Mitochondrial mechanisms and signaling in manganese exposure

摘要： 本申请的重点是一种新的致病机制，包括金属动态平衡， 线粒体蛋白稳定和神经系统对认知和运动的不利影响的开始。我们 已经确定线粒体rna颗粒是神经元对环境反应的关键启动者。 和/或使个人易患疾病的基因侮辱。这条RNA加工途径在 线粒体位于锰中毒效应的上游，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，线粒体中的锰积累 破坏线粒体rna颗粒的功能，并诱导dsrna积累，作为毒性的一部分。 机制。这种干扰导致dsRNA的积累和OXPHOS功能的降低 氧化应激增加。因此，线粒体RNA颗粒为我们提供了一个分子报告程序 暴露和对锰的敏感性，以及神经保护干预的潜在目标。 具体目标： 目的1：确定线粒体中锰的分子靶点。 目的2：评价锰诱导的线粒体功能障碍是否导致dsRNA积聚 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、野生型和SLC30a10KO小鼠的脑内。 研究设计：我们将采用锰外排中存在遗传缺陷的细胞模型 转运蛋白SLC30A10，这足以在人类中诱导帕金森综合症，以及 急性和慢性锰处理细胞鉴定线粒体中的锰结合蛋白 并用质谱仪分析线粒体RNA颗粒的组成。 通过分析线粒体多顺反子的加工过程来研究RNA颗粒的功能 用直接探针分子计数法检测锰刺激细胞的线粒体RNA 在成绩单中未处理的结合点。通过敲除和过度表达RNA颗粒 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。 我们将询问人脑有机化合物和小鼠脑组织的分子机制 在细胞系模型中被识别。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的加工和下游 代谢对过量的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.