This study will identify the role of a Poly (ADP-ribose) polymer (PAR)-dependent pathological process in stroke that we believe is a central pathway to induce bioenergetic defects, oxidative stress and mitochondrial permeability transition in stroke. Stroke remains a major cause of mortality and morbidity in United States and worldwide and lacks effective therapeutic innervations. There is a critical need to identify the molecular pathways of cell death in stroke so that novel therapeutic targets can be identified. Excessive activation of poly (ADP-ribose) polymerase-1 (PARP-1) activation is strongly implicated to induce cell death in stroke. Studies have shown that PAR is a death signaling molecule in PARP-1 activation and that binding of PAR to hexokinase (HK-1) may cause bioenergetic collapse. However, the role of this PAR/HK-1 interaction in stroke is not known. HK-1 is an essential enzyme in the brain to regulate cellular bioenergetics via glycolysis and mitochondrial function, and maintains redox homeostasis via pentose phosphate pathway. In addition, protection of mitochondria against permeability transition is an important function of HK-1 in the brain. Preliminary data in this application supports the view that in cortical neurons exposed to OGD (an in vitro ischemia / stroke model), PAR binds HK-1 and alters its functions. Collapse of the HK-1 dependent cell survival functions namely bioenergetic balance, redox homeostasis and mitochondrial membrane protection are well known to induce cell death in stroke. Therefore, it is conceivable that binding of PAR to HK-1 is a central pathological pathway to induce cell death in stroke and therefore, may be a credible target for therapeutic intervention in stroke. To directly and rigorously identify the role of this novel pathway in stroke, we will use viral-mediated knockdown of endogenous HK-1 and replacement with PAR-binding mutant form of HK-1 (pbmHK-1) in combination with OGD in neuronal cultures and an MCAO model of stroke in adult mice. We propose the following 4 aims:
Aim #1 : Does PAR-dependent inhibition of HK-1 mediate bioenergetic defects in OGD-treated neurons? Aim #2: Are oxidative stress and redox imbalance in OGD-subjected neurons the consequence of PAR/HK-1 interaction? Aim # 3: Does PAR-binding to HK-1 lead to mitochondrial permeability transition after OGD in neurons? Aim # 4: What is the role of PAR-binding to HK-1 on cell death in stroke in vivo? Each of the proposed aims are supported by a set of important preliminary data, which strongly support the hypothesis that PAR to HK-1 is a central pathological process to induce cell death in stroke. Combining viral-mediated expression of pbmHK-1 with Seahorse Flux analysis, metabolic tracer studies, live-cell imaging and biochemical analysis in experimental models of stroke represent a novel approach to understand PAR- dependent cell death signaling in stroke in greater depth. These studies are crucial to further reveal the role of this novel HK-1/PAR pathway in stroke and to determine whether this important pathological pathway is a credible target for therapeutic intervention in stroke.
In stroke Poly (ADP-ribose) polymer (PAR), formed by poly (ADP-ribose) polymerase-1 inhibits hexokinase-1 (HK-1), which is an essential enzyme to maintains cellular bioenergetics, oxidative balance and mitochondrial integrity in the brain. Here we propose that inhibition of HK-1 by PAR is an upstream pathological event that leads to bioenergetic collapse, redox failure, and mitochondrial permeability transition in stroke. This study will reveal novel strategies for pharmacological intervention in stroke.
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