Investigating mechanisms of pathogenesis in TBK1-associated amyotrophic lateral sclerosis

该指导职业发展奖的目的是推动早期PI成为一名独立的医生-科学家，研究肌萎缩侧索硬化症（ALS）的机制和神经肌肉医学的临床重点。肌萎缩侧索硬化症是一种以运动神经元退化为特征的快速进展的神经系统疾病，可在3-5年内导致死亡，目前尚无有效的治疗方法。TANK结合激酶1 （TBK1）最近被发现是散发性和家族性ALS和额颞叶痴呆（FTD）的一个新的致病基因。然而，TBK1变异导致疾病的机制尚不清楚。在Timothy Miller博士的指导下，我提议研究TBK1的破坏是如何导致疾病发病的。TBK1是一种普遍表达的激酶，在涉及ALS发病机制的两个过程（自噬和先天免疫）的交叉点起作用。虽然TBK1突变的一个子集导致该蛋白的截断和表达减少，但许多错义变异的意义和致病性仍未被表征。值得注意的是，小胶质源性基因如前颗粒蛋白（PGRN）和髓样细胞2上表达的触发受体（TREM2）的单倍性不足已被证明通过改变炎症信号和吞噬功能导致或增加阿尔茨海默病、额颞叶痴呆和ALS[10]的风险。鉴于非细胞自主机制在ALS患者运动神经元细胞死亡中的作用，以及TBK1在神经元和非神经元细胞类型中的已知表达，我还将研究TBK1功能丧失（LoF）对星形胶质细胞和小胶质细胞（中枢神经系统的初级免疫细胞）的影响。为了了解TBK1 LoF如何影响疾病的表现，动物疾病模型将是一个非常有价值的工具。不幸的是，先前描述的TBK1敲除小鼠是胚胎致死的，这妨碍了对神经表型的分析。基于这些考虑，我提出了一个包含以下目标的研究计划：1)通过生化和组织病理学方法确定TBK1错义和LoF变异对自噬的功能影响2)确定TBK1 LoF对星形胶质细胞和小胶质细胞吞噬、自噬和炎症信号的影响3)通过检测运动神经元、星形胶质细胞中TBK1选择性缺失小鼠的表型，确定TBK1是否通过细胞自主或非细胞自主机制导致ALS。上述实验有望阐明TBK1在ALS发病机制中的作用，并揭示新的治疗靶点。该奖项支持的研究培训将加强我的技能和知识基础，将基础科学发现转化为ALS和其他潜在神经退行性疾病的新的床边治疗和诊断标记。

The purpose of this mentored career development award is to propel an early stage PI towards independence as a physician-scientist with a research program on mechanisms of amyotrophic lateral sclerosis (ALS) and clinical focus on neuromuscular medicine. ALS is a rapidly progressive neurologic disorder characterized by motor neuron degeneration that leads to death in 3-5 years and for which there are no effective treatments. TANK binding kinase 1 (TBK1) was recently uncovered as a novel causative gene in sporadic and familial ALS and frontotemporal dementia (FTD). However, the mechanisms by which variants in TBK1 because disease remain unclear. Under the guidance of Dr. Timothy Miller, I propose to investigate how disruptions in TBK1, a ubiquitously expressed kinase that functions at the intersection of two processes implicated in ALS pathogenesis, autophagy and innate immunity, lead to disease pathogenesis. Although a subset of TBK1 mutations cause truncation and reduced expression of the protein, the significance and pathogenicity of many missense variants remain uncharacterized. Notably, haploinsufficiency of microglial- derived genes such as progranulin (PGRN) and triggering receptor expressed on myeloid cells 2 (TREM2) has been shown to cause or increase risk for Alzheimer's disease, frontotemporal dementia, and ALS [10] by altering inflammatory signaling and phagocytic function. Given the contribution of non-cell-autonomous mechanisms in motor neuron cell death in ALS and known expression of TBK1 in neuronal and non-neuronal cell types, I will also examine the impact of TBK1 loss of function (LoF) in astrocytes and microglia, the primary immune cells of the central nervous system. In order to understand how TBK1 LoF influences manifestation of disease, an animal model of disease would be an incredibly valuable tool. Unfortunately, previously described TBK1 knock out mice are embryonic lethal which precluded an analysis of neurological phenotypes. Based on these considerations, I propose the a research program incorporating the following aims: 1) Determine the functional consequences of TBK1 missense and LoF variants on autophagy using biochemical and histopathological methods 2) Determine the effect of TBK1 LoF on phagocytosis, autophagy, and inflammatory signaling in astrocytes and microglia 3) Determine whether TBK1 causes ALS via cell autonomous or non-cell autonomous mechanisms by examining phenotypes of mice with selective loss of TBK1 in motor neurons, astrocytes, and microglia The experiments proposed above are expected to clarify the role of TBK1 in ALS pathogenesis and reveal novel therapeutic targets. The research training supported by the award will fortify my skill set and knowledge base for translating basic science discoveries to novel bedside treatment and diagnostic markers for ALS and potentially other neurodegenerative diseases.