Ca2+/Calcineurin‐mediated release of exosomal miR‐23a regulates astrocyte‐neuron cross‐talk and protects against neuroinflammation and neurodegeneration

Abstract

AbstractBackgroundAstrocyte dysfunction and associated neuroinflammation is well implicated in various neurodegenerative disorders like Alzheimer’s (AD) and Parkinson’s diseases (PD). Importantly, Ca2+/Calcineurin signaling is reported to induce astrocyte dysfunction in AD (1) but very little is known regarding PD. So, our objective was to study the role of Ca2+/Calcineurin signaling in astrocyte‐neuron cross‐talk during PD‐related neuroinflammation which is unreported till date.Method1.Mammalian cell culture 2.Immunoblotting. 3.qRT‐PCR. 4.Exosome isolation. 5.Intracellular Ca2+ measurement. 6.Transfection (miRNA mimics). 7. 3’UTR cloning. 8.Luciferase assay.ResultWe treated human astrocytic 1321N1 cells with Rotenone, a neuro‐toxin known to induce PD‐associated neuroinflammation (2). At the dose of Rotenone which caused about 40% death in 24 hrs, simultaneous activation of the Ca2+‐dependent protein phosphatase Calcineurin and intracellular surge in the Ca2+ levels were also observed. Now, miR‐23a is an anti‐apoptotic miRNA and Calcineurin/NFAT‐mediated upregulation of miR‐23a has been reported in cardiac hypertrophy (3). Surprisingly, in our study, Rotenone caused a Calcineurin‐mediated decrease of intracellular miR‐23a levels in astrocytes. Now this decrease, we found, was due to a concomitant release of miR‐23a via exosomes. Can this anti‐apoptotic miR‐23a released by astrocytes protect the neurons? To answer this, we treated neuronal SH‐SY5Y cells with Rotenone and found that the pro‐apoptotic protein PUMA was upregulated although the miR‐23a levels remained unchanged. Interestingly, over‐expression of miR‐23a caused attenuation of the Rotenone‐mediated death in neurons. PUMA being a predicted target of miR‐23a, we cloned the 3’UTR of PUMA and subsequent luciferase assay revealed that indeed PUMA is a direct target of miR‐23a under our experimental conditions.ConclusionOur results suggest a novel mechanism by which Ca2+/Calcineurin‐mediated release of miR‐23a through exosomes regulates astrocyte‐neuron cross‐talk and plays a probable role in protection against neuroinflammation and neurodegeneration.