Pericyte phenotypic switching in diabetic post-stroke cognitive impairment (PSCI)

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ABSTRACT Diabetes increases the risk of developing stroke, exacerbates brain injury, worsens stroke recovery and predisposes to post-stroke cognitive impairment (PSCI). The goal of this competitive renewal is to investigate the role of impaired endothelial-pericyte signaling in the pro-inflammatory phenotypic transformation of pericytes after stroke and the development of PSCI in diabetic mice. The parent R01 investigated the role of disrupted endothelial pericyte crosstalk in the acute microvascular dysfunction (“no-reflow”) that is common after diabetic stroke, and that exacerbates acute ischemic brain damage. Our findings indicate that loss of endothelial signaling to pericytes, mediated via endothelial derived epoxyeicosatrienoates (EETs) acting on G protein coupled receptor GPR39 on pericytes, leads to pericyte contraction and reduced capillary blood flow. Along the way, we observed that in the subacute/chronic phases of stroke, pericytes retract from capillaries and undergo phenotypic transformation consistent with cell activation, followed by migration to and proliferation within the ischemic region. We will test the hypothesis that persistent disruption of EETs/GPR39 signaling under diabetic conditions transforms pericytes to a pro-inflammatory phenotype that contributes to PSCI. Aim 1 will determine if endothelial EETs protect against diabetic PSCI in a sex- and age-dependent manner. Male and female, young and old, diabetic and non-diabetic mice with an inducible endothelial specific deletion of EETs-metabolizing enzyme soluble epoxide hydrolase endothelial sEH (iEC-sEHKO) will undergo transient MCA occlusion (MCAO), followed by cognitive testing at 1 and 3 months after MCAO. In Aim 2, we will determine the effect of T2D on post-stroke pericyte heterogeneity and phenotype. We will use single-cell RNAseq and single-cell mass cytometry (scCyTOF) to characterize pericyte heterogeneity and phenotypes on days 1, 7 and 30 after MCAO. Pericytes will be isolated from male and female, diabetic and no-diabetic NG2-DsRed mice, which label pericytes in red fluorescence. Novel genes and markers of neuroinflammation will be localized in pericytes using immunohistochemistry (IHC) and RNAscope. Aim 3 will determine the role of GPR39 in PSCI and pericyte phenotypic switching after stroke under diabetic and non-diabetic conditions. We will use GPR39 knockout (KO) mice to test the hypothesis that GPR39 activation by endothelial EETs is protective against the development of PSCI in non-diabetic brain, and that diabetes decreases endothelial EETs, which promotes a pro-inflammatory pericyte phenotype, leading to persistent neuroinflammation and PSCI. We will use unbiased lipidomics to profile oxylipins (oxidized fatty acids, including EETs), and IHC and RNAscope to evaluate expression, distribution and cellular localization of GPR39 and genes identified in Aim 2 in diabetic and non-diabetic brain. The highly translational proposal uses innovative approaches to investigate the role of a novel receptor (GPR39) in pericyte phenotypic switching and its role in diabetic PSCI. If confirmed, the results will pave the way for the use of GPR39 agonists as therapeutic agents to prevent diabetic PSCI by modulating the pericyte response to stroke.