UC Irvine

Ischemic stroke is a major cause of death and disabilities worldwide, and it has been long hoped that improved understanding of relevant injury mechanisms would yield targeted neuroprotective therapies. While Ca2+ overload during ischemia-induced glutamate excitotoxicity has been identified as a major contributor, failures of glutamate targeted therapies to achieve desired clinical efficacy have dampened early hopes for the development of new treatments. However, additional studies examining possible contributions of Zn2+, a highly prevalent cation in the brain, have provided new insights that may help to rekindle the enthusiasm. In this thesis, we discuss findings yielding clues as to sources of the Zn2+ that accumulates in many forebrain neurons after ischemia, and mechanisms through which it mediates injury, specifically highlighting the growing evidence of important Zn2+ effects on mitochondria. First, using brief high (90 mM) K+/Zn2+ exposures to mimic neuronal depolarization and extracellular Zn2+ accumulation as may accompany ischemia in vivo, we examined effects of disrupted cytosolic Zn2+ buffering and/or the presence of Ca2+, and made several observations: 1. Mild disruption of cytosolic Zn2+ buffering—while having little effects alone—markedly enhanced mitochondrial Zn2+ accumulation and dysfunction (including loss of ∆Ψm, ROS generation, swelling and respiratory inhibition) caused by relatively low (10 – 50 µM) Zn2+ with high K+. 2. The presence of Ca2+ during the Zn2+ exposure decreased cytosolic and mitochondrial Zn2+ accumulation, but markedly exacerbated the consequent dysfunction. 3. Paralleling effects on mitochondria, disruption of buffering and presence of Ca2+ enhanced Zn2+-induced neurodegeneration. We also use recently available MCU knockout mice to examine how the deletion of this channel impacts deleterious effects of Zn2+. In cultured cortical neurons from MCU knockout mice, we find significantly reduced mitochondrial Zn2+ accumulation. Correspondingly, these neurons were protected from both acute and delayed Zn2+-triggered mitochondrial dysfunction, including mitochondrial reactive oxygen species generation, depolarization, swelling and inhibition of respiration. Furthermore, when toxic extramitochondrial effects of Ca2+ entry were moderated, both cultured neurons (exposed to Zn2+) and CA1 neurons of hippocampal slices (subjected to prolonged oxygen glucose deprivation to model ischemia) from MCU knockout mice displayed decreased neurodegeneration. Finally, to examine the therapeutic applicability of these findings, we added a Zn2+ chelator or MCU blocker after Zn2+ exposure in wildtype neurons (to induce post-insult MCU blockade). This significantly attenuated the delayed evolution of both mitochondrial dysfunction and neurotoxicity. Taken together, these data support the hypothesis that mitochondrial Zn2+ accumulation is a critical contributor to ischemic neurodegeneration that could be targeted for neuroprotection.