Spreading Depolarization in Relation to Cytotoxic Edema and Cell Death

Spreading Depolarization (SD) is associated with migraine aura and is recognized as a novel mechanism of injury in stroke and brain trauma patients. SDs are waves of sustained depolarization of neurons and glia that propagate the breakdown of transmembrane ion gradients, distortion of synaptic circuitry, and cytotoxic edema. Yet, the fundamental question of the molecular mechanism of rapid water entry into depolarized neurons remains an enigma. Passive osmotically obligated water flux following cations influx during SD is problematic because pyramidal neurons are highly resilient to osmotic swelling due to a lack of aquaporins in their membrane. Based on our preliminary data and the literature, it is plausible that volume-regulated Cl-/anion channels (VRAC) are involved in SD-induced neuronal swelling and recovery. In neurons lacking aquaporins, VRAC may either promote neuronal swelling during strong depolarization as a route for swelling-aggravating Cl- influx or assist in neuronal volume recovery during repolarization, providing a conduit for Cl- efflux. Thus, by serving as a major anionic pathway, VRAC plays a dual reciprocal role in neuronal volume regulation, and it conducts water. Aim 1 will reveal the role of VRAC in SD-induced neuronal edema and recovery. SD could lead to cell death in the energy-deprived cortex, but not all neurons die. Little is known about the cause of this variability across depolarized but viable neurons. It is feasible that the variation in the increased levels and duration of mitochondrial Ca2+ during SD could underlie this variability. However, in vivo mitochondrial Ca2+ levels in SD were never quantified, and cells were never followed in real-time until their death or recovery. Aim 2 will address these unresolved questions. Blood and plasma are released into the brain parenchyma during neurologic emergencies, and even without SD, many blood components can contribute to cell injury. The role of excitatory amino acids in triggering excitotoxicity cascades has been extensively studied. Surprisingly, our novel results reveal that non-excitatory amino acids induce severe damage to neurons in hypoxic brain tissue. Astroglial VRAC appears to mediate this injury, and this hypothesis will be tested in Aim 3. The specific aims are: 1) To test the hypothesis that the activation of neuronal VRAC is the mechanism implicated in SD-induced neuronal swelling and recovery. 2) To test the hypothesis that the increase in mitochondrial Ca2+ caused by SD is the mechanism underlying the “commitment point” marking the switch between cell death and recovery from SD. 3) To test the hypothesis that astroglial VRAC activity mediates neuronal injury by non-excitatory amino acids during hypoxic- ischemic conditions. Various classic and state-of-the-art technologies such as viral expression, mouse genetics, intravital imaging, and in vivo FRET-based 2-photon quantitative mitochondrial Ca2+ imaging will be used while simultaneously monitoring the occurrence of SD with electrophysiology. When applicable, intravital 2-photon imaging will be followed by ultrastructural analyses with serial section transmission electron microscopy. The results will bring new insight into mechanisms of acute cellular injury in SD-associated neurologic emergencies.