Determining the role of FoxO in TDP-43 toxicity

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种致命的神经退行性疾病，以进行性肌肉萎缩和运动神经元死亡为特征。迄今为止已知的90% ALS病例的一个标志性特征是TAR DNA结合蛋白（TDP-43）的错误定位及其与运动神经元和周围胶质细胞的细胞质聚集体的关联，而不考虑病因。此外，在家族性和散发性ALS病例中都发现了TDP-43突变。总之，这些发现证明了TDP-43在疾病中的核心作用。我们的实验室已经开发了一种基于TDP-43的ALS果蝇模型，该模型表现出年龄依赖性神经退行性变、运动缺陷和生存率下降，类似于人类疾病的表现。利用这个模型，我们发现TDP-43和果蝇FoxO之间存在强大的遗传相互作用，FoxO是应激反应的调节因子。我们发现FoxO过表达增强了果蝇视网膜中由TDP-43引起的神经退行性变，而FoxO表达的降低挽救了这种表型和运动神经元中由TDP-43表达引起的运动缺陷。这些发现使我们假设FoxO至少在某些方面是TDP-43毒性的基础。该研究的首要目标是确定FoxO在TDP-43毒性中的作用，并开始阐明这种功能相互作用的机制。在Aim 1中，我们将调节运动神经元中的FoxO表达，并使用一系列已建立的检测方法，确定其对tdp -43依赖性表型的影响，包括NMJ形态异常、微管组织改变、突触标记分布缺陷、生存能力和寿命降低以及运动功能受损。我们将通过细胞分离来确定FoxO是否会影响TDP-43的聚集。我们还将测试FoxO对初级运动神经元中TDP-43与RNA应激颗粒关联的影响，并进行光漂白后荧光恢复（FRAP）分析，以确定TDP-43在神经突内的流动性。初步结果表明，减少FoxO表达可缓解NMJ特异性TDP-43依赖性异常，并增加TDP-43的溶解度。在Aim 2中，我们将使用源自果蝇胚胎的初级运动神经元模型来确定FoxO对表达TDP-43的神经元对氧化应激反应的影响，以及应激去除后的恢复。我们将使用共聚焦荧光显微镜测量应力诱导的TDP-43定位、应力颗粒形成、微管组织、神经元形态和细胞死亡的变化。还将对应力诱导的TDP-43点进行实时成像和FRAP分析，以测量TDP-43在整个应力响应过程中的动力学。使用这种方法，我们也将能够测试新兴的范式，即tdp -43阳性应激颗粒可能在长期应激后产生病理聚集。这些实验将确定FoxO在TDP-43毒性中的作用，并有助于确定其作为ALS和相关神经退行性疾病的新治疗靶点的潜力。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by progressive muscle atrophy and motor neuron death. A hallmark feature of >90% of ALS cases known to date is the mislocalization of TAR DNA Binding Protein (TDP-43) and its association with cytoplasmic aggregates in motor neurons and surrounding glia regardless of etiology. In addition, TDP-43 mutations have been found in both familial and sporadic ALS cases. Together, these findings demonstrate a central role for TDP-43 in disease. Our lab has developed a Drosophila model of ALS based on TDP-43 that exhibits age dependent neurodegeneration, locomotor defects, and decreased survival, resembling the presentation of the human disease. Using this model, we identified a robust genetic interaction between TDP-43 and Drosophila FoxO, a regulator of stress response. We found that FoxO overexpression enhances neurodegeneration caused by TDP-43 in the fly retina while reduction of FoxO expression rescued both this phenotype and the locomotor defects caused by TDP-43 expression in motor neurons. These findings led us to hypothesize that FoxO underlies at least some aspects of TDP-43 toxicity. The overarching objective of the proposed research is to determine FoxO's role in TDP-43 toxicity and begin to elucidate the mechanisms underlying this functional interaction. In Aim 1, we will modulate FoxO expression in motor neurons and, using a battery of established assays, determine its effect on TDP-43-dependent phenotypes including abnormal NMJ morphology, altered microtubule organization, defects in distribution of synaptic markers, reduced viability and lifespan, as well as impaired locomotor function. We will determine whether FoxO affects TDP-43 aggregation by performing cellular fractionations. We will also test FoxO's effect on TDP-43's association with RNA stress granules in primary motor neurons and perform Fluorescence Recovery After Photobleaching (FRAP) analyses to determine TDP-43's mobility within neurites. Preliminary results indicate that reducing FoxO expression rescues specific TDP-43-dependent abnormalities at the NMJ and increases the solubility of TDP-43. In Aim 2 we will use a primary motor neuron model derived from Drosophila embryos to determine the effects of FoxO on the response of TDP-43 expressing neurons to oxidative stress, as well as recovery following removal of stress. We will measure stress-induced changes in TDP-43 localization, formation of stress granules, microtubule organization, neuronal morphology, and cell death using confocal fluorescence microscopy. Live imaging and FRAP analyses will also be performed on stress-induced TDP-43 puncta to measure the kinetics of TDP-43 throughout stress response. Using this approach, we will also be able to test the emerging paradigm that TDP-43-positive stress granules may seed pathological aggregates following prolonged stress. These experiments will establish FoxO's role in TDP-43 toxicity and help determine its potential as a novel therapeutic target for ALS and related neurodegenerative diseases.