There is an urgent need to improve upon the safety and treatment window of current stroke therapies. Mitochondria play key roles during both early (minutes) and late (days) stages of ischemic brain injury, and are thus recognized as promising targets for neuroprotective therapy. Mitochondrial architecture, as determined by opposing fission and fusion reactions, has recently emerged as a critical determinant for survival of both neuronal and non-neuronal cells. Mitochondrial fission catalyzed by the mechanoenzyme dynamin-related protein 1 (Drp1) facilitates cytochrome C release and apoptosis, in part through promoting Bax oligomerization. In addition, mitochondria fragment during stroke and pathological Drp1 activation occurs in neurodegenerative disorders. On the other hand, we and others have shown that fusion of mitochondria into an interconnected network has a neuroprotective effect, which may involve increased energy production, ROS and calcium sequestration, and sparing of the organelle from autophagic degradation. Despite the widely appreciated disease relevance of mitochondrial dynamics, our understanding of regulatory mechanisms controlling mitochondrial architecture is still in its infancy. In the previous funding cycle, we identified a pivotal phosphorylation site in Drp1. Conserved in all metazoans, S656 is phosphorylated by protein kinase A (PKA) to inhibit the fission enzyme, leading to unopposed fusion of mitochondria. Opposite PKA is the calcium-dependent protein phosphatase calcineurin (CaN), which dephosphorylates S656 to promote mitochondrial fragmentation. Phospho-Drp1 protects from, while dephospho-Drp1 sensitizes PC12 cells to apoptosis. We also uncovered a potent neuroprotective activity of mitochondria-localized A kinase anchoring protein 1 (AKAP1) in hippocampal neurons, which is mediated by Drp1 phosphorylation at S656 and stabilization of the mitochondrial network. Intriguingly, a PKA binding- deficient AKAP1 mutant fragmented mitochondria, suggesting that some of the signaling molecules reported to also associate with AKAP1 may oppose mitochondrial stabilization by PKA. We propose to continue with this line of inquiry in three specific aims (SA). In SA1, we will characterize AKAP1 knockout mice for changes in mitochondrial morphology, Drp1 phosphorylation, and injury severity following focal ischemia. We also propose to examine the role of other AKAP1-interacting proteins (PP1, CaN) in mitochondrial remodeling and neuronal survival. SA2 proposes to elucidate molecular mechanisms of CaN recruitment to Drp1 in calcium-mediated mitochondrial fission and excitotoxic/ischemic death. Finally, SA3 explores regulation of the recently discovered Drp1 adaptor protein Mff (mitochondrial fission factor) by alternative splicing and phosphorylation. The proposal addresses the overall hypothesis that AKAP1 assembles a signalosome at the outer mitochondrial membrane, which integrates death and survival signals from the cytosol and from within mitochondria to restructure the organelle via reversible phosphorylation of the Drp1::Mff fission complex. The proposed studies will increase our understanding of mitochondrial fragmentation and its regulation by reversible phosphorylation in neurons, which may lead to better therapies of neurodegeneration in stroke and other disorders of the brain.
Mitochondria produce energy, buffer calcium, and are often dysregulated when the brain is damaged by disease or injury, such as ischemic stroke. Mitochondrial dysregulation is frequently associated with fragmentation carried out by the enzyme Drp1. This proposal seeks to identify regulatory mechanisms that converge on Drp1 and its receptor protein in the outer mitochondrial membrane, Mff. Results may lead to better treatments for stroke and neurodegenerative disorders.
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|Strack, Stefan; Wilson, Theodore J; Cribbs, J Thomas (2013) Cyclin-dependent kinases regulate splice-specific targeting of dynamin-related protein 1 to microtubules. J Cell Biol 201:1037-51|
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