Transient global ischemia in rodents (4 vessel occlusion, 4VO) and humans induces death of hippocampal CA1 neurons and is a model for delayed (non-necrotic) cell death after ischemic brain injury. Events that occur long before neuronal death include caspase activation, cleavage of anti-death Bcl-2 family proteins and large mitochondrial channel activity. We have found that the small molecule Bcl-xL inhibitor ABT-737, which enhances death of tumor cells by inhibition of Bcl-xL, paradoxically protects against neuronal death after 4VO in rats. Bcl-xL is prominently expressed in adult neurons and can be cleaved by caspases to generate a pro- death fragment, DeltaN-Bcl-xL that forms in the hippocampus after global ischemia. We find that ABT-737 administered before or after ischemia inhibits mitochondrial membrane channel activity and downstream cell death. Knock-in mice expressing a caspase-resistant form of Bcl-xL exhibit markedly reduced mitochondrial channel activity and reduced vulnerability to ischemia-induced neuronal death, establishing a causal role for DeltaN-Bcl-xL. We have recently described that, in addition to its role in the outer mitochondrial membrane, full length Bcl-xL also affects inner membrane processes by interacting directly with the F1FO ATP synthase, enhancing metabolic efficiency, increasing the rate of enzymatic activity and increasing inner membrane coupling. We further determined that the site of uncoupling is a leak channel made up of the c-subunit ring of the ATP synthase. We now suggest that the c-subunit leak channel forms the inner membrane component of the calcium-sensitive mitochondrial permeability transition pore (mPTP) and in this proposal we plan to test if, during delayed neuronal death after global ischemia, pro-apoptotic DeltaN-Bcl-xL binds to the F1FO ATP synthase causing uncoupling. In an in vitro model of delayed cell death caused by glutamate toxicity, we find that although high dose ABT-737 enhances cell death and worsens metabolic compromise, in contrast low concentrations of ABT-737 prevent such injurious changes, suggesting that low dose ABT-737 is sufficient for sequestration of DeltaN-Bcl-xL but not full length Bcl-xL. We will further test this hypothesis with an aim to determine how the interaction between DeltaN-Bcl-xL and the F1FO ATP synthase regulates ischemic cell death. We will attempt to define this interaction as a novel therapeutic target in ischemic brain injury.

Public Health Relevance

Stroke is a major cause of morbidity and mortality. We have previously described an anti-cell death system in neurons in the brain that promotes cell survival, but that can be high-jacked by enzyme systems to act as a pro-death system. We will study a pharmacological inhibitor whose dual interaction with the pro- and anti-death machinery makes it a useful tool for understanding cell death and opening avenues for the design of future therapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
2R37NS045876-11
Application #
8760518
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Bosetti, Francesca
Project Start
2003-04-01
Project End
2018-06-30
Budget Start
2014-09-01
Budget End
2015-06-30
Support Year
11
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Yale University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
New Haven
State
CT
Country
United States
Zip Code
06510
Mnatsakanyan, Nelli; Beutner, Gisela; Porter, George A et al. (2016) Physiological roles of the mitochondrial permeability transition pore. J Bioenerg Biomembr :
Jonas, Elizabeth A; Porter Jr, George A; Beutner, Gisela et al. (2015) Cell death disguised: The mitochondrial permeability transition pore as the c-subunit of the F(1)F(O) ATP synthase. Pharmacol Res 99:382-92
Park, Han-A; Licznerski, Pawel; Alavian, Kambiz N et al. (2015) Bcl-xL is necessary for neurite outgrowth in hippocampal neurons. Antioxid Redox Signal 22:93-108
Alavian, Kambiz N; Dworetzky, Steven I; Bonanni, Laura et al. (2015) The mitochondrial complex V-associated large-conductance inner membrane current is regulated by cyclosporine and dexpramipexole. Mol Pharmacol 87:1-8
Licznerski, Pawel; Duric, Vanja; Banasr, Mounira et al. (2015) Decreased SGK1 Expression and Function Contributes to Behavioral Deficits Induced by Traumatic Stress. PLoS Biol 13:e1002282
Jonas, Elizabeth A; Porter, George A; Alavian, Kambiz N (2014) Bcl-xL in neuroprotection and plasticity. Front Physiol 5:355
Jonas, Elizabeth A (2014) Contributions of Bcl-xL to acute and long term changes in bioenergetics during neuronal plasticity. Biochim Biophys Acta 1842:1168-78
Park, Han-A; Jonas, Elizabeth A (2014) Mitochondrial membrane protein Bcl-xL, a regulator of adult neuronal growth and synaptic plasticity: multiple functions beyond apoptosis. Neural Regen Res 9:1706-7
Alavian, Kambiz N; Beutner, Gisela; Lazrove, Emma et al. (2014) An uncoupling channel within the c-subunit ring of the F1FO ATP synthase is the mitochondrial permeability transition pore. Proc Natl Acad Sci U S A 111:10580-5
Jonas, Elizabeth A (2014) Impaired import: how huntingtin harms. Nat Neurosci 17:747-9

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