Astrocytes are the most abundant cell type in brain. They are responsible for clearing extracellular glutamate, the predominant excitatory neurotransmitter, from the synapse to maintain crisp signaling and prevent excitotoxicity. In forebrain, the astrocytic glutamate transporter, GLT1, is responsible for the vast majority of glutamate uptake. Mitochondria are invested throughout fine astrocytic processes where they colocalize with GLT1. We recently discovered physical and functional interactions between GLT1, multiple glycolytic enzymes and mitochondria. In completing Aim 1 of my original grant submission, we concluded that mitochondria in astrocytic processes are retained near glutamate transporters and synapses. Our data suggest that this distribution is regulated by neuronal glutamate release, astrocytic glutamate uptake, and reversal of the Na+/Ca2+ exchanger. Mitochondria can support glutamate uptake by providing ATP and buffering ions, and there is growing evidence suggesting that a portion of transported glutamate is oxidized in mitochondria to generate energy in these compartments. Mitochondrial dysfunction and excitotoxicity from failure of astrocytic glutamate uptake are at the core of the delayed cell deat that persists after an ischemic insult. Aside from inducing hypothermia, this pathology is currently untreatable. I have observed a loss of mitochondrial density in astrocytic processes in response to oxygen glucose deprivation (OGD) that precedes the delayed neuronal cell death that is common to this ex vivo model and to stroke in vivo. In the first aim I will characterize th OGD-induced loss of mitochondria from astrocytic processes, and determine if it is preceded by changes in mitochondrial membrane potential and reactive oxygen species. I will pharmacologically block or activate NMDA receptors to investigate the relationship between excitotoxicity and reduced mitochondrial occupancy of astrocytic processes. In a preliminary study, I found that cyclosporin A reduced cell death and attenuated the loss of mitochondrial density in astrocytic processes after OGD. Cyclosporin A increases mitochondrial capacity for calcium buffering. Treatment with cyclosporin A will be evaluated as mechanism of intervention for attenuating the loss of mitochondria from processes. I have also observed increased mitophagy (a mechanism for degradation of dysfunctional mitochondria) in astrocytic processes after OGD.
In aim two I will characterize changes in mitophagy after OGD. I will also evaluate the effects of pharmacological and genetic inhibition of mitophagy on mitochondrial occupancy of astrocytic processes and delayed neuronal death after OGD. By providing the first ever examination of the role of mitochondrial dynamics in astrocytic processes during ischemic injury, execution of this project could help lead to new therapeutic targets for a field of medicine that desperately needs them.
Mitochondrial dysfunction and failure of astrocytic glutamate clearance are core contributors to damage from stroke that continues for days or even weeks after the initial injury. Aside from inducing hypothermia, there are currently no treatments to attenuate this damage. This project will for the first time examine the role of astrocytic mitochondrial dynamics in ischemic injury to identify desperately needed therapeutic targets for reducing damage after stroke.