TDP43 mRNA complex transport in physiologic and pathologic states

 描述(由申请人提供)：本建议书描述了Pallavi Gopal博士的5年职业发展计划，作为向成功的独立内科医生-科学家的过渡。Gopal博士在宾夕法尼亚大学(Penn)完成了解剖病理学和神经病理学的临床培训，目前正在开发一项独立的研究和培训计划，使她能够获得生理条件下和疾病中信使核糖核酸运输的空间和时间动力学方面的专业知识。这项建议汇集了RNA新陈代谢、分子神经科学和神经病理学方面的各种资源，将为戈帕尔博士发展成为一名独立的内科科学家提供极好的培训。研究将在Erika Holzbaur博士的指导下进行，Erika Holzbaur博士是国际公认的微管马达和实时轴突运输动力学专家。这笔赠款将为戈帕尔博士提供受保护的时间，让他通过正式的课程、科学研讨会和会议获得神经元细胞骨架、细胞器和RNA-蛋白质动力学方面的专业知识。宾夕法尼亚大学的协作环境将促进利用新技术开展拟议的项目，并将为戈帕尔博士提供迈向成功学术生涯所需的培训。肌萎缩侧索硬化症(ALS)和前颞叶变性(FTLD)位于临床病理图谱的两端，但具有共同的临床、遗传和病理特征。泛素化的胞质包涵体由43 kDa的Tran的活性反应DNA结合蛋白(TDP-43)组成，是散发性ALS的共同特征，也是FTLD的最常见形式；伴随着正常核TDP-43表达的丧失。此外，TDP-43和其他RNA加工蛋白中与疾病相关的突变的发现突显了RNA新陈代谢改变是神经退化的常见致病机制。然而，我们对TDP-43错误定位如何扰乱其核和细胞质RNA处理功能和/或在细胞质中介导毒性的了解仍然不完整。该研究计划将利用创新的方法和原代神经元的实时成像技术来检验两个主要假设：核TDP-43的丢失导致TDP-43靶mRNA和蛋白在轴突中的动态流量减少，以及病理条件下TDP-43在细胞质的重新分布导致TDP-43相关的mRNA错误定位。其具体目的是：1)确定核TDP-43功能的丧失是否减少突触蛋白的轴突运输和细胞器翻转；2)确定(A)细胞质TDP-43 RNA结合功能的丧失和(B)应激诱导的细胞质TDP-43聚集如何影响轴突和树突中mRNA的定位和运输。这些研究将提供神经元中单个RNA转录本的时间和空间分辨率，以便更清楚地了解ALS/FTLD发病机制中RNA代谢的变化。

 DESCRIPTION (provided by applicant): This proposal describes a 5 year career development plan for Dr. Pallavi Gopal to serve as a transition to successful independent physician-scientist. Dr. Gopal completed her clinical training in Anatomic Pathology and Neuropathology at the University of Pennsylvania (Penn), and is now developing an independent research and training program that will allow her to gain expertise in spatial and temporal dynamics of mRNA transport under physiological conditions and in disease. This proposal brings together diverse resources in RNA metabolism, molecular neuroscience and neuropathology and will provide superb training for Dr. Gopal to develop into an independent physician-scientist. Research will be performed under the mentorship of Dr. Erika Holzbaur, an internationally recognized expert in microtubule-based motors and real-time axon transport dynamics. This grant will provide protected time for Dr. Gopal to gain expertise in neuronal cytoskeletal, organelle, and RNA-protein dynamics through formal coursework, scientific seminars and meetings. The collaborative environment at Penn will foster utilization of novel techniques to conduct the proposed project and will provide Dr. Gopal with the training required to proceed towards a successful academic career. Amyotrophic lateral sclerosis (ALS) and front temporal lobar degeneration (FTLD) exist on two ends of a clinic pathologic spectrum but share clinical, genetic, and pathologic features. Ubiquitinated cytoplasmic inclusions composed of Tran's active response DNA-binding protein of 43 kDa (TDP-43) are a shared feature of sporadic ALS and the most common form of FTLD; there is concomitant loss of normal nuclear TDP-43 expression. Moreover, the discovery of disease-linked mutations in TDP-43 and other RNA processing proteins highlights altered RNA metabolism as a common pathogenic mechanism of neurodegeneration. However, our knowledge of how TDP-43 mislocalization disrupts its nuclear and cytoplasmic RNA processing functions and/or mediates toxicity in the cytoplasm is still incomplete. The research plan will utilize innovative approaches with real-time imaging techniques in primary neurons to test two main hypotheses: that loss of nuclear TDP-43 results in reduced dynamic flux of TDP-43 target mRNA and proteins in axons and that cytoplasmic redistribution of TDP-43 under pathological conditions results in mislocalization of TDP-43- associated mRNA. The specific aims are to: 1) Determine whether loss of nuclear TDP-43 function reduces axonal trafficking of synaptic proteins and organelle turn over and 2) Determine how (A) loss of cytoplasmic TDP-43 RNA binding function and (B) stress-induced cytoplasmic TDP-43 aggregation affect localization and trafficking of mRNA in axons and dendrites. These studies will provide temporal and spatial resolution of individual RNA transcripts in neurons in order to gain a clearer understanding of altered RNA metabolism in ALS/FTLD pathogenesis.