Melissa Trotman-Lucas, PhD
Neuroprotective treatments aim to prevent cellular death, particularly neuronal cell death, during ischemic/hypoxic conditions such as in acute ischemic stroke. Maintenance of neuroprotection long-term requires a return of blood and oxygen to the ischemic territory area, not regularly achieved in previous clinical trials. Yet, with recanalization rates of greater than 80%, afforded by the advent of mechanical thrombectomy following ischemic stroke, the return of flow to the ischemic territory is now more consistent. Nevertheless, following the return of blood flow to this area, cellular damage continues to spread and grow due to the phenomenon of “ischemic-reperfusion injury.” This progressive increase in damage indicates a continued need to investigate neuroprotective treatments that could be used alongside recanalization, both thrombolysis and thrombectomy, to recover/prevent long-term cellular damage.
It is well established that calcium (Ca2+) overload plays a key role in neuronal death during ischemic/hypoxic events. In addition to voltage-gated and receptor-mediated Ca2+ influx, there is a further route of entry into the cell, a route switched on in response to depleted intracellular Ca2+ stores. This additional entry route is called capacitative or store operated Ca2+ entry and is thought to contribute to the stabilization of both cytosolic Ca2+ concentration and intracellular Ca2+ stores.
Stegner et al. reported previously that capacitative Ca2+ entry (CCE) contributes to hypoxic neuronal cell death and that this is regulated by endoplasmic reticulum resistant Ca2+ sensor STIM2 (stromal interaction molecule 2). The channel mediating CCE is unsolved; therefore, the group has investigated what role the protein Orai2, known to form functional CCE channels alongside the other Orai isoforms 1 and 3, may play in the CCE-mediated contribution to ischemic cell death.
The findings show that the membrane located Orai2 mediates CCE in cortical and hippocampal neurons, regulated by STIM2. Also reported was that the absence of Orai2 leads to altered Ca2+ homeostasis within the cell. It appears the STIM2-Orai2 pathway is a major route for CCE in cerebral neurons, contrary to other non-excitable cells, where the STIM1-Orai1 axis mediates CCE. Neurons may rely on this disparity due to functional differences in Ca2+ homeostasis; they are less dependent on Ca2+ store refilling and have a higher activity associated basal cytosolic Ca2+ load alongside increased sensitivity to Ca2+ overload, whereas non-excitable cells undergo large changes in Ca2+ concertation and require rapid refilling of intracellular stores. The reliance on Orai2 over Orai1 in neurons may also act as a safeguarding mechanism against increased Ca2+ concentrations, due to Orai2’s fast Ca2+ dependent inactivation. When taken in combination with STIM2’s activation near resting endoplasmic reticulum Ca2+ levels and low efficiency for Orai activation, the STIM2-Orai2 pathway may provide fine-tuned regulation of Ca2+ for neurons compared to the STIM1-Orai1 pathway in non-excitable cells.
The group show that CCE is a critical factor in ischemic neuronal damage, making it an attractive neuroprotective target. Orai2 knockout (KO) mice showed injury protection during acute ischemia and from ischemia/reperfusion injury, these KO mice also showed improved functional outcome following reperfusion. Inhibition of neuronal CCE similarly resulted in reduced infarct, confirming the protective outcome and Ca2+ changes by in vitro cultured cells exposed to oxygen glucose deprivation. Yet, although the KO mice showed no morphological alterations, altered Ca2+ homeostasis or aberrant development in these mice may lead to cell vulnerability or ischemic preconditioning of cells that cannot be ruled out. Regardless, targeting Orai2-mediated CCE remains attractive as a novel therapeutic neuroprotective treatment.
It must be noted that use of the CCE inhibitor resulted in hemorrhagic transformation, an outcome not seen in the Orai2 KO mice using the same protocol, suggesting a role for the inhibitor used. This emphasizes the need for improved specificity of CCE targeting inhibitors, to better determine any potential neuronal protection afforded following administration at reperfusion. Administering a potential neuroprotective treatment during the reperfusion period would have clinical impact, allowing patients to be assessed and treated with thrombolysis treatment and still benefit from additional neuroprotective treatment. There are remaining questions surrounding CCE inhibition, and now particularly focused on Orai2-mediated CCE. With the advent of mechanical thrombectomy and that middle cerebral artery occlusion, in vivo, replicates the reperfusion surge seen in thrombectomy, this presents an opportunity to revisit pharmacological neuroprotective treatments as an avenue for clinical treatment. It’s a possible new focus for the field, and a drive towards potential transference from the bench to the bedside.