Kristen L Zuloaga - US grants

DESCRIPTION (provided by applicant): Women have lower stroke risk and mortality compared to age-matched men. While some of this protection appears to be mediated via ovarian estrogen, ovarian hormones do not account for all of the protective effects seen in females since female protection persists even after menopause when ovarian estrogen is lost. In post- menopausal women, the major source of estrogen becomes local (extra-gonadal) synthesis of estradiol by the enzyme aromatase. Aromatase is expressed in numerous cell types, including vascular endothelial cells (EC). In female mice, both aromatase gene deletion and pharmacological inhibition lead to worse outcome following cerebral ischemia, suggesting that aromatase plays a protective role. Therefore, the hypothesis to be tested is that females are protected from cerebrovascular dysfunction following cerebral ischemia compared to males due to higher expression and activity of endothelial-specific aromatase. In order to determine if there are sex differences in cerebrovascular endothelial dysfunction following cerebral ischemia in mice, responses to the endothelium-dependent vasodilator acetylcholine (ACh) will be compared in male and female mice before and after transient middle cerebral artery occlusion (tMCAO, 1h) or sham surgery using an in vivo cranial window preparation and optical microangiography imaging. To determine if sex differences in endothelium-dependent dilation after tMCAO are mediated by aromatase, ACh responses before and after tMCAO or sham surgery will be compared between wild-type and aromatase knockout mice of both sexes. Finally, to determine if there are sex differences in EC-specific aromatase expression and cerebrovascular aromatase activity, EC-specific aromatase mRNA and protein expression and cerebrovascular aromatase activity will be compared in cerebral vessels isolated from male and female mice at baseline or after tMCAO or sham surgery. Understanding EC specific regulation of aromatase may lead to therapeutic strategies aimed at enhancing local estradiol production specifically within endothelial cells, thus avoiding the negative side effects associated with global estrogen administration, but maintaining the protective effects of estrogen on the vasculature. PUBLIC HEALTH RELEVANCE: Aromatase inhibitors, which are in use clinically for the treatment of hormone positive breast cancer, have been linked to adverse cardiovascular effects. The proposed research will use a rodent model of stroke, the middle cerebral artery occlusion, to determine the protective effect of aromatase (the enzyme that produces estradiol) against endothelial dysfunction following cerebral ischemia, especially in females. Understanding endothelial cell specific regulation of aromatase may lead to therapeutic strategies for stroke aimed at enhancing local estradiol production specifically within endothelial cells, thus avoiding the negative side effects associated with global estrogen administration, but maintaining the protective effects of estrogen on the vasculature.

PROJECT SUMMARY/ABSTRACT Half of all dementia patients have evidence of vascular contributions to cognitive impairment and dementia (VCID), either as a single pathology or as a mixed dementia. Diabetic women have a 2.3-fold greater risk to develop VCID than non-diabetic women. However, even though prediabetes causes cognitive deficits and is 3 times more common than diabetes, little is known regarding the effects of prediabetes on VCID. Further, why metabolic disease is a larger risk factor for VCID in women than in men is unknown. Women are unique in that they go through menopause. Menopause accelerates mid-life risk factors for dementia, by increasing risk for cardiovascular and metabolic diseases. Despite the fact that nearly all women with VCID are post-menopausal, the influence of menopause on VCID is a critical gap in knowledge. The objective of this proposal is to understand the important interaction between menopause and prediabetes on VCID pathology and identify a novel therapeutic approach to treat VCID in post-menopausal females. Our preliminary data in a mouse model of VCID demonstrate that prediabetes reduces blood flow and accelerates cognitive deficits in peri- menopausal females, the underlying mechanism is unknown but may be related to neuroinflammation or vascular damage; effects in post-menopausal females have not been assessed. In post-menopausal women, the major source of estrogen is local synthesis of 17-? estradiol by the enzyme aromatase in a variety of tissues. Brain aromatase and brain estradiol levels are drastically decreased in women with other forms of dementia, however, the role of aromatase in VCID has yet to be explored. We have discovered that inhibition of aromatase severely impairs cerebrovascular function in female mice suggesting that targeting aromatase in females may be an effective strategy for increasing blood flow to the brain, thereby reducing VCID pathology. We hypothesize that cerebral hypoperfusion, cognitive deficits, and neuroinflammation induced by prediabetes will be exacerbated in post-menopausal females and will be reversed by increasing production of brain-derived estradiol. In Aim 1, using a mouse model of VCID, we will determine if menopause exacerbates cerebral hypoperfusion, neuroinflammation, and cognitive deficits induced by prediabetes. In Aim 2, we will determine if loss of endothelial or astrocytic aromatase causes cerebral hypoperfusion and exacerbates cognitive deficits and neuroinflammation in post-menopausal female mice. In Aim 3, we will determine if increasing estradiol specifically in the brain improves vascular function and reduces cognitive deficits and neuroinflammation in prediabetic post-menopausal female mice. We anticipate these studies will provide mechanistic insight that will facilitate future interventions to decrease the burden of dementia by identifying a novel approach to treat VCID in post-menopausal females: enhancement of brain estradiol. Without knowledge of the effects of menopause on VCID in prediabetic females, development of therapies to treat this high-risk population remains unlikely.

PROJECT SUMMARY/ABSTRACT Cerebrovascular pathology is present throughout stages of Alzheimer?s Disease and is correlated with cognitive changes. There is strong evidence that vascular dysfunction is a significant driver of neuropathology. Our long- term objective is to understand the function of Alzheimer?s Disease-associated risk genes in vascular cells, their contribution to the development of cerebrovascular pathology and the opportunities to use this information in therapeutic development. There are over 27 Alzheimer?s Disease-associated risk (AD-risk) loci encompassing numerous genetic variants in non-coding and coding regions and hundreds of linked genes. Our overarching hypothesis is that a subset of AD-risk genes impairs vascular function, causing release of inflammatory factors, blood brain barrier (BBB) impairment, and reduced perfusion, thus contributing to neurodegeneration. To address this, we have assembled a multi-disciplinary team with a proven track record of collaboration, including with ADSP and ADGP members, who bring expertise in vascular pathology in dementia, endothelial cell (EC) signaling and EC functional testing, Alzheimer?s Disease genomics, single cell and nuclear transcriptomics, bioinformatics, CRISPR-based gene editing for large scale screening and AD mouse models for in-depth functional assessment in vivo. Notably, we will address differences in gene effects related to the important biological variables, sex and metabolic disease. Men and women differ in their genetic risk for Alzheimer?s Disease, with sex-specific polygenic risk scores providing better prediction of onset, progression, and pathology than pooled-sex scores. Over 80% of individuals with Alzheimer?s Disease have co-morbid metabolic disease, which exacerbates vascular pathology. We have identified the top 50 AD-risk SNPs and 600 AD-associated genes, and these will be targeted for induced pluripotent stem cell (iPSC)-derived endothelial cell (EC) screens by prime editing and CRISPR-based gene inhibition and activation approaches respectively. iPSC-based production of human ECs and mural cells in 2D and 3D models has been optimized and scaled to enable efficient functional testing of the impact of gene changes, including on neuro-vascular interactions in cerebral organoids. Discoveries made in these human cell systems will be validated by an in-depth investigation of gene expression changes in individual ECs and mural cells across a large collection of Alzheimer?s Disease brain samples using single nuclear sequencing. The EC translatome will also be obtained from mouse Alzheimer?s Disease models that incorporate sex and metabolic disease. These diverse datasets will be harmonized and integrated in order to map vascular phenotypes of AD-risk genes and identify critical molecular pathways that are targetable drivers of AD cerebrovascular pathology. These data will add to the breadth of knowledge being gathered by other groups to further elucidate underlying neuronal, glial, microglial, endothelial and mural cell-cell interactions that contribute in a substantial way to the complex architecture of Alzheimer Disease pathology. 1