Role of N6-Methyladenosine (m6A) epitranscriptomic mechanisms in manganese-induced astrocyte inflammation

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Malovic, Emir
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Kanthasamy, Anumantha G
Sashital, Dipali
Thippeswamy, Thimmasettapp
Tuteja, Geetu
Mair, Gunnar
Jernigan, Robert
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Interdisciplinary Graduate Studies
Manganese (Mn) is considered an environmental toxicant, known to cause a type of Parkinsonism called manganism. First observed in Mn ore crushers, the overexposure of Mn produces neurological signs and symptoms, predominantly motor impairment but can also elicit neuropsychiatric effects. The motor impairment is resulting from neuronal dysfunction and inflammation within the extrapyramidal motor circuitry of basal ganglia. Although these observations are known, it remains unclear as to how the etiological process ensues. The death of globus pallidus neurons within basal ganglia (neurodegeneration) is intimately linked to the signs and symptoms, but how and when these neurons begin to die is a more complex inquiry. Astrocytes are considered the other major players, glial cells tasked with numerous homeostatic functions in the central nervous system such as blood-brain barrier regulation, reuptake and recycling of neurotransmitters such as glutamate and GABA, and even maintenance of synapses. Astrocytes are also known to be potent inflammatory glia, neurotoxic in certain diseases and disorders. In manganese toxicity, evidence for preferential accumulation in astrocytes over other cell types like neurons exists, especially in vitro data, but in vivo data is remains unclear, mostly because of the inefficiencies of known methodologies and insufficient cell-specific quantitative measures. Nonetheless, it would stand to reason astrocytes could present as crucial candidates in the etiological process of manganese neurotoxicity. As such, investigations into astrocytic inflammation have surged in the manganese toxicological discipline. Herein, we explore novel molecular mechanisms by which astrocytes may elicit their pro-inflammatory responses to provide the field momentum in the deeper exploration of specific avenues that may eventually lead to a better understanding of manganism etiology. The first mechanism, chapter 2, attempts to evidentially bridge the connection between mitochondrial dysfunction and pro-inflammatory responses by evaluating the efficacy of mito-apocynin, a mitochondria-targeting NOX inhibitor, in preventing mitochondrial dysfunction. Our results suggest that preventing mitochondrial dysfunction may attenuate the exerted pro-inflammatory responses to Mn. In chapters 3 and 4, we explore a relatively unexplored avenue at the time of this writing, namely that of N6-methyladenosine (m6A) epitranscriptomics and its regulatory tiers that control the writing, erasing, and reading of m6A modified RNAs. We find that the global m6A modifications are significantly affected in Mn exposed astrocytes. More specifically, our findings suggest Mn perturbs the reader and eraser regulatory tiers, in which we find YTHDF2, the m6A decay-promoting reader, decreases upon Mn exposure, while ALKBH5 and FTO, two m6A demethylases, are increased. Our deeper inquiry into YTHDF2 function revealed that it could act as a potential anti-inflammatory protein in Mn exposed astrocytes by negatively regulating the MAP2K4 (SEK1)-JNK-JUN pathway. The evidence suggested YTHDF2 could decrease total SEK1 levels to severely reduce the downstream activation of JNK and cJUN phosphorylation of Mn exposed astrocytes, and thus, attenuating their pro-inflammatory, proliferative, and survival signaling responses. Collectively, the exploration of this dissertation suggests astrocytes have multiple mechanisms that are requisite for their pro-inflammatory responses in Mn toxicity, in which m6A reader, YTHDF2, could be exploited to exert a potent control over their gene expression responses that eventually affect their protein synthesis and subsequent signaling. Because of the observed potency YTHDF2 exerts upon astrocyte gene expression and overall survival, this research endeavor provides an enticing exploratory avenue for other neurodegenerative and neuroinflammatory conditions where astrocytes have major roles, especially Parkinson’s disease, for which Mn has been posited as a risk factor.