Molecular, Cellular and Behavioral Impact of the R203W PACS1 Syndrome Mutation

项目摘要 PACS1综合征是最近被发现的一种神经发育障碍，由反复发生的从头认识错误引起 PACS1(p.Arg203Trp)突变。携带这种错义突变的患者有几个发育缺陷， 包括智力残疾、癫痫发作和自闭症。PACS1R203W引起PACS1的机制 症状未知，也没有根治方法可用。PACS1是一种多功能分选蛋白， 促进从内体到跨高尔基网络的逆行运输，以便将蛋白质输送到初级 纤毛和基因组的完整性。这一多功能依赖于PACS1的一小部分，称为Furin- 结合区(FBR)，它结合广泛的客户蛋白和信号分子。R203W突变 位于快堆中，我们的生物物理研究揭示了当R203W 替换的存在，表明PACS1与一个或多个 其客户蛋白在PACS1综合征中的作用我们的初步研究强烈表明PACS1R203W增加了结合 去乙酰酶HDAC6严重扰乱膜运输，损害神经元发育。因此， PACS1R203W减少已知的HDAC6底物的乙酰化，包括α-微管蛋白，破坏中心体 定位并导致锥体神经元高尔基体碎裂和树突分枝增加。这 L2/3皮质神经元中树突状过度分支与抑制电流减少偶联，导致 兴奋性：抑制性(E：I)比率，与其他神经发育障碍相似，表明 PACS1R203W严重影响神经功能和行为。我们的长期目标是了解 PACS1R203W导致疾病，并利用这些信息开发有效的治疗方法。这样做的目的是 具体的应用是确定PACS1R203W和HDAC6如何结合来调节神经元的树枝状结构 和突触传递。我们假设PACS1R203W和HDAC6之间的异常相互作用发生了改变 细胞器定位，导致过多的树突分枝和突触活动失调。 在强大的初步数据的指导下，我们将通过追求三个具体目标来检验我们的假设：1)确定如何 R203W突变改变PACS1结构和动力学以影响客户蛋白相互作用，2) 确定PACS1R203W和HDAC6如何结合以失调高尔基体定位和树突分枝， 3)确定PACS1R203W如何改变突触活动和行为。这种方法是创新的，因为 我们将从原子结构到整个有机体，描述循环的机制 R203W替代导致神经元功能障碍。这项研究具有重要意义，因为它可能确定新的目标 以及治疗这种衰弱障碍的方法。

Project Summary PACS1 Syndrome is a recently identified neurodevelopmental disorder caused by a recurrent de novo missense mutation in PACS1 (p.Arg203Trp). Patients carrying this missense mutation share several developmental deficits, including intellectual disability, seizures and autism. The mechanism by which PACS1R203W causes PACS1 Syndrome is unknown and no curative treatment is available. PACS1 is a multifunctional sorting protein that facilitates retrograde trafficking from endosomes to the trans-Golgi network, for delivery of proteins to the primary cilium and for genome integrity. This multifunctionality depends on a small segment of PACS1 called the furin- binding region (FBR), which binds a broad range of client proteins and signaling molecules. The R203W mutation is located in the FBR, and our biophysical studies reveal a change in the FBR dynamics when the R203W substitution is present, suggesting the possibility of an altered interaction between PACS1 and one or more of its client proteins in PACS1 Syndrome. Our preliminary studies strongly suggest PACS1R203W increases binding to the deacetylase HDAC6 to profoundly disturb membrane traffic and impair neuron development. Consequently, PACS1R203W reduces acetylation of known HDAC6 substrates, including α-tubulin, disrupting centrosome positioning and leading to Golgi fragmentation and increased dendritic arborization in pyramidal neurons. This dendritic overbranching is coupled to reduced inhibitory currents in L2/3 cortical neurons resulting in an increased excitatory:inhibitory (E:I) ratio, similar to that found in other neurodevelopmental disorders, suggesting that PACS1R203W severely affects neuronal function and behavior. Our long-term goal is to understand how PACS1R203W causes disease and to use this information to develop effective therapies. The objective of this particular application is to determine how PACS1R203W and HDAC6 combine to dysregulate neuronal arborization and synaptic transmission. We hypothesize that the aberrant interaction between PACS1R203W and HDAC6 alters organellar positioning, which contributes to excessive dendrite arborization and dysregulated synaptic activity. Guided by strong preliminary data, we will test our hypothesis by pursuing three specific aims: 1) Determine how the R203W mutation alters PACS1 structure and dynamics for influencing client protein interactions, 2) Determine how PACS1R203W and HDAC6 combine to dysregulate Golgi positioning and dendrite arborization, and 3) Determine how PACS1R203W alters synaptic activity and behavior. The approach is innovative because we will characterize, from the atomic structure to the whole-organism, the mechanism by which the recurrent R203W substitution causes neuronal dysfunction. This research is significant because it may identify new targets and therapeutic approaches to treat this debilitating disorder.