Date of Award
Doctor of Philosophy (PhD)
Michael B. Robinson
GLUTAMATE TRANSPORT AFFECTS MITOCHONDRIA AND CALCIUM SIGNALING IN ASTROCYTIC PROCESSES UNDER NORMAL AND PATHOLOGICAL CONDITIONS
John Charles O’Donnell
Michael B. Robinson
Mitochondria are responsible for synthesis and metabolism of the primary excitatory neurotransmitter, glutamate, which is cleared from synapses via Na+-dependent transporters on astrocytes. Astrocytic clearance of glutamate is required to prevent excitotoxic neuronal death. Mitochondria also participate in calcium signaling in various cell types. Astrocytic calcium signaling is implicated in neurovascular coupling. Glutamate transport and calcium signaling are central to the function of astrocytic processes that are in turn vital for normal brain function. We recently confirmed that mitochondria are present throughout astrocytic processes. Using confocal microscopy and hippocampal slice cultures along with a variety of biochemical assays, we sought to elucidate the physiological and pathological interactions between mitochondria, glutamate transport, and calcium signaling in astrocytic processes. We found that glutamate uptake is reduced after displacing hexokinase from the voltage-dependent anion channel on the outer mitochondrial membrane, but coimmunoprecipitations between transporter and mitochondrial proteins are not changed. As in neurons, we found that some astrocytic mitochondria are mobile. We provide evidence that neuronal activity, activation of astrocytic glutamate transporters, and subsequent reversal of the Na+/Ca2+ exchanger leads to immobilization of mitochondria near transporters and synapses, where they can oxidize glutamate, buffer ions, and provide ATP. Finally, I found that following transient oxygen/glucose deprivation (a model of ischemia/reperfusion injury), mitochondria in astrocytic processes undergo fragmentation and autophagic degradation, culminating 24 h after insult with an ~50% reduction in mitochondrial size and the percentage of process length occupied by mitochondria. This loss of mitochondria is independent of the accompanying excitotoxic neuropathology, and seems to instead be driven by an extended period of high glutamate uptake. I also identified a previously overlooked distinction between Ca2+ signals in astrocytic processes, showing two populations with different properties based on their anatomical relationship to mitochondria. These Ca2+ signals were greatly increased after mitochondrial loss and were no longer spatially restricted by the remaining mitochondria. In summary, we found that glutamate transport positions mitochondria at sites of activity in astrocytic processes where they shape calcium signals; but glutamate uptake under excitoxic conditions leads to mitochondrial loss and dramatically altered calcium signaling, potentially impacting neuronal injury and recovery.
O'donnell, John Charles, "Glutamate Transport Affects Mitochondria And Calcium Signaling In Astrocytic Processes Under Normal And Pathological Conditions" (2016). Publicly Accessible Penn Dissertations. 2504.
Available for download on Monday, January 13, 2020