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，其特征在于进行性肌肉萎缩和运动神经元死亡。迄今为止已知>90%的ALS病例的标志性特征是TAR DNA结合蛋白（TDP-43）的错误定位及其与运动神经元和周围神经胶质细胞中的细胞质聚集体的相关性，无论病因如何。此外，在家族性和散发性ALS病例中均发现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变性，运动缺陷和存活率降低，类似于人类疾病的表现。使用这个模型，我们确定了一个强大的TDP-43和果蝇FoxO，应激反应的调节基因之间的相互作用。我们发现，FoxO过表达增强了由TDP-43引起的果蝇视网膜神经变性，而FoxO表达的减少拯救了这种表型和由TDP-43表达引起的运动神经元缺陷。这些发现使我们假设FoxO至少是TDP-43毒性的某些方面的基础。拟议研究的总体目标是确定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。在目标1中，我们将调节FoxO在运动神经元中的表达，并使用一系列已建立的测定法，确定其对TDP-43依赖性表型的影响，包括异常NMJ形态、微管组织改变、突触标记物分布缺陷、活力和寿命降低以及运动功能受损。我们将通过进行细胞分级来确定FoxO是否影响TDP-43聚集。我们还将测试FoxO对TDP-43与初级运动神经元中的RNA应激颗粒的关联的影响，并进行光漂白后荧光恢复（FRAP）分析以确定TDP-43在神经突内的移动性。初步结果表明，减少FoxO表达挽救了NMJ处的特定TDP-43依赖性异常，并增加了TDP-43的溶解度。在目标2中，我们将使用来自果蝇胚胎的初级运动神经元模型来确定FoxO对TDP-43表达神经元对氧化应激的反应以及去除应激后的恢复的影响。我们将使用共聚焦荧光显微镜测量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应力诱导变化。还将对应激诱导的TDP-43斑点进行实时成像和FRAP分析，以测量TDP-43在整个应激反应中的动力学。使用这种方法，我们也将能够测试新出现的范例，TDP-43阳性应激颗粒可能在长时间应激后播种病理性聚集体。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.