Non-canonical mechanisms of excitotoxicity

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PROJECT SUMMARY This project addresses the underlying mechanisms that progress ischemic damage of vulnerable tissue that surround acute brain injuries such as stroke. Our long-term goal is to identify the specific mechanisms that transition metabolically compromised tissue to damaged, pro-death penumbra and to develop clinically relevant therapeutic targets to improve patient survival after brain injury. While understanding of ischemic methods of neuronal injury has been the primary focus, exploring specific mechanisms of consequence and potential therapeutic targets has lagged far behind. This project looks specifically at Spreading Depolarizations (SDs) which have recently been identified as contributing significantly to the progression of injury in vulnerable penumbra. The main focus of these events has been on their contribution to prolonged NMDA-mediated Ca2+ influx and subsequent damage. However, SDs present a much more complex and underexplored surge of pre- synaptic cation release and post-synaptic uptake through alternate pathways. Specifically both post-synaptic activation of voltage-gated Ca2+ channels and increase in extracellular Zn2+ release and uptake have been linked to the initiation and subsequent propagation of SD. Our central hypothesis is that SD-induced injury progression in metabolically depleted tissue is mediated by dysregulation of non-NMDA-centric cation homeostasis. Furthermore, agents that are selective to reduce alternative Ca2+ channel activation and/or decrease post- synaptic Zn2+ uptake will reduce the downstream mediated damage that occurs following SD. We will use brain slice and animal models to explore Zn2+ and Ca2+ specific mechanisms of injury as well as pharmacological intervention to support compromised tissue during and after onset of SD. Specific Aim 1 focuses on the hypothesis that neuronal voltage-gated Ca2+ channels contribute to post-synaptic uptake of intracellular Ca2+ and lead to downstream cell execution. Neuronal Ca2+ loading will be assessed using a specific genetically modified model and pharmacological intervention will be used to assess recovery in a metabolically compromised tissue setting. Specific Aim 2 tests the hypothesis that disruption of Zn2+ homeostasis contributes to mechanisms of SD-induced injury in vulnerable setting. Zn2+ wave in vulnerable tissue and specific stores of Zn2+ will be assessed to explore where damaging levels are released. Specific Aim 3 then assess these mechanisms in an in vivo setting. In all aims, both electrophysiological and imaging techniques will be used to assess specific mechanisms in brain slice (Aim 1 and 2) and then translate into in vivo model (Aim 3). Pharmacological intervention and specific knockdown models will explore where these cations contributions to damage and identify where damaging levels of these cations originate. Completion of these aims should ascertain specific mechanisms of Ca2+ and Zn2+-mediated injury following SD in vulnerable tissue and propose potential targets for clinical prevention paradigms.