Numerous neuronal ultrastructural and biochemical abnormalities develop during brain ischemia and reperfusion; however, the causal interrelationships of these phenomena are not well understood. Dr.Neumar has recently found that (1) brain eIF-4E levels decline sharply during ischemia and (2) eIF-4E is rapidly degraded in vitro by Ca2+-activated mu- calpain. Proteolysis of eIF-4E during ischemia could be due to calcium- induced mu-calpain activation. mRNA binding by eIF-4E is a rate limiting step in translation initiation, and degradation of eIF-4E by mu-calpain could connect the recognized phenomena of calcium influx during ischemia and subsequent depressed protein synthesis. The hypotheses proposed are that (1) ischemia in vivo and Ca2+ overloading in vitro cause mu-calpain activation and eIF-4E degradation in neurons, (2) ischemia-induced activation of mu-calpain and loss of eIF-4E are most prominent in selectively vulnerable neurons, and (3) pretreatment with a calpain inhibitor reduces both mu-calpain autoproteolytic activation and degradation of eIF-4E. Dr. Neumar will study these hypotheses utilizing ((i) an in vivo model of complete brain ischemia and reperfusion by cardiac arrest and resuscitation in rats and (ii) inophore-induced Ca2+ overload in neuronally-differentiated NB-104 cells. Antibodies specific to eIF-4E and activated mu-calpain will be used in Western blots to characterize (i) in cultured neurons Ca2+-induced and (ii) in rat brains ischemia and reperfusion-induced mu-calpain activation and eIF-4E degradation. Light and electron microscopy with immunohistochemistry will provide regional and ultrastructural localization of mu-calpain activation and eIF-4E degradation with simultaneous ultrastructural evaluation for neuronal injury. The effect of a cell-permeant mu-calpain inhibitor will be evaluated in all of the above experiments.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08NS001832-03
Application #
2431080
Study Section
NST-2 Subcommittee (NST)
Program Officer
Jacobs, Tom P
Project Start
1995-08-01
Project End
1997-08-31
Budget Start
1997-06-01
Budget End
1997-08-31
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Wayne State University
Department
Emergency Medicine
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Neumar, R W; Hagle, S M; DeGracia, D J et al. (1996) Brain mu-calpain autolysis during global cerebral ischemia. J Neurochem 66:421-4