Investigating mechanisms of pathogenesis in TBK1-associated amyotrophic lateral sclerosis

这个指导职业发展奖的目的是推动早期PI成为一名独立的内科科学家，拥有肌萎缩侧索硬化症(ALS)机制的研究计划，并将临床重点放在神经肌肉医学上。肌萎缩侧索硬化症是一种进展迅速的神经系统疾病，以运动神经元变性为特征，可导致3-5年内死亡，目前尚无有效的治疗方法。坦克结合蛋白1(TANK BINDING KINK 1，TBK1)是近年来发现的一种新的致病基因，与家族性ALS和额颞叶痴呆(FTD)有关。然而，由于疾病而导致的TBK1变异的机制仍不清楚。在Timothy Miller博士的指导下，我提议调查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ALS发病机制中的两个过程--自噬和先天免疫的交叉点发挥作用。尽管TBK1基因突变的一个子集会导致蛋白质的截断和表达减少，但许多错义变体的重要性和致病性仍未确定。值得注意的是，小胶质衍生基因，如原颗粒蛋白(PGRN)和髓样细胞上表达的触发受体2(TREM2)的单倍性不足已被证明通过改变炎症信号和吞噬功能而导致或增加阿尔茨海默病、额颞叶痴呆和ALS的风险[10]。鉴于非细胞自主机制在肌萎缩侧索硬化症运动神经元死亡中的作用，以及已知的TBK1在神经元和非神经细胞类型中的表达，我还将研究TBK1功能丧失(LoF)对中枢神经系统的主要免疫细胞-星形胶质细胞和小胶质细胞的影响。为了了解TBK1 LOF如何影响疾病的表现，疾病的动物模型将是一个非常有价值的工具。不幸的是，之前描述的TBK1基因敲除小鼠是胚胎致死的，这排除了对神经表型的分析。基于这些考虑，我提出了一个包含下列目标的研究计划：1)使用生化和组织病理学方法确定TBK1错义和LOF变体对自噬的功能后果2)确定TBK1 LOF对星形胶质细胞和小胶质细胞的吞噬、自噬和炎症信号的影响3)通过检测选择性丢失TBK1在运动神经元、星形胶质细胞和小胶质细胞中的表型，确定TBK1是通过细胞自主机制还是非细胞自主机制导致ALS。该奖项支持的研究培训将加强我的技能和知识库，将基础科学发现转化为ALS和潜在的其他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.