Gary M Fiskum

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Novel delivery of Bcl-2 for neuroprotection

Gary M Fiskum

1 December 2002 - 30 November 2005
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE

The overall goal of this exploratory project is to develop a novel strategy for in vivo neuroprotection based on the new concept of protein transduction. This model will be used to deliver full-length, anti-death Bcl-2 proteins into the brain to test the hypothesis that postischemic neuroprotection by Bcl-2 can be achieved by a realistic method for delivering exogenous protein into brain cells. A second objective is to elucidate the contribution of the anti-oxidant vs. anti-Bax mechanisms in different neural cell death paradigms and to investigate the specific role of phosphorylation in regulating these mechanisms and in neuroprotection. The specific aims of the project are to; 1) Test the hypothesis that the anti-death activity of Bcl-2 is inhibited by post-translational mechanisms activated in response to chemical hypoxia and glucose deprivation in vitro. 2) Determine the role of phosphoQrlation in regulating the mechanisms by which Bcl-2 protects against mitochondrial dysfunction caused by Ce plus oxidative stress compared to the interaction of proapoptotic proteins, i.e., Bax plus BH3 death domain only protein. 3) Test the hypothesis that delivery of full-length Bcl-2, as a TAT-Bcl-2 fusion protein, into the brain is protective in a rat transient focal cerebral ischemia model. The methods of approach to these aims will utilize cloning of both the normal Bcl-2 gene and the gene with mutations in specific phosphorylation sites, each ligated to the TAT protein transduction domain. TATBcl-2 fusion proteins will be isolated and tested for their ability to inhibit hypoxic neural cell death in vitro and to reduce cerebral infarct volume in a rat reversible focal ischemia model. The effects of exogenous Bcl-2 constructs on mitochondrial dysfunction will also be assessed using measures of mitochondrial cvtochrome c release, membrane potential, redox potential, and reactive 07 species production. The influence of phosphorylation state on the ability of endogenous and exogenous Bcl-2 to protect against mitochondrial dysfunction and cell death will also be investigated. The significance of these studies is 1. They could establish the foundation for a novel neuroprotective treatment strategy targeting both necrotic and apoptotic cell death and, 2. They will provide completely new insight into the molecular mechanisms by which the antideath activities of proteins like Bcl-2 are regulated under pathological conditions.

5 Resulting Publications

• 1.

  2005

  Lucian Soane; Gary Fiskum

  TAT-mediated endocytotic delivery of the loop deletion Bcl-2 protein protects neurons against cell death.

  Journal of neurochemistry 2005;95(1):230-43.

• 2.

  2005

  Lucian Soane; Gary Fiskum

  Inhibition of mitochondrial neural cell death pathways by protein transduction of Bcl-2 family proteins.

  Journal of bioenergetics and biomembranes 2005;37(3):179-90.

• 3.

  2005

  Alicia J Kowaltowski; Gary Fiskum

  Redox mechanisms of cytoprotection by Bcl-2.

  Antioxidants & redox signaling 2005;7(3-4):508-14.

• 4.

  2004

  Brian M Polster; Gary Fiskum

  Mitochondrial mechanisms of neural cell apoptosis.

  Journal of neurochemistry 2004;90(6):1281-9.

• 5.

  2004

  Gary Fiskum; Robert E Rosenthal; Viktoria Vereczki; Erica Martin; Gloria E Hoffman; Christos Chinopoulos; Alicia Kowaltowski

  Protection against ischemic brain injury by inhibition of mitochondrial oxidative stress.

  Journal of bioenergetics and biomembranes 2004;36(4):347-52.

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