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A novel therapeutic target for stroke

Institution: The Burnham Institute for Medical Research
Investigator(s): Li-Tao Zhong, M.D., Ph.D.
Award Cycle: 1997 (Cycle 6) Grant #: 6FT-0157 Award: $72,310
Subject Area: Cardiovascular Disease
Award Type: Postdoctoral Fellowship Awards

Initial Award Abstract
Stroke kills 150,000 Americans each year and is the third leading cause of death in the United States. Cigarette smoking is firmly established as a risk factor for stroke. In stroke, severe, irreversible brain tissue destruction occurs which results from extensive neuronal cell death. There are two types of cell death, necrosis and apoptosis. Apoptosis is an inducible process of gene-directed cellular self-destruction. Accumulated evidence shows that apoptosis plays an important part in neuronal cell death found in stroke. It is becoming increasingly clear that many cells display surface molecules known as receptors, some of which act to trigger apoptosis. Such apoptosis-inducing receptors play important roles in many disease processes, including possibly stroke. Therefore, determin-ing the mechanism(s) by which such receptors induce apoptosis, and identifying novel apoptosis-inducing receptors, is likely to lead to more effective treatments for stroke.

This laboratory recently produced a mono-clonal antibody which is able to induce apoptosis in cortical neurons (this is the first such antibody). This antibody was used to isolate a member of a novel class of apoptosis-inducing cell surface molecules (made of sugar and fat--called a "glycolipid”), demonstrated that this is the first "portable" cell death receptor by demon-strating cell to cell migration and subsequent killing of cells via activity of the transferred receptor.

The current proposal is to (1) utilize the same approach that was successful in identifying this molecule to identify additional surface molecules that initiate apoptosis in other brain regions; (2) study the mechanism of apoptosis induction in cortical neuron as transduced by the target molecule; (3) further define the “portable” nature of the death signal. The long-term objective of this proposal is to identify cell surface molecules that control apoptosis in central brain cells, and to understand the mechanism(s) by which specific surface molecules induce apoptosis in central neurons. Given recent demonstrations of apopt-osis in ischemia, it is hoped that this work will lead to improved treatments for stroke.

Final Report
Tobacco smoking has been associated with more than 50% of deaths resulted from cerebrovascular disease, including stroke, among men and women aged less than 65 years. Discoveries have also demonstrated that apoptosis, a form of cell death resulted from the active expression of certain genes, plays an important role in the pathogenesis of stroke. The long-term objectives of this fellowship project are to identify cell surface molecules that control apoptosis in central neurons, and to understand the mechanism(s) by which specific cell surface molecules induce apoptosis in central neurons. The specific aims of the project are: (1) To identify neuronal cell death receptors from hippoeampus and cerebellum (2) To study the mechanism of apoptosis induction in cortical neurons as transduced by NAIM-1, an apoptosis-inducing monoclonal antibody, and its associated antigen isoglobotetrasylceramide (isoGB4) (3) To further define the "portable" nature of the death signal of isoglobotetrasylceramide.

Progress from the work to date includes: (1) Over 10,000 monoclonal antibody hybridoma clones have been produced against a group of hippocampal neural cells, HS2. These hybridoma clones have been screened for the capabilities to (a) induce or suppress cell death (b) induce or promote neuronal differentiation. So far, two interesting clones have been identified. One was found to be able to induce hippocampal neuronal differentiation while the other induced detachment of the neurons from the culture dishes. (2) It was found, by the study of mechanism of apoptosis transduced by NAIM-1/isoGB4, that (a) The Fab fragment of NAIM-1 antibody did not induce cell death (b) NAIM 1 elevated the intracellular ceramide level (c) NAIM-1 did not cause sphingomyelin hydrolysis (d) Fumonisin B 1, a ceramide synthase inhibitor, inhibits ceramide production induced by NAIM-1 antibody (e)Daunorubicin induced apoptosis in CSM25 cells from which isoGB4 was originally identified as a death receptor. These results suggest that (a) the crass-linking of the receptors is required to initiate the cell death, thus a signal transduction pathway is involved (b) ceramide synthase may be a key player in the NAIM-1/isoGB4 signal transduction pathway. (3)NAM 1 antigen was found to be able to migrate directly from cell to cell, further demonstrated its portable nature.

We believe that the studies done in the project yielded significant advances in the understanding of neural apoptosis, which would further lead to better understanding of pathogenesis of stroke and novel therapy for the disease.