A growing interest has been recently devoted to the role of intracellular Ca2+ stores in brain ischemia. For instance, disturbances of Ca2+ content in the endoplasmic reticulum (ER) has been reported as one of the main mechanisms underlying the neurological disease. Interestingly, lysosomes are emerging as other important Ca2+-storing organelles, cooperating with ER in the handling of intracellular Ca2+ concentration ([Ca2+]i). One of the main regulators of lysosomal Ca2+ level is represented by Mucolipin TRP channel 1 (TRPML1), a non-selective cation channel releasing lysosomal Ca2+ into the cytosol. Here we investigated the role of ER/lysosome Ca2+ coupling and the contribution of TRPML1 in brain ischemia. Our results showed that under physiological conditions TRPML1 activation induced by its specific agonist ML-SA1 or by lysosomal v-ATPase inhibitor bafilomycin A1 significantly increased [Ca2+]i in cortical neurons. ML-SA1-induced Ca2+ leak from lysosomes strongly reduced ER Ca2+ content, whereas theTRPML1 inhibitor trans-Ned19 or channel knocking down increased ER Ca2+ level. However, this interplay was disrupted under hypoxic conditions produced by exposing cortical neurons to oxygen and glucose deprivation (OGD) followed by reoxygenation (Rx). Indeed, during OGD/Rx both ER and lysosomal Ca2+ levels were significantly impaired. Interestingly, the administration of trans-Ned19 during the Rx phase prevented dysfunctional lysosomal Ca2+ homeostasis and neuronal death. In consideration to the role played by lysosomes in autophagy regulation, we showed that trans-Ned19 hampered the autophagic flux during hypoxia thus protecting neurons. Collectively, our data demonstrate that a disruption of the functional interplay between lysosomes and ER underlied neuronal loss occurring in brain ischemia.
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