测试在Fmr 1-KO树突棘中肌动蛋白调节蛋白的异常基础水平是否导致TBS诱导的肌动蛋白细胞骨架信号传导的畸变。研究将采用去卷积免疫荧光技术来测试θ爆发传入刺激对KO和WT小鼠脊髓中靶蛋白水平的影响。目的3将使用急性切片来测试MPEP和ampakine治疗在Fmr 1-KO小鼠中海马LTP的拯救中是否具有相加或协同作用（3A）。随后的急性切片实验将测试挽救LTP的治疗是否也使海马区CA 1中锥体细胞棘测量和棘肌动蛋白调节蛋白的水平和活性诱导的变化正常化（3B）。目标4中的研究补充了目标3中的研究，以测试拯救海马LTP的药物是否也恢复了Fmr 1-KO小鼠体感新皮层切片中的稳定增强（4A）和棘测量（4 B）。然后，Aim 4C将测试用安巴金、MPEP或两者的体内治疗是否使体感皮层和海马区CA 1中的脊柱测量标准化。目的3和4将使用在分散的锥体细胞中组成型表达黄色荧光蛋白（YFP）的Fmr 1-KO和WT小鼠，以提供树突棘的明亮标记。这些研究有望对脆性X突变干扰脊柱可塑性和结构的原因做出具体解释，并产生纠正缺陷的潜在疗法。公共卫生相关性：努力确定与脆性X综合征相关的精神发育迟滞的原因，导致在该疾病的小鼠模型中发现突触可塑性损伤。目前的研究将测试的假设，损伤是由于异常水平的肌动蛋白调节蛋白，这是至关重要的突触功能的变化在学习过程中。研究还将测试纠正这些突触缺陷的潜在疗法，这些疗法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.