Role of EPS8-mediated RAC activation in disease-related protein aggregation

衰老是与蛋白质聚集有关的神经退行性疾病的主要风险因素，包括肌萎缩性侧索硬化症、亨廷顿舞蹈症、帕金森病和阿尔茨海默病。减缓衰老过程的途径的发现已经彻底改变了分子老年学领域，表明创造治疗方法来预防与年龄相关的神经退行性疾病的现实可能性。然而，衰老的机制及其对年龄相关疾病的影响才刚刚开始在分子水平上被揭示。在之前的DFG研究中，我们发现在衰老过程中泛素化蛋白质组的重塑，这可以通过饮食限制和减少胰岛素信号传导等长寿模式得到改善。值得注意的是，衰老导致泛素化的全局损失，这是由去泛素化酶（DUB）活性增加引发的。由于泛素化可以标记蛋白质以供蛋白酶体识别，因此一个基本问题是靶向降解的缺陷是否会影响寿命。通过整合具有缺陷蛋白酶体的蠕虫的数据，我们确定了由于泛素化减少和随后的降解而随着年龄积累的蛋白酶体靶标。降低年龄失调的蛋白酶体水平旨在延长寿命，而防止其降解则会缩短寿命。在蛋白酶体靶点中，我们发现了IFB-2中间丝和RAC信号的EPS-8调节剂。虽然IFB-2水平的升高促进了肠道完整性的丧失，但肌肉和神经元中EPS-8过度激活的RAC上调，并导致肌动蛋白细胞骨架和蛋白激酶JNK的改变。这些结果提出了一个令人兴奋的假设，即随着年龄的增长而逃避蛋白酶体降解的调节蛋白是否在涉及蛋白质聚集的年龄相关疾病的发展中起作用。事实上，我们的初步结果表明，降低EPS-8/RAC信号，而不是IFB-2，可以阻止秀丽隐杆线虫疾病模型中与亨廷顿氏病和肌萎缩性侧索硬化症等神经退行性疾病相关的蛋白质聚集。在这项更新申请中，我们建议使用不同的秀丽隐杆线虫和人类细胞模型来定义EPS-8/RAC在疾病相关蛋白聚集和随后的生理后果中的影响。然后，我们将确定过度激活的EPS8/RAC信号是否通过其在肌动蛋白和/或JNK调控中的作用来调节疾病相关蛋白聚集。最后，我们将定义去泛素化和抑制EPS-8蛋白酶体降解的dub。重要的是，我们的初步数据表明，USP4（一种随着年龄增长而上调的DUB）的敲低可以在秀丽隐杆线虫和人类细胞中表现出降低EPS8/RAC信号的有益作用。总之，我们的发现可能会导致不同年龄相关的神经退行性疾病的治疗方法的收敛。

Aging is a major risk factor for neurodegenerative diseases that involve protein aggregation, including amyotrophic lateral sclerosis, Huntington’s, Parkinson’s and Alzheimer’s. The discovery of pathways that slow down the aging process has revolutionized the field of molecular gerontology, suggesting the realistic possibility of creating therapeutics to prevent age-related neurodegenerative diseases. However, the mechanisms underlying aging and their impact on age-related diseases are only beginning to be unraveled at the molecular level. In the previous DFG grant, we found a remodeling of the ubiquitinated proteome during aging, which was ameliorated by longevity paradigms such as dietary restriction and reduced insulin signaling. Notably, aging caused a global loss of ubiquitination that was triggered by increased deubiquitinase (DUB) activity. Because ubiquitination can tag proteins for recognition by the proteasome, a fundamental question was whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identified proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolonged longevity, whereas preventing their degradation shortened lifespan. Among the proteasomal targets, we found the IFB-2 intermediate filament and the EPS-8 modulator of RAC signaling. While increased levels of IFB-2 promoted the loss of intestinal integrity, upregulation of EPS-8 hyperactivated RAC in muscle and neurons, and led to alterations in the actin cytoskeleton and protein kinase JNK. An exciting hypothesis raised by these results is whether regulatory proteins that escape proteasomal degradation with aging have a role in the development of age-related disorders that involve protein aggregation. Indeed, our preliminary results indicate that lowering EPS-8/RAC signaling, but not IFB-2, prevents aggregation of proteins linked with neurodegenerative diseases such as Huntington’s and amyotrophic lateral sclerosis in C. elegans disease models. In this renewal application, we propose to define the impact of EPS-8/RAC in disease-related protein aggregation and subsequent physiological consequences using different C. elegans and human cell models. Then, we will define whether hyperactivated EPS8/RAC signaling modulates disease-related protein aggregation through its role in actin and/or JNK regulation. Finally, we will define the DUBs that deubiquitinate and suppress proteasomal degradation of EPS-8. Importantly, our preliminary data indicate that knockdown of USP4, a DUB that becomes upregulated with age, phenocopies the beneficial effects of lowering EPS8/RAC signaling in C. elegans and human cells. Together, our findings could lead to converging therapeutic approaches for distinct age-related neurodegenerative disorders.