Mitochondrial stress responses in neurodegeneration

神经退行性变是我们老龄化人口中日益严重的疾病负担。受损的线粒体在有丝分裂后细胞如神经元中积累，并且与常见的神经退行性疾病相关，最显著的是散发性帕金森病（PD）。此外，受损的线粒体是单基因形式的PD（由于PINK 1、PRKN和CHCHD 26的突变）以及单基因形式的肌萎缩侧索硬化（ALS）、额颞叶痴呆（FTD）和肌病（由于CHCHD 10的突变）的神经变性的驱动因素。 为了防止（或在某些情况下加剧）神经变性，细胞已经进化出应激反应来处理受损的线粒体。这些包括通过自噬（线粒体自噬）的选择性降解，通过抑制它们的融合进行分离，以及通过整合应激反应（ISR）向细胞质和细胞核发出信号。在哺乳动物中，这些反应是由线粒体内的应激传感蛋白启动的，包括PINK 1和DELE 1，它们在线粒体输入停滞的情况下稳定，以及OMA 1，一种由线粒体去极化激活的内膜肽酶。对这些线粒体应激反应的更全面理解可能会发现治疗神经退行性疾病的新靶点。 我们计划的总体目标是表征神经变性中的线粒体应激反应，并通过研究单基因疾病开发精确治疗，重点是致病基因PINK 1，PRKN，CHCHD 2和CHCHD 10。为此，我们提出了三个互补的目标，利用校内计划的优势和我作为细胞生物学家和神经学家的观点： 具体目的1：确定CHCHD 2和CHCHD 10相关神经变性中的线粒体应激反应并开发治疗方法（60%努力）。CHCHD 2和CHCHD 10旁系同源物中的突变共享线粒体嵴内蛋白质错误折叠的发病机制。在基因敲入（KI）小鼠模型和工程细胞系和患者细胞系（包括iPSC）中，我们正在确定CHCHD 2和CHCHD 10的正常功能以及这些疾病的发病机制。此外，在与NCATS的合作中，我们正在开发等位基因特异性反义寡核苷酸（阿索）治疗ALS和其他形式的神经退行性疾病（由于CHCHD 10突变）。我们一直在评估一个家庭与ALS由于突变CHCHD 10在临床中心内。最后，我们已经确定了一个保护线粒体应激反应对突变CHCHD 10蛋白质错误折叠，这是介导的应激诱导的肽酶OMA 1。这种反应包括通过OMA 1切割DELE 1激活ISR，但也通过OPA 1切割抑制线粒体融合。我们正在确定mito-ISR在防止线粒体损伤中的具体作用。我们还产生了KO小鼠模型，以剖析哪些OMA 1底物介导体内这种保护。 具体目标2：鉴定PINK 1-Parkin通路的调节剂和致病突变（25%努力）。通过对NIH PD临床患者和公共数据集中PRKN的综合分析，我们发现PRKN错义变体很常见，但大多数没有功能特征。我们已经开发了一种新的基于流式细胞术的帕金活性报告，我们正在使用它来确定哪些PRKN变体是良性的，哪些是合并形式的功能丧失。此外，我们还使用该报告基因在全基因组筛选中鉴定PINK 1-Parkin通路的调节剂。最后，我们正在NIH和U Penn建立早发性和/或家族性PD患者的分子诊断。 具体目标3：开发新的方法来测量神经变性模型中的蛋白质周转（15%努力）。利用动态蛋白质组学和纳米代谢成像，我们正在开发新的方法来评估神经退行性疾病和肌病模型中的线粒体自噬和其他线粒体应激反应。这些方法将补充其他目标的努力，除了其更广泛的应用。

Neurodegeneration is an increasing and unmitigated disease burden in our aging population. Damaged mitochondria accumulate in post-mitotic cells such as neurons and are associated with the common neurodegenerative disorders, most notably sporadic Parkinsons disease (PD). Additionally, damaged mitochondria are drivers of neurodegeneration in monogenic forms of PD (due to mutations in PINK1, PRKN, and CHCHD26), as well as monogenic forms of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and myopathy (due to mutations in CHCHD10). Preventing (or in some cases exacerbating) neurodegeneration, the cell has evolved stress responses to handle damaged mitochondria. These include selective degradation by autophagy (mitophagy), isolation by inhibition of their fusion, and signaling to the cytosol and nucleus through the integrated stress response (ISR). In mammals, these responses are initiated by stress sensing proteins within mitochondria, including PINK1 and DELE1, which are stabilized in the setting of mitochondrial import arrest, and OMA1, an inner membrane peptidase that is activated by mitochondrial depolarization. A more complete understanding of these mitochondrial stress responses may uncover novel targets for the treatment of neurodegenerative disorders. The overall goal of our program is to characterize mitochondrial stress responses in neurodegeneration and develop precision therapy through the study of monogenic disorders, with an emphasis on the disease-causing genes PINK1, PRKN, CHCHD2, and CHCHD10. To this end, we propose three complementary aims that draw on strengths of the intramural program and my perspective as a cell biologist and neurologist: SPECIFIC AIM 1: To determine mitochondrial stress responses in and develop therapies for CHCHD2 and CHCHD10 related neurodegeneration (60% Effort). Mutations in the CHCHD2 and CHCHD10 paralogs share a pathogenetic mechanism of protein misfolding within mitochondrial cristae. In knock-in (KI) mouse models and engineered and patient cell lines (including iPSC), we are determining the normal function of CHCHD2 and CHCHD10 and the pathogenesis of these disorders. Additionally, in a collaboration with NCATS we are developing allele specific antisense oligonucleotide (ASO) treatments for ALS and other forms of neurodegeneration due to mutations in CHCHD10. We have been evaluating a family with ALS due to a mutation in CHCHD10 within the clinical center. Finally, we have identified a protective mitochondrial stress response against mutant CHCHD10 protein misfolding, which is mediated by the stress-induced peptidase OMA1. This response includes activation of the ISR through cleavage of DELE1 by OMA1, but also inhibits mitochondrial fusion through cleavage of OPA1. We are determining the specific role of the mito-ISR in protecting against mitochondrial damage. We have additionally generated KO mouse models to dissect which OMA1 substrates mediate this protection in vivo. SPECIFIC AIM 2: To identify modulators of and pathogenic mutations in the PINK1-Parkin pathway (25% Effort). Through comprehensive analysis of PRKN in NIH PD clinic patients and public datasets, we have found that PRKN missense variants are common, but most are not functionally characterized. We have developed a novel FACS based reporter of Parkin activity, which we are using to determine which PRKN variants are benign, and which are loss of function in a pooled format. We are additionally using this reporter to identify modulators of the PINK1-Parkin pathway in genome-wide screens. Finally, we are establishing the molecular diagnosis of patients with early onset and/or familial PD at the NIH and U Penn. SPECIFIC AIM 3: To develop novel methods to measure protein turnover in models of neurodegeneration (15% Effort). Using dynamic proteomics and nanoscale metabolic imaging, we are developing novel methods to assess mitophagy and other mitochondrial stress responses in models of neurodegenerative disease and myopathy. These methods will complement efforts in other aims, in addition to their broader applications.