Novel mechanisms of metabolic reprogramming of reactive astrocytes and brain repair after focal cerebral ischemia

University Of Missouri-Columbia, Columbia MO

Investigators

Abstract

Project Summary Focal ischemic ischemia (FCI) is a leading cause of human death, however, functional recovery after FCI has been observed and can be remarkable in both patients and animal models. Due to limited therapeutic choices for clinical treatment, studying its mechanisms has been an important topic and could provide therapeutic insights. Astrocytes in normal adult brains referred to as resting astrocytes (RestAs) are quiescent, but they undergo rapid proliferation, display a `hypertrophic' morphology, exhibit an altered transcriptional profile, and eventually form glial scars in the peri-infarct region (PIR) after FCI. These phenomena are known as reactive astrogliosis, and the activated astrocytes are called reactive astrocytes (ReAs). The phenomena of reactive astrogliosis indicate that the microenvironment constraining the proliferation capacity of astrocytes is removed and ReAs must adapt to a more metabolically active phenotype to meet the increased biosynthetic and bioenergetic demands. Accordingly, the current application will study the metabolic reprogramming of ReAs and its effects on brain repair after FCI. Our preliminary studies discovered that nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage pathway, is undetectable in RestAs but is highly upregulated in ReAs after photothrombosis (PT)-induced FCI and oxygen-glucose deprivation (OGD) of cell cultures. Metabolomic analysis indicated increased metabolites in major metabolic pathways including glycolysis and pentose phosphate pathway (PPP) in ReAs. Furthermore, we found that hypoxia inducible factor 1 (HIF1) is an upstream regulator of NAMPT and conditional deletion of NAMPT in astrocytes caused increased brain damage, neuronal death, and motor function deficits after PT. Because NAD+ and its derivative NADP+ are important determinants of glycolysis and PPP, we hypothesize that NAMPT upregulation is necessary and sufficient for metabolic reprogramming of ReAs after FCI, thereby mediating reactive astrogliosis, promoting brain repair and improve stroke outcomes after FCI. Our overall project goal is to conduct an in-depth study on the metabolic reprogramming of ReAs and its effect on post-stroke brain repair after FCI. To accomplish our goals, we will use integrated technologies including astrocyte-specific inducible and conditional NAMPT knockout and overexpression mice and HIF1 knockout mice, stable isotope labeling (SIL), LC-MS analysis of metabolomic profile, RNA-seq, in vivo two-photon imaging of metabolism, histology and behavioral tests. We propose three specific aims. Aim 1 will characterize metabolic phenotype and determine the effect of NAMPT on metabolic reprogramming of ReAs after FCI. Aim 2 will investigate the regulatory mechanism of NAMPT induction by HIF1 and other potential transcriptional factors (TFs) in ReAs. Aim 3 will study the effect and mechanisms by which ReAs promote brain repair and functional recovery through metabolic reprogramming after FCI. We expect our work will elucidate novel mechanisms of ReAs in post-stroke brain repair in the context of glia-neuron interactions and provide potential therapeutic insights for FCI.