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

Astrocyte 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.1.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.We 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.Our 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. References: 1.Sompol P. et al. Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in Aβ-Bearing Mice. J Neurosci. 2017 Jun 21;37(25):6132-6148. 2.Javed, H., Azimullah, S., Abul Khair, S.B. et al. Neuroprotective effect of nerolidol against neuroinflammation and oxidative stress induced by rotenone. BMC Neurosci 17, 58 (2016). https://doi.org/10.1186/s12868-016-0293-4. 3.Lin Z. et al. miR-23a functions downstream of NFATc3 to regulate cardiac hypertrophy. Proc Natl Acad Sci U S A. 2009 Jul 21;106(29):12103-8.