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 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 i 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, bioenergetics, Drp1 phosphorylation, and injury severity following focal ischemia. SA2 examines the role of AKAP1-interacting protein phosphatases (PP1, CaN) in mitochondrial remodeling and neuronal survival. Finally, SA3 proposes to elucidate molecular mechanisms of CaN recruitment to Drp1 in calcium-mediated mitochondrial fission and ischemic death. 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 Drp1 at S656. The proposed studies will increase our mechanistic understanding of mitochondrial fragmentation and its regulation by reversible phosphorylation in neurons, which may lead to better therapies for neurodegeneration in stroke and disease.
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. Results may lead to better treatments for stroke and neurodegenerative disorders.
|Nguyen, Emily K; Koval, Olha M; Noble, Paige et al. (2018) CaMKII (Ca2+/Calmodulin-Dependent Kinase II) in Mitochondria of Smooth Muscle Cells Controls Mitochondrial Mobility, Migration, and Neointima Formation. Arterioscler Thromb Vasc Biol 38:1333-1345|
|Flippo, Kyle H; Gnanasekaran, Aswini; Perkins, Guy A et al. (2018) AKAP1 Protects from Cerebral Ischemic Stroke by Inhibiting Drp1-Dependent Mitochondrial Fission. J Neurosci 38:8233-8242|
|Flippo, Kyle H; Strack, Stefan (2017) Mitochondrial dynamics in neuronal injury, development and plasticity. J Cell Sci 130:671-681|
|Ji, Wei-Ke; Chakrabarti, Rajarshi; Fan, Xintao et al. (2017) Receptor-mediated Drp1 oligomerization on endoplasmic reticulum. J Cell Biol 216:4123-4139|
|Mapuskar, Kranti A; Flippo, Kyle H; Schoenfeld, Joshua D et al. (2017) Mitochondrial Superoxide Increases Age-Associated Susceptibility of Human Dermal Fibroblasts to Radiation and Chemotherapy. Cancer Res 77:5054-5067|
|Flippo, Kyle H; Strack, Stefan (2017) An emerging role for mitochondrial dynamics in schizophrenia. Schizophr Res 187:26-32|
|Gupte, Raeesa P; Kadunganattil, Suraj; Shepherd, Andrew J et al. (2016) Convergent phosphomodulation of the major neuronal dendritic potassium channel Kv4.2 by pituitary adenylate cyclase-activating polypeptide. Neuropharmacology 101:291-308|
|Hatch, Anna L; Ji, Wei-Ke; Merrill, Ronald A et al. (2016) Actin filaments as dynamic reservoirs for Drp1 recruitment. Mol Biol Cell 27:3109-3121|
|Ji, Wei-ke; Hatch, Anna L; Merrill, Ronald A et al. (2015) Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites. Elife 4:e11553|
|Jin, Zhigang; Chung, Jin Wei; Mei, Wenyan et al. (2015) Regulation of nuclear-cytoplasmic shuttling and function of Family with sequence similarity 13, member A (Fam13a), by B56-containing PP2As and Akt. Mol Biol Cell 26:1160-73|
Showing the most recent 10 out of 26 publications