Dysregulation of mTORC2 and cofilin signaling in Fragile X Syndrome

脆性X染色体综合征是智力残疾最常见的遗传形式，也是导致智力残疾的主要遗传原因。 自闭症与FXS相关的认知和社会互动缺陷的有效治疗是一个未满足的需求。 哺乳动物雷帕霉素靶蛋白（mTOR）途径是细胞代谢、生长、增殖 存活、帽依赖性翻译和肌动蛋白细胞骨架。而mTOR复合物1的失调 尽管脆性X染色体中的mTORC 1已经被很好地确定，但mTORC 2的作用还不清楚。mTORC 2是一种中枢调节因子， 肌动蛋白聚合和棘结构，并作用于肌动蛋白解聚因子cofilin， 可塑性和记忆。我们发现，cofilin及其上游调节因子Rac 1，一个小的Rho GTdR， 涉及肌动蛋白重塑和脊柱结构mTORC 2靶点在脆性X染色体小鼠模型中受损 提供了FMRP、mTOR和cofilin信号传导之间的功能联系，并强调了这一点的临床相关性。 工作拟议研究的总体目标是检查过度激活的mTORC 2信号传导是否是 在Fmr 1基因敲除小鼠中，与cofilin信号传导、棘结构和突触成熟有因果关系， mTORC 2作为改善FXS的新治疗靶点。潜在的假设是， 导致mTORC 2和cofilin信号过度激活，从而诱导脊柱异常，受损的突触 成熟，感觉处理和自闭症相关行为。我们试图在下面测试这个假设 目的：1.检查在神经细胞中cofilin信号传导失调和突触表型之间的因果关系。 年轻脆性X小鼠的躯体感觉皮层。实验将检查1）组成型活性cofilin 突变体（S3 A）通过慢病毒表达系统直接递送到Fmr 1 KO小鼠的体感皮层中 2）cofilinS 3A拯救脊髓损伤后第V层神经元延迟的突触成熟的能力; 在关键期期间，Fmr 1 KO小鼠的躯体感觉皮层; 3）cofilinS 3A拯救受损的锋电位的能力， Fmr 1基因敲除小鼠躯体感觉皮层兴奋性突触的LTP计时：4）磷酸化cofilin 肽，抑制内源性cofilin，表型异常肌动蛋白聚合，脊柱缺陷和受损 WT小鼠体感皮层中的突触成熟。2.识别异常cofilin上游的信号通路 信号传导以及药理学和遗传操作mTORC 2信号传导以拯救FXS表型的能力。 实验将确立1）mTORC 2作为异常cofilin信号传导的关键上游效应子; 2）Rac 1/PAK 作为cofilin上游和mTORC 2下游的潜在途径的信号传导和PAK抑制的能力， 挽救受损的脊柱结构、突触成熟和锋电位计时LTP; 3）FXS中感觉知觉的缺陷 小鼠和PAK抑制拯救受损感知的能力; 4）shRNA对Rictor的能力， 通过慢病毒表达系统对Fmr 1 KO小鼠的躯体感觉皮层进行重组，以纠正神经缺陷。这些 实验将记录靶向mTORC 2、Rac 1/PAK和cofilin信号传导的治疗策略的能力 挽救年轻的Fmr 1基因敲除小鼠的脊柱缺陷、受损的突触成熟和自闭症相关行为。

Fragile X syndrome is the most common heritable form of intellectual disabilities and a leading genetic cause of autism. An effective treatment for the cognitive and social interaction deficits associated with FXS is an unmet need. The mammalian target of rapamycin (mTOR) pathway is a central regulator of cell metabolism, growth, proliferation, survival, cap-dependent translation and the actin cytoskeleton. Whereas dysregulation of mTOR Complex 1 (mTORC1) in Fragile X is well established, a role for mTORC2 is, as yet, unclear. mTORC2 is a central regulator of actin polymerization and spine structure and acts on the actin-depolymerizing factor cofilin implicated in synaptic plasticity and memory. Our finding that cofilin and its upstream regulator Rac1, a small Rho GTPase and direct target of mTORC2 implicated in actin remodeling and spine structure, are impaired in a mouse model of Fragile X provides a functional link between FMRP, mTOR, and cofilin signaling and underscore the clinical relevance of this work. The overall goals of the proposed research are to examine whether overactivated mTORC2 signaling is causally linked to cofilin signaling, spine structure, and synaptic maturation in Fmr1 KO mice, and establish mTORC2 as a novel therapeutic target for the amelioration of FXS. The underlying hypothesis is that loss of FMRP leads to overactivated mTORC2 and cofilin signaling, which induce spine abnormalities, impaired synaptic maturation, sensory processing and autism-relevant behaviors. We seek to test this hypothesis in the following Aims: 1. Examine a causal relation between dysregulation of cofilin signaling and the synaptic phenotype in the somatosensory cortex of young Fragile X mice. Experiments will examine 1) ability of a constitutively active cofilin mutant (S3A) delivered directly into the somatosensory cortex of Fmr1 KO mice via the lentivirus expression system to rescue spine defects; 2) ability of cofilinS3A to rescue delayed synaptic maturation of layer V neurons in the somatosensory cortex of Fmr1 KO mice during the critical period; 3) ability of cofilinS3A to rescue impaired spike- timing LTP at excitatory synapses in the somatosensory cortex of Fmr1 KO mice: 4) ability of a phospho-cofilin peptide, which inhibits endogenous cofilin, to phenocopy aberrant actin polymerization, spine defects and impaired synaptic maturation in somatosensory cortex of WT mice. 2. Identify signaling pathways upstream of aberrant cofilin signaling and ability of pharmacologic and genetic manipulation of mTORC2 signaling to rescue the FXS phenotype. Experiments will establish 1) mTORC2 as an upstream effector critical to aberrant cofilin signaling; 2) Rac1/PAK signaling as a potential pathway upstream of cofilin and downstream of mTORC2 and ability of PAK inhibition to rescue impaired spine structure, synaptic maturation and spike timing LTP; 3) deficits in sensory perception in FXS mice and ability of PAK inhibition to rescue impaired perception; 4) ability of shRNA to Rictor delivered into the somatosensory cortex of Fmr1 KO mice via the lentivirus expression system to correct neurologic defects. These experiments will document the ability of therapeutic strategies targeting mTORC2, Rac1/PAK and cofilin signaling to rescue spine defects, impaired synaptic maturation and autism-relevant behaviors in young Fmr1 null mice.