Role of Autism Susceptibility Gene, TAOK2 kinase, and its novel substrates in Synaptogenesis

 描述(申请人提供)：树突棘是一种特殊的富含肌动蛋白的突起，是大脑中大多数兴奋性突触的主要接受者。脊椎是非常有活力的，在发育过程中表现出不同的结构和功能变化，以响应刺激，以及学习和记忆。虽然有大量证据表明，几种神经发育和精神疾病集中在一个共同的主题上，即脊柱形成异常，但脊柱形成的机制及其功能障碍与疾病的关系尚不清楚。TAOK2是一种丝氨酸/苏氨酸激酶，与神经元发育有关，是16p11.2基因组中存在的基因之一。该区域的缺失是与自闭症谱系障碍(ASD)相关的最常见的危险因素。尽管它与神经元发育和ASD相关，但TAOK2激酶的生理神经元底物尚不清楚。目前尚不清楚TAOK2信号如何介导脊柱发育，以及TAOK2基因剂量的不平衡如何导致与ASD相关的神经元和行为改变。结合多种创新方法，本研究旨在阐明TAOK2激酶在突触形成过程中的机制作用，并了解该信号通路的功能障碍如何导致疾病。作为加州大学旧金山分校Jan博士实验室的博士后研究员，除了细胞生物学和生物化学的研究生背景外，我还获得了神经生物学、质谱学和化学遗传学方面的技能。对于本申请中提出的实验，我寻求在干细胞技术、超分辨率显微镜和定量蛋白质组学方面的跨学科指导培训。我组建了一个导师团队，他们的专长是：质谱学和蛋白质组规模的数据采集和分析(Al Burlingame博士)，IPSC技术(Kathryn Ivey和Lauren Weiss博士)，神经发育和自闭症(John Rubenstein博士)，以及超分辨率显微镜(黄波博士)。这次培训将使我能够实现以下目标：1)使用超分辨率风暴成像系统地研究TAOK2在脊柱发育过程中的定位；2)使用化学遗传学方法识别和表征TAOK2激酶的直接新靶点；以及3)使用16p11.2缺失患者的IPSC来源的神经元来表征ASD病理中的TAOK2通路。有了K99阶段获得的新培训和信息，我将扩大我在R00阶段的研究范围。我将分析已识别的TAOK2磷酸化靶标在神经元发育过程中的作用。我还将对16p11.2缺失的IPSC来源的神经元进行磷酸蛋白质组学分析，以确定TAOK2剂量失衡造成的全局变化，这可能揭示ASD发病机制的临床相关见解。K99/R00奖项提供的培训时间将极大地促进我作为一名独立研究员的长期目标，阐明正常和疾病状态下树突棘形成所涉及的信号通路。

 DESCRIPTION (provided by applicant): Dendritic spines are specialized actin-rich protrusions that serve as primary recipients of most excitatory synapses in the brain. Spines are extremely dynamic, exhibiting diverse structural and functional changes during development, in response to stimuli, as well as in learning and memory. While there is substantial evidence that several neurodevelopmental and psychiatric diseases converge on a common theme of aberrant spine formation, the mechanisms of spine formation and how its dysfunction relates to disease is unclear. TAOK2 is a serine/threonine kinase implicated in neuronal development, and is one of the genes present in the 16p11.2 genomic locus. Deletion of this region is the most common risk factor associated with autism spectrum disorder (ASD). Despite its relevance in neuronal development and to ASD, the physiological neuronal substrates of TAOK2 kinase are not known. It is unclear how TAOK2 signaling mediates spine development and how an imbalance in TAOK2 gene dosage might contribute to neuronal and behavioral alterations associated with ASD. Using a combination of innovative approaches, this proposal aims to delineate the mechanistic role of TAOK2 kinase during synaptogenesis and to understand how dysfunction in this signaling pathway might contribute to disease. As a postdoctoral fellow in Dr. Yuh-Nung Jan's laboratory at UCSF, I have gained skills in neurobiology, mass spectrometry, and chemical-genetics in addition to my graduate background in cell biology and biochemistry. For the experiments proposed in this application, I seek interdisciplinary mentored training in stem cell technology, super-resolution microscopy, and quantitative proteomics. I have assembled a team of mentors with expertise in: mass spectrometry and proteome-scale data acquisition and analysis (Dr. Al Burlingame), iPSC technology (Drs. Kathryn Ivey and Lauren Weiss), neurodevelopment and ASD (Dr. John Rubenstein), and super-resolution microscopy (Dr. Bo Huang). This training will allow me to achieve the following aims: 1) systematic investigation of TAOK2 localization during spine development using super-resolution STORM imaging, 2) identification and characterization of direct novel targets of TAOK2 kinase using a chemical-genetic approach, and 3) characterization of the TAOK2 pathway in ASD pathology using iPSC derived neurons from 16p11.2 deletion patients. With the new training and information acquired in the K99 phase, I will extend the scope of my research in the R00 phase. I will analyze the role of the identified TAOK2 phosphorylation targets during neuronal development. I will also perform phosphoproteomic analysis of 16p11.2 deletion iPSC-derived neurons to identify global changes caused by imbalances in TAOK2 dosage, which may reveal clinically relevant insights into the pathogenesis of ASD. The training period afforded by the K99/R00 award will greatly facilitate my long-term goal as an independent investigator to elucidate signaling pathways involved in dendritic spine formation during normal and disease states.