Molecular mechanisms of abnormal dendritic spine development and function in human neurons with TSC2 disease mutations

项目摘要/摘要 越来越多的证据表明，结节性硬化症(TSC)患者与其他神经元病患者一样， 发育障碍，有功能失调的突触连接。这些神经元连接的缺陷 可能导致TSC的症状，如认知障碍、自闭症和癫痫。然而，有缺陷的 人类神经元的突触发育和功能只是从动物模型研究中提出的 以及最近几项使用人类神经元的研究，导致了相互矛盾的报告。要直接解决这些问题 基本问题并帮助解决相互矛盾的发现，我们将比较 TSC患者神经元与其校正对照神经元的人脑皮质神经元突触。大脑皮层 TSC患者的人诱导多能干细胞(HiPSCs)分化为神经元 细胞和它们的工程同源对应物。使用一系列形态测量和分子信号 通过分析，我们将研究突触前和突触后的控制和 TSC2突变体突触在混合基因培养中的表达。我们令人惊讶的最新发现表明，TSC2功能 不依赖于生长锥体中的mTORC直接调节细胞骨架和控制轴突引导， 促使我们考虑类似的机制可能在树突棘的发育中起作用。我们还将测试 如何改变TSC2患者神经元的突触传递和网络活动。从长远来看， 我们相信我们的研究可能有助于确定TSC患者的关键可用药靶点。

Project Summary/Abstract Growing evidence suggests that patients with Tuberous Sclerosis Complex (TSC), like other neuro- developmental disorders, have dysfunctional synaptic connections. These defects in neuronal connectivity likely contribute to symptoms of TSC, such as cognitive deficits, autism and epilepsy. However, defective synaptic development and function by human neurons has only been suggested from animal model studies and a few recent studies using human neurons, which have led to conflicting reports. To directly address these fundamental questions and help resolve conflicting findings, we will compare the development and function of human cortical neuron synapses of TSC patient neurons with their corrected control counterparts. Cortical neurons are differentiated from human induced pluripotent stem cells (hiPSCs) generated from TSC patient cells and their engineered isogenic counterparts. Using a series of morphometric and molecular signaling assays, we will examine the structure and function of pre- and post-synaptic compartments of control and TSC2 mutant synapses in mixed genotype cultures. Our surprising recent findings suggest that TSC2 functions independent of mTORC in growth cones to directly regulate the cytoskeleton and control axon guidance, prompting us to consider similar mechanisms may function in dendritic spine development. We will also test how synaptic transmission and network activity may be altered in TSC2 patient neurons. Over the long term, we believe our research may help identify key druggable targets in patients with TSC.