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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01NS056244-07
Application #
8619667
Study Section
Neural Oxidative Metabolism and Death Study Section (NOMD)
Program Officer
Bosetti, Francesca
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Iowa
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Iowa City
State
IA
Country
United States
Zip Code
52242
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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
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
Merrill, Ronald A; Strack, Stefan (2014) Mitochondria: a kinase anchoring protein 1, a signaling platform for mitochondrial form and function. Int J Biochem Cell Biol 48:92-6
Wilson, Theodore J; Slupe, Andrew M; Strack, Stefan (2013) Cell signaling and mitochondrial dynamics: Implications for neuronal function and neurodegenerative disease. Neurobiol Dis 51:13-26
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
Merrill, Ronald A; Slupe, Andrew M; Strack, Stefan (2013) N-terminal phosphorylation of protein phosphatase 2A/Bβ2 regulates translocation to mitochondria, dynamin-related protein 1 dephosphorylation, and neuronal survival. FEBS J 280:662-73
Slupe, Andrew M; Merrill, Ronald A; Flippo, Kyle H et al. (2013) A calcineurin docking motif (LXVP) in dynamin-related protein 1 contributes to mitochondrial fragmentation and ischemic neuronal injury. J Biol Chem 288:12353-65
Oberg, Elizabeth A; Nifoussi, Shanna K; Gingras, Anne-Claude et al. (2012) Selective proteasomal degradation of the B'β subunit of protein phosphatase 2A by the E3 ubiquitin ligase adaptor Kelch-like 15. J Biol Chem 287:43378-89

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