Effects of 16p11.2 copy number variation on neuronal development and pathology

项目摘要/摘要 人类16p11.2基因座的拷贝数变异(CNV)包含29个编码基因 患有多种神经发育和精神障碍。删除(16pdel)和复制(16pdup) 这一区域的变异对多效性的影响知之甚少。尽管自闭症在患者中更为常见 有缺失，精神分裂症在有重复的人中更常见，潜在的机制不是 安全。来自16p11.2区域的几个分子通路调节神经元的分化、迁移、轴突 发育、突触形成以及能量和脂类代谢。16P-动物模型的研究 提示KCTD13-RhoA通路激活、神经元迁移、轴突发育和 行为。反过来，FAM57B基因座16p11.2 CNV对神经酰胺稳态的破坏会改变血脂 丰度、细胞膜动力学、突触蛋白表达和突触运输，表明 脂质体失调会影响神经元的功能和活性。然而，确切的分子 兴奋性神经元对抑制性神经元功能障碍的机制尚不清楚。 此外，不同的动物和人类细胞模型也报道了相互矛盾的结果。致信地址 在这一知识缺口中，我们开发了人类IPSC衍生的16p11.2 CNV神经元模型，并展示了 即1)KCTD13调节RhoA途径的激活，RhoA表达增加导致细胞过度活跃。 16pdel人类皮质神经元网络，以及ii)关键线粒体和脂质发生显著变化 酶转录本，包括FAM57B介导的神经酰胺合成酶表达的降低，直接 与观察到的代谢组和脂体的变化相关。这些数据表明，16pdel导致 复杂的代谢紊乱和神经酰胺表达不足可能与观察到的功能性 神经网络表型。这些数据让我们假设16p11.2个CNV导致了调控失调 谷氨酸和氨基丁酸能神经元中神经酰胺丰度的变化，进而促进突触的缺陷 发展和功能导致网络解体和过度激活。在这里，我们将对此进行调查 16p11.2 CNV对人IPSC来源皮层神经元发育和功能的影响及其假说 二维兴奋性-抑制性神经元共培养与人IPSC来源的三维前脑 有机化合物。为了减少不同基因背景引起的变异性，我们将研究CRISPR-Cas9诱导的 16p11.2 CNV IPSC系，以及来源于患者和健康对照的IPSC系。我们将利用国家- 最先进的分子方法来揭示兴奋性-突触功能障碍的潜在机制- 16p11.2 CNV中的抑制神经元，包括单细胞转录基因表达谱和 脂体/代谢组谱。最后，我们将研究兴奋-抑制网络的功能、连通性、 以及具有多电极阵列和膜片钳制的振荡模式。我们预计这项研究将揭示 16p11.2 CNV疾病中与皮质神经元功能障碍相关的新分子靶点。

Project Summary/Abstract Copy number variations (CNVs) of the human 16p11.2 genetic locus, containing 29 coding genes, are associated with a number of neurodevelopmental and psychiatric disorders. The deletion (16pdel) and duplication (16pdup) variants of this region have poorly understood pleiotropic effects. Although autism is more common in patients with deletions, and schizophrenia is more common in those with duplications, underlying mechanisms are not clear. Several molecular pathways from the 16p11.2 region modulate neuronal differentiation, migration, axonal development, and synapse formation, as well as energy and lipid metabolism. Studies of 16p-animal models have suggested deficits in the KCTD13-RhoA pathway activation, neuronal migration, axonal development, and behavior. In turn, disruption of ceramide homeostasis due to 16p11.2 CNVs at FAM57B locus altered lipid abundance, cell membrane dynamics, synaptic protein expression, and synaptic transport, suggesting that lipidome dysregulation could contribute to neuronal function and activity. However, the exact molecular mechanisms underlying these neuronal dysfunctions in excitatory versus inhibitory neurons are lacking. Moreover, contradictory results have been reported from different animal and human cell models. To address this gap of knowledge, we developed human iPSC-derived neuronal models of 16p11.2 CNVs and demonstrated that i) KCTD13 regulates RhoA pathway activation, and increased RhoA expression leads to hyperactivity of the 16pdel human cortical neuron networks, and ii) there are significant changes in key mitochondrial and lipid enzyme transcripts, including decrease in FAM57B-mediated ceramide synthase expression, that directly correlate with observed changes in the metabolome and lipidome. These data suggest that 16pdel leads to complex metabolic disruptions and deficient ceramide expression that might contribute to the observed functional neuronal network phenotypes. These data have led us to hypothesize that 16p11.2 CNVs cause dysregulation of ceramide abundance in glutamatergic and GABAergic neurons that in turn promotes deficits in synaptic development and function leading to network disorganization and hyperactivation. Here, we will investigate this hypothesis and the effects of 16p11.2 CNVs on cortical neuron development and function in human iPSC-derived 2-dimensional excitatory-inhibitory neuron co-cultures and human iPSC-derived 3-dimensional forebrain organoids. To reduce variability caused by different genotypic backgrounds, we will study CRISPR-Cas9 induced 16p11.2 CNV iPSC lines in addition to iPSC lines derived from patients and healthy controls. We will utilize state- of-the-art molecular methodologies to uncover mechanisms underlying the synaptic dysfunction of the excitatory- inhibitory neurons in 16p11.2 CNVs, including single cell transcriptional gene expression profiling and lipidome/metabolome profiling. Finally, we will investigate the excitatory-inhibitory network function, connectivity, and oscillation patterns with multi-electrode arrays and patch clamping. We anticipate that this study will uncover new molecular targets related to cortical neuron dysfunction in 16p11.2 CNV disorders.