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

项目摘要 PACS 1综合征是最近发现的一种神经发育障碍，由反复出现的新生错义引起 PACS 1突变（p.Arg203Trp）。携带这种错义突变的患者有几个共同的发育缺陷， 包括智力残疾、癫痫和自闭症。PACS 1 R203 W导致PACS 1的机制 综合征是未知的，没有治愈性治疗是可用的。PACS 1是一种多功能分选蛋白， 促进从核内体到高尔基体网络的逆行运输，以将蛋白质递送到初级细胞。 纤毛和基因组完整性。这种多功能性依赖于PACS 1的一小部分，称为弗林蛋白酶。 结合区（FBR），其结合广泛的客户蛋白和信号分子。R203 W突变 位于FBR中，我们的生物物理研究揭示了当R203 W 存在取代，表明PACS 1与以下一种或多种之间的相互作用可能改变： PACS 1综合征中的客户蛋白。我们的初步研究强烈表明PACS 1 R203 W增加结合 去乙酰化酶HDAC 6严重干扰膜运输并损害神经元发育。因此，委员会认为， PACS 1 R203 W减少已知HDAC 6底物的乙酰化，包括α-微管蛋白，破坏中心体 定位并导致高尔基体碎裂和锥体神经元中树突分支增加。这 树突过度分支与L2/3皮层神经元中抑制电流的减少相耦合，导致L2/3皮层神经元中抑制电流的增加。 兴奋：抑制（E：I）比，类似于其他神经发育障碍，表明， PACS 1 R203 W严重影响神经元功能和行为。我们的长期目标是了解 PACS 1 R203 W导致疾病，并利用这些信息开发有效的治疗方法。的目的 具体应用是确定PACS 1 R203 W和HDAC 6如何联合收割机来失调神经元分支 和突触传递。我们假设PACS 1 R203 W和HDAC 6之间的异常相互作用改变了 细胞器定位，这有助于过度树突树枝化和突触活动失调。 在强有力的初步数据的指导下，我们将通过追求三个具体目标来测试我们的假设：1）确定如何 R203 W突变改变了PACS 1的结构和动力学，从而影响了客户蛋白的相互作用，2） 确定PACS 1 R203 W和HDAC 6如何联合收割机来调节高尔基体定位和树突树枝化， 以及3）确定PACS 1 R203 W如何改变突触活动和行为。这种方法是创新的，因为 我们将描述，从原子结构到整个有机体，循环的机制， R203 W取代导致神经元功能障碍。这项研究意义重大，因为它可能会确定新的目标 和治疗方法来治疗这种使人衰弱的疾病。

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.