Stroke is a major cause of morbidity and mortality in the United States and presents challenges in the development of disease prevention strategies. Deprivation of nutrients and oxygen supply to brain cells produces immediate death in the most severely affected cells and delayed cell death in some cells that are placed at risk at the first onset of ischemia. The latter form of neuronal death is orchestrated by BCL-2 family proteins, which also regulate cell death during nervous system development. BCL-2 family proteins are present in mitochondrial membranes and become activated to form large conductance ion channel activity in response to many death stimuli, including a low concentration of cellular oxygen. The relationship of the channel activity to the onset of cell death is as yet poorly understood. We have developed patch clamp techniques to record mitochondrial ion channel activity within living neurons and to study the ion channel activity of BCL-2 family proteins in mitochondria isolated from brain. In comparing the channel activity of recombinant BCL-2 family proteins to endogenous activity, we find that the recombinant N-truncated form of BCL-xL, (?N BCL-xL) that is produced by acute proteolytic processing of full length BCL-xL in response to death stimuli, has biophysical and pharmacological similarities to an endogenous mitochondrial channel that appears during transient global ischemia in mammalian brain. The onset of channel activity of ?N BCL-xL may initiate a series of molecular events that leads to selective, delayed cell death in vulnerable CA1 neurons of the hippocampus. We hypothesize that inhibition of the ?N BCL-xL channel activity with the specific inhibitor of BCL-xL, ABT-737, will block cell death in these sensitive neurons after ischemia. Furthermore, if proteolytic processing of full length BCL-xL is necessary for cell death, then hippocampal neurons of a mouse that lacks a cleavable form of BCL-xL may fail to form ?N BCL-xL and fail to die after ischemic insult. These studies will test the hypothesis that ?N BCL-xL is the key regulator of cell death in hippocampal neurons. In addition, these studies will attempt to elucidate the molecular mechanisms underlying cell death in neurons after ischemic brain injury with an eye to developing strategies to combat stroke.

Public Health Relevance

Stroke is a major cause of morbidity and mortality in the United States. We have found that mitochondrial ion channel activity of the pro- and anti-death BCL-2 protein family contributes to the physiological and pathological function of neurons. We hypothesize that in the setting of brain ischemia, such channel activity leads to cell death in the brain and that this death can be prevented by a pharmacological inhibitor of BCL-2-induced mitochondrial ion channels.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS045876-09
Application #
8231377
Study Section
Neural Oxidative Metabolism and Death Study Section (NOMD)
Program Officer
Bosetti, Francesca
Project Start
2003-04-01
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
9
Fiscal Year
2012
Total Cost
$354,791
Indirect Cost
$140,416
Name
Yale University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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