Dysregulation of mTORC2 and cofilin signaling in Fragile X Syndrome

脆性X综合征是最常见的可遗传的智力残疾形式，也是导致 自闭症。与FXS相关的认知和社会互动缺陷的有效治疗是一种未得到满足的需求。 哺乳动物靶标雷帕霉素(MTOR)通路是细胞新陈代谢、生长、增殖、 存活、依赖帽子的翻译和肌动蛋白细胞骨架。而mTOR复合体1的调节失调 (MTORC1)在脆性X中已经确立，但mTORC2的作用尚不清楚。MTORC2是一种中央调节因子 肌动蛋白聚合和脊柱结构及其对突触中涉及的肌动蛋白解聚因子cofilin的作用 可塑性和记忆力。我们发现cofilin及其上游调控因子rac1，一种小的Rho GTP酶和直接 涉及肌动蛋白重塑和脊柱结构的mTORC2靶基因在脆性X小鼠模型中受损 提供了FMRP、mTOR和cofilin信号之间的功能联系，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mTORC2信号是否 与Fmr1 KO小鼠的cofilin信号、脊柱结构和突触成熟有关，并建立 MTORC2作为改善FXS的新治疗靶点。潜在的假设是FMRP的损失 导致mTORC2和cofilin信号过度激活，导致脊柱异常，突触受损 成熟、感觉加工和自闭症相关行为。我们试图从以下几个方面来检验这一假设 目的：1.研究COFILIN信号异常与突触表型之间的因果关系。 幼年脆性X小鼠的体感皮质。实验将检验1)成分活性胶合的能力 通过慢病毒表达系统将突变体(S3A)直接导入Fmr1 KO小鼠的体感皮质 2)Cofilin S3A挽救延迟性突触成熟的能力。 3)Cofilin S3A抢救受损棘波的能力。 Fmr1KO小鼠躯体感觉皮层兴奋性突触的LTP计时：4)磷酸化胶体蛋白的能力 抑制内源性粘连蛋白的多肽，对表型异常的肌动蛋白聚合、脊柱缺陷和损伤 WT小鼠躯体感觉皮层的突触成熟。2.识别异常胶着蛋白上游的信号通路 MTORC2信号转导及药理学和遗传操作能力拯救FXS表型。 实验将建立1)mTORC2作为上游效应因子，对异常的cofilin信号转导至关重要；2)rac1/PAK 作为cofilin上游和mTORC2下游的潜在信号通路和PAK抑制能力 抢救受损的脊柱结构、突触成熟和棘波计时LTP；3)FXS的感觉知觉缺陷 小鼠与PAK抑制修复受损知觉的能力；4)shRNA对Rictor的传递能力 通过慢病毒表达系统纠正神经缺陷的Fmr1KO小鼠的体感皮层。这些 实验将记录针对mTORC2、rac1/PAK和cofilin信号转导的治疗策略的能力 挽救年轻Fmr1基因缺失小鼠的脊柱缺陷、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.