Mitochondrial mechanisms and signaling in manganese exposure

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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.