Background: Dysfunctional RNA binding proteins (RBPs) have been shown to contribute to the pathogenesis of neurodegeneration (NDG) in neurologic diseases. In Multiple Sclerosis (MS), the mechanisms of NDG are incompletely understood and the role that dysfunctional RBPs play in its pathogenesis have not been thoroughly explored. Our lab has shown pathogenic features of an RBP known as heterogeneous nuclear ribonucleoprotein A1 (A1) in neurons from MS patients. Hallmark features of A1 dysfunction include mislocalization of A1 from its homeostatic nuclear location to the cytoplasm and reduced nuclear expression, suggesting that loss of A1 nuclear function may contribute to the pathogenesis of NDG in MS. However, the exact mechanism of how A1 dysfunction may affect neuronal health is largely unknown.
Objectives: To determine whether decreased A1 nuclear expression, a characteristic of A1 dysfunction, impacts neuronal health and viability.
Methods: We established an in vitro model of decreased A1 nuclear expression using differentiated Neuro-2a cells (N2a; a neuronal cell line) and siRNA to A1 (siA1). Molecular consequences of decreased A1 expression were analyzed using immunostaining to assess neurite morphology and western blotting for the cell death markers, active caspase-3 (apoptosis) and phospho-mixed lineage kinase domain-like protein (p-MLKL; necroptosis). Cytotoxicity assays were performed to assess the effect of decreased A1 nuclear expression on cell viability and cytotoxicity.
Results: SiA1 entered neurons and significantly reduced A1 protein expression compared to siControl (p=0.0003; p<0.0001). SiA1 treated neurons showed a significant reduction in neurite branching and neurite length as compared to siControl (p<0.0001), as well as increased levels of p-MLKL (necroptosis; p=0.02), but no change in active caspase-3 levels (apoptosis). Cellular cytotoxicity assays revealed that decreased A1 nuclear expression resulted in increased cellular cytotoxicity as compared to siControl (p=0.04).
Conclusions: These findings suggest that A1 knockdown may result in cell death through the necroptotic pathway. Additionally, it suggests that there is a direct link between reduced A1 nuclear expression and decreased neuronal health and viability. Overall, this system models decreased A1 nuclear expression observed in MS brains and provides a relevant system to assess the consequences of A1 dysfunction on neuronal health and viability in the context of NDG.
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