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走向独立，作为一个医生，科学家与肌萎缩侧索硬化症（ALS）的机制和临床重点神经肌肉医学的研究计划。ALS是一种以运动神经元变性为特征的快速进行性神经系统疾病，其导致3-5年内死亡，并且没有有效的治疗方法。TANK结合激酶1（TBK 1）是近年来发现的一种新的与散发性和家族性ALS和额颞叶痴呆（FTD）相关的致病基因。然而，TBK 1变异导致疾病的机制仍不清楚。蒂莫西米勒博士的指导下，我建议调查如何中断TBK 1，一个无处不在的表达激酶，在涉及ALS发病机制，自噬和先天免疫两个过程的交叉点，导致疾病的发病机制。虽然TBK 1突变的一个子集导致蛋白质的截短和表达减少，但许多错义变体的意义和致病性仍然没有得到表征。值得注意的是，小胶质细胞衍生的基因如颗粒蛋白原（PGRN）和髓样细胞2上表达的触发受体（TREM 2）的单倍不足已显示通过改变炎症信号传导和吞噬功能引起或增加阿尔茨海默病、额颞叶痴呆和ALS的风险[10]。鉴于ALS中运动神经元细胞死亡的非细胞自主机制的贡献以及TBK 1在神经元和非神经元细胞类型中的已知表达，我还将研究TBK 1功能丧失（LoF）对星形胶质细胞和小胶质细胞（中枢神经系统的主要免疫细胞）的影响。为了了解TBK 1 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，先前描述的TBK 1敲除小鼠是胚胎致死的，这排除了神经表型的分析。基于这些考虑，我提出了一个研究计划，包括以下目标：1）使用生物化学和组织病理学方法确定TBK 1错义和LoF变体对自噬的功能后果2）确定TBK 1 LoF对吞噬作用、自噬、3）确定TBK 1是否通过细胞自主或非细胞自主信号传导引起ALS。通过检查运动神经元、星形胶质细胞和小胶质细胞中TBK 1选择性缺失的小鼠表型的细胞自主机制预期上述实验将阐明TBK 1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型床边治疗和诊断标志物。

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.