Abstract
Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP3Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes.
Highlights
Mitochondria are crucial to intracellular Ca2 þ homeostasis and accumulation of Ca2 þ induced by excitotoxic insults leads to mitochondrial membrane depolarization, increased production of oxygen free radicals and caspase-dependent or -independent oligodendrocyte death.[6]
These results indicate that the three isoforms of IP3Rs and releasing Ca2 þ mainly through ryanodine (RyR) are present in our in vitro oligodendrocytes derived from rat optic nerve
Inhibition of IP3Rs by 2APB (10 mM) did not attenuate [Ca2 þ ]i significantly (Figure 2c). These results indicate that Ca2 þ release through RyRs but not through IP3Rs contributes to AMPA-induced cytosolic Ca2 þ overload and excitotoxicity in oligodendrocytes
Summary
Mitochondria are crucial to intracellular Ca2 þ homeostasis and accumulation of Ca2 þ induced by excitotoxic insults leads to mitochondrial membrane depolarization, increased production of oxygen free radicals and caspase-dependent or -independent oligodendrocyte death.[6] In turn, the endoplasmic reticulum (ER) is critical to Ca2 þ homeostasis and its stress contributes to demyelinating disorders.[7] ER serves as a rapidly exchanging Ca2 þ store and contributes to the cytosolic Ca2 þ signalling cascade by releasing Ca2 þ mainly through ryanodine (RyR) and inositol triphosphate (IP3R) receptors.[8] The RyR family has three isoforms, all expressed in the brain, and has multiple allosteric Ca2 þ -binding sites responsible for triggering Ca2 þ -induced Ca2 þ release (CICR) to the cytosol.[9] IP3Rs (isoforms I, II and III) are expressed in the brain and activated by Ins(1,4,5)P3, a metabolic product of phospholipase C (PLC) activity, and are regulated by IP3-independent pathways.[10] These RyR/IP3R have a central role in cell survival as well as in apoptotic cell death.[11]. We show that a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor activation induces ER stress and the subsequent activation of caspase-12, as well as cytosolic Ca2 þ overload and mitochondrial dysfunction, which are all dependent on ER-Ca2 þ release through RyRs
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