Analysis of autophagy and mitochondrial homeostasis in a human iPS model of NCL

描述(申请人提供)：作为一名经过临床和分子病理学培训的内科科学家，我计划继续我对遗传性神经变性形式的长期兴趣，并在马萨诸塞州综合医院(MGH)发展一项独立的、由R01资助的学术生涯。我在蛋白质加工缺陷和神经退行性变之间的关系的研究生背景为我提出的关于自噬在神经元蜡样脂褐素增多症(NCLS；也称为巴顿病)中的作用的工作提供了坚实的基础。MGH-CHGR(人类基因研究中心)NCL疾病联合项目将作为我职业发展和培训活动的焦点。该联合计划包括(1)我的主要导师Susan Cotman博士和我的共同导师Marcy MacDonald博士的基础研究实验室，(2)由我的共同导师Katherine Sims博士指导的临床部分(MGH Batten疾病卓越中心)，(3)NCLS和其他罕见神经疾病的参考实验室，以及(4)组织培养核心设施，其中包括IRB批准的来自NCL先证者及其家庭成员的成纤维细胞和淋巴母细胞系的生物库。NCL包括一组至少12种不同的、目前无法治疗的溶酶体储存疾病，其临床特征包括进行性运动和认知能力下降，大多数情况下视网膜退化和视力丧失，癫痫发作，运动障碍，以及最终过早死亡。我建议建立从NCL的基因精确敲入小鼠模型中获得的见解，该模型复制了NCL疾病中最常见的突变。从这个模型衍生的小脑细胞系已经证明，在终末期病理之前，自噬受损以及线粒体形态改变，基础ATP水平降低，对氧化应激的敏感性增加，以及基础条件下线粒体氧化磷酸化的转录调节改变。在此应用程序中， 我建议使用新的人类诱导性神经干细胞来源的NCL神经元培养模型来解决这一假说，即线粒体自噬翻转受损(有丝分裂)，从而削弱线粒体质量控制，是NCL患者病理生理学的关键早期组成部分。具体地说，我将通过电子显微镜、有丝分裂吞噬的动态成像以及包括基础和最大呼吸能力在内的生物能量学参数的测量来研究这个神经系统中的自噬和线粒体生物学。此外，我将使用我开发的一个广泛的数据库来识别具有线粒体疾病早期临床病理证据的不典型和分子上未明确的NCL患者。我将使用下一代测序技术在这些患者的核心亚群中识别潜在的因果变异，这种方法可能会产生关于NCL和线粒体生物学之间的遗传相互作用的新假说，并为治疗干预提出新的靶点。

DESCRIPTION (provided by applicant): As a physician-scientist with training in clinical and molecular pathology, I plan to pursue my long-standing interest in inherited forms of neurodegeneration and develop an independent, R01-funded academic career at the Massachusetts General Hospital (MGH). My graduate background on the relationship between protein processing defects and neurodegeneration provides a solid foundation for my proposed work on the role of autophagy in the neuronal ceroid lipofuscinoses (NCLs; also known as Batten Disease). The MGH-CHGR (Center for Human Genetic Research) Joint Program in NCL Disorders will serve as the focal point of my career development and training activities. The Joint Program includes (1) the basic research labs of my primary mentor Dr. Susan Cotman and my co-mentor Dr. Marcy MacDonald, (2) a clinical component (MGH Batten Disease Center of Excellence) directed by my co-mentor Dr. Katherine Sims, (3) a reference lab for NCLs and other rare neurologic disorders, and (4) a tissue culture core facility that includes an IRB-approved bio repository of fibroblast and lymphoblastic cell lines from NCL probands and family members. The NCLs comprise a group of at least 12 distinct, currently untreatable lysosomal storage diseases with clinical features that include progressive motor and cognitive decline, retinal degeneration and visual loss in most cases, seizures, movement disorder, and eventual premature death. I propose to build on insights gained from a genetically precise knock-in murine model of NCL that replicates the most common mutation among the NCL disorders. Cerebellar cell lines derived from this model have demonstrated that end-stage pathology is preceded by impairments in autophagy as well as by altered mitochondrial morphology, decreased basal ATP levels, increased sensitivity to oxidative stress, and altered transcriptional regulation of mitochondrial oxidative phosphorylation under basal conditions. In this application, I propose to use novel, human iPS-derived neuronal culture models of NCL to address the hypothesis that impaired autophagic turnover of mitochondria (mitophagy), and thus impaired mitochondrial quality control, is a critical early component of pathophysiology in NCL patients. Specifically, I will investigate autophagy and mitochondrial biology in this neuronal system by electron microscopy, dynamic imaging of mitophagy, and measurement of bioenergetics parameters, including basal and maximal respiratory capacity. In addition, I will use an extensive database that I developed to identify atypical and molecularly undefined NCL patients with early clinic pathologic evidence of mitochondrial disease. I will use next- generation sequencing technology to identify potential causal variants in a core subset of these patients, an approach that will likely generate new hypotheses about the genetic interaction between NCL and mitochondrial biology and suggest novel targets for therapeutic intervention.