Mitochondrial mechanisms and signaling in manganese exposure

总结： 本申请集中于一种新的致病机制，包括金属稳态， 线粒体蛋白质稳态和对认知和运动的神经系统不良影响的发生。我们 已经确定了线粒体RNA颗粒，作为神经元对环境反应的关键起始物， 和/或使个体易患疾病的遗传损伤。这种RNA加工途径在 线粒体位于锰毒性效应的上游，包括 呼吸链活性降低，线粒体膜去极化，线粒体氧化 压力导致细胞死亡我们的总体假设是线粒体中锰的积累 破坏线粒体RNA颗粒功能并诱导dsRNA积累，作为毒性的一部分 机制这种破坏导致dsRNA的积累和OXPHOS功能的降低， 增加氧化应激。因此，线粒体RNA颗粒为我们提供了 暴露和敏感性锰和神经保护干预的潜在目标。 具体目标： 目的1：确定锰在线粒体中的作用靶点。 目的2：评估锰诱导的线粒体功能障碍是否诱导dsRNA积累 和促炎反应。 目的3：检测线粒体RNA颗粒功能受损在多大程度上调节锰敏感性 在人脑类器官和来自野生型和SLC 30 a10 KO小鼠的脑中。 研究设计：我们将采用具有锰外排遗传缺陷的细胞模型， 转运蛋白SLC 30 A10，其足以在人类中诱导帕金森综合征，以及 急性和慢性锰处理的细胞，以鉴定线粒体中的锰结合蛋白 并通过质谱方法分析线粒体RNA颗粒的组成。 RNA颗粒的功能将通过分析线粒体多顺反子的加工来研究。 用探针定向分子计数法检测锰攻击细胞中的线粒体RNA 在转录本中未处理的连接处。通过RNA颗粒的敲除和过表达 因此，我们将评估线粒体RNA颗粒功能障碍是有害的还是适应性的。 我们将询问人脑类器官和小鼠脑组织的分子机制 在细胞系模型中识别。鉴于RNA颗粒对线粒体的显著潜在影响， 功能，我们将评估药物是否影响线粒体RNA加工和下游 锰代谢对过量锰暴露具有神经保护作用或加剧遗传脆弱性。

Summary: This application focuses on a novel pathogenic mechanism that includes metal homeostasis, mitochondrial proteostasis and onset of neurological adverse effects for cognition and movement. We have identified the mitochondrial RNA granule, as a key initiator of neuronal responses to environmental and/or genetic insults that predispose an individual to disease. This RNA processing pathway in mitochondria resides upstream of the effects that have been attributed to manganese toxicity, including reduced respiratory chain activity, mitochondrial membrane depolarization, mitochondrial oxidative stress, leading to cell death. Our overall hypothesis is that manganese accumulation in mitochondria disrupts the mitochondrial RNA granule function and induces dsRNA accumulation as part of the toxicity mechanism. This disruption leads to the accumulation of dsRNA and reduced OXPHOS function and increased oxidative stress. Thus, the mitochondrial RNA granule offers us a molecular reporter of exposure and sensitivity to manganese and a potential target for neuroprotective interventions. Specific Aims: Aim 1: Identify molecular targets of manganese in mitochondria. Aim 2: Evaluate whether manganese-induced mitochondrial dysfunction induces dsRNA accumulation and pro-inflammatory responses. Aim 3: To test to what degree impaired mitochondrial RNA granule function modulates manganese sensitivity in human brain organoids and brain from wild type and SLC30a10 KO mouse. Study Design: We will employ cell models with genetic deficiencies in the manganese efflux transporter SLC30A10, which is sufficient to induce a parkinsonian syndrome in humans, as well as acutely and chronically manganese treated cells to identify Manganese binding proteins in mitochondria and to analyze the composition of the mitochondrial RNA granule by mass spectrometry approaches. RNA granule function will be studied by analyzing the processing of the mitochondrial polycistronic mitochondrial RNA in manganese challenged cells employing molecular counting with probes directed at unprocessed junctions in the transcripts. By knockout and over-expression of RNA granule components, we will assess whether mitochondrial RNA granule dysfunction is deleterious or adaptive. We will interrogate human brain organoids and mouse brain tissue for the molecular mechanisms identified in cell line models. Given the significant potential impact of RNA granule on mitochondrial function, we will evaluate whether drugs affecting mitochondrial RNA processing and downstream metabolism are neuroprotective to excess manganese exposure or exacerbate genetic vulnerability.