Cerebral white matter is highly vulnerable to cerebral ischemia13 and white matter injury (WMI) is a major cause of functional disability in stroke, the nation's third leading cause of death and the leading cause of permanent disability. Thus, interventions targeted at WMI, an area that remains relatively understudied and poorly understood, may significantly improve post-stroke quality of life. Nicotinamide phosphoribosyltransferase (NAMPT) primarily localizes in the cytosol and functions as the rate-limiting enzyme for the biosynthesis of nicotinamide adenine dinucleotide (NAD)14. NAMPT is neuroprotective against cerebral ischemia15,16. In non-CNS systems, NAMPT can be found in the extracellular space (eNAMPT) and circulates systemically in the blood. Whether the CNS also has eNAMPT and the role of this eNAMPT remains unknown. In our pilot studies, we first detected that neural cells secrete NAMPT into the extracellular space and that eNAMPT, but not its intracellular counterpart (iNAMPT), is selectively induced after ischemia, implying that extracellular NAMPT may play a role in ischemia. Increased NAMPT levels are present in the extracellular space and in white matter after ischemia in neuron-specific transgenic mice. Most importantly, in addition to gray matter protection, we found that NAMPT transgenic overexpression protects against WMI after ischemia. These findings lead us to speculate that extracellular NAMPT may mediate white matter protection after ischemia. In line with this, we found that recombinant NAMPT protein protects oligodendrocytes against ischemia-like injury induced by oxygen glucose deprivation (OGD) and AMPA toxicity. More importantly, we found that exogenously administered NAMPT protein can cross the blood brain barrier and protect against ischemic WMI in vivo. Further studies found that recombinant NAMPT protein protects against cell death of oligodendrocytes and its progenitor cells (OPCs) induced by ischemia, and enhances the differentiation of OPCs into mature oligodendrocytes. These findings suggest that NAMPT may directly protect against WMI via inhibiting oligodendrocyte death and enhancing the maturation of OPCs. We found that NAMPT also enhances microglial myelin phagocytosis. This is important because damaged myelin is believed to suppress the maturation of OPCs and cause demyelination in many pathological conditions. Thus NAMPT may indirectly enhance oligodendrogenesis via modulating microglial myelin phagocytosis. The objective of this project is to further test the white matter protective effect of NAMPT and its underlying mechanisms, and to develop a novel and clinically relevant therapeutic approach for white matter protection after cerebral ischemia. The central hypothesis to be tested is that NAMPT protects against WMI via enhancing oligodendrocytic protection/ regeneration and microglia-mediated myelin phagocytosis. Focal ischemia will be induced in mice by middle cerebral artery occlusion (MCAO) The following Specific Aims are proposed:
Aim 1 : Test the hypothesis that systemic administration of NAMPT protein protects against WMI induced by focal ischemia. The proposed study will test the potential translational value of NAMPT in treating WMI induced by focal cerebral ischemia. NAMPT recombinant protein will be administrated intraperitoneally to mice and the effect of NAMPT on ischemic brain injury will be quantitatively evaluated. The endpoints for assessment include infarct volume, WMI, and neurological outcomes.
Aim 2 : Test the hypothesis that NAMPT protects against WMI by inhibiting oligodendrocytic death and enhancing oligodendrogenesis after cerebral ischemia. The proposed study will test whether systemic administration of NAMPT protein reduces oligodendrocyte death after focal ischemia. We will also test whether NAMPT stimulates the regeneration of oligodendrocytes after ischemia.
Aim 3 : Test the hypothesis that NAMPT protects against WMI by enhancing microglial myelin phagocytosis. Two approaches will be used to elevate NAMPT levels: systemic administration of NAMPT protein and microglia-targeted NAMPT overexpression via an AAV9 vector driven by microglia/macrophage specific F4/80 promoter. The effect of NAMPT on microglial myelin phagocytosis, oligodendrogenesis, WMI, and functional outcomes after ischemia will be evaluated. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page
Guodong Cao, Ph.D Narrative White matter (WM) is severely affected in most cases of human stroke, accounting for up to half of the lesion volume. Because WM is involved in the nerve signal transduction to and from the cerebral cortex, white matter injury (WMI) is a major cause of long term functional disability in stroke. Thus, interventions targeted at WMI may significantly improve post-stroke quality of life. Despite its importance, WMI has been largely neglected in animal studies as well as in clinical treatment/trials, perhaps explaining the many failed or ineffective treatments of stroke. Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme that regulates the production of nicotinamide mononucleotide (NAD), the major source of cellular energy. Previous studies have found that NAMPT is an important endogenous neuroprotective molecule against ischemic stroke. The mechanisms underlying the protection afforded by NAMPT may be the supply of extra energy and the activation of NAD- related signaling pathways. NAMPT normally localizes inside cells, where it functions as an NAD producer. However, we have discovered a new form of NAMPT which is secreted by neural cells to form extracellular NAMPT (eNAMPT) in the brain. Interestingly, we have also found that recombinant eNAMPT protein can cross the blood-brain barrier and protect both gray and white matter after cerebral ischemia. Thus, this proposal will study the white matter protective effect o this novel form of NAMPT and the underlying mechanisms. Our long term goal is to fill the remaining gaps in our understanding of WMI in ischemic stroke and gain deeper insights into WMI. This project serves a dual role;not only will it impact our understanding of the basic molecular mechanisms of ischemic WMI but also will shed light on NAMPT as a potential therapeutic target for stroke. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page
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