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PURPOSE. Glaucoma is a complex disease with major risk factors including advancing age and increased intraocular pressure (IOP). Dissecting these earliest events will likely identify new avenues for therapeutics. Previously, we performed transcriptional profiling in DBA/2J (D2) mice, a widely used mouse model relevant to glaucoma. Here, we use these data to identify and test regulators of early gene expression changes in DBA/2J glaucoma. METHODS.Upstream regulator analysis (URA) in Ingenuity Pathway Analysis was performed to identify potential master regulators of differentially expressed genes. The function of one putative regulator, mesenchyme homeobox 2 (Meox2), was tested using a combination of genetic, biochemical, and immunofluorescence approaches.RESULTS. URA identified Meox2 as a potential regulator of early gene expression changes in the optic nerve head (ONH) of DBA/2J mice. Meox2 haploinsufficiency did not affect the characteristic diseases of the iris or IOP elevation seen in DBA/2J mice but did cause a significant increase in the numbers of eyes with axon damage compared to controls. While young mice appeared normal, aged Meox2 haploinsufficient DBA/2J mice showed a 44% reduction in MEOX2 protein levels. This correlated with modulation of age-and diseasespecific vascular and myeloid alterations. CONCLUSIONS.Our data support a model whereby Meox2 controls IOP-dependent vascular remodeling and neuroinflammation to promote axon survival. Promoting these earliest responses prior to IOP elevation may be a viable neuroprotective strategy to delay or prevent human glaucoma.

Introduction: Apolipoprotein E (APOE) ε4 is the strongest genetic risk factor for Alzheimer's disease and related dementias (ADRDs), affecting many different pathways that lead to cognitive decline. Exercise is one of the most widely proposed prevention and intervention strategies to mitigate risk and symptomology of ADRDs. Importantly, exercise and APOE ε4 affect similar processes in the body and brain. While both APOE ε4 and exercise have been studied extensively, their interactive effects are not well understood. Methods:To address this, male and female APOE ε3/ε3, APOE ε3/ε4, and APOE ε4/ε4 mice ran voluntarily from wean (1 month) to midlife (12 months). Longitudinal and cross-sectional phenotyping were performed on the periphery and the brain, assessing markers of risk for dementia such as weight, body composition, circulating cholesterol composition, murine daily activities, energy expenditure, and cortical and hippocampal transcriptional profiling.Results: Data revealed chronic running decreased age-dependent weight gain, lean and fat mass, and serum low-density lipoprotein concentration dependent on APOE genotype. Additionally, murine daily activities and energy expenditure were significantly influenced by an interaction between APOE genotype and running in both sexes.Transcriptional profiling of the cortex and hippocampus predicted that APOE genotype and running interact to affect numerous biological processes including vascular integrity, synaptic/neuronal health, cell motility, and mitochondrial metabolism, in a sex-specific manner.Discussion: These data in humanized mouse models provide compelling evidence that APOE genotype should be considered for population-based strategies that incorporate exercise to prevent ADRDs and other APOE-relevant diseases.

Background Human data suggest cerebrovascular dysfunction precedes and exacerbates Alzheimer’s disease (AD). APOEε4 contributes to cerebrovascular decline with age, and APOE status interacts with angiogenesis pathways including the VEGF family to modify cognitive decline. However, the precise nature of this relationship is not known. Research suggests that running provides substantial health benefits that reduce risk for AD. Previous data from our lab showed that, transcriptionally, cerebrovascular processes such as vascular remodeling and angiogenesis terms can be altered short term running in young mice. Here, we are combining human and mouse studies to (i) understand the relationship between APOE status and the VEGF pathway in AD, and (ii) determine whether the effects of exercise on the cerebrovasculature are modified by APOE status. Method To assess cerebrovascular health, we took several approaches: (i) transcriptional profiling, (ii) immunofluorescence of basement membrane, and (iii) immunofluorescence of leakage (fibrin). To assess exercise and metabolic differences, B6.APOEε4/ε4 (risk) and B6.APOEε3/ε3 (neutral) mice were run from 1‐4 months (mo) and 1‐12mos, as well as metabolic phenotyping including circulating plasma lipid levels, food intake, water loss, running speed, and gas exchange. Heterozygous B6.APOEε3/ε4 mice are included to evaluate the potential hypomorphic/ hypermorphic function of the APOEε4 allele on cerebrovascular health. Human data from ROSMAP and ADNI cohorts is being compared to mouse data. Result Cerebrovascular dysfunction was evaluated in 2mo and 12mo B6.APOEε3/ε3 and APOEε4/ ε4 mice for basement membrane coverage and fibrin leakage which revealed an increase in COL4 and increased leakage, in 12mo female APOEε4/ε3 compared to B6.APOEε3/ε3 mice. Analysis of 1‐4mo running data showed no transcriptional evidence that the cerebrovasculature was altered in young APOEε4 or APOEε3 carriers, suggesting longer term interventions are necessary. Human data shows patients that suffer a more severe cognitive decline have increasing levels of angiogenesis markers in the brain and blood. Our ongoing work aims to identify the mechanism between APOEε4 and angiogenesis/cerebrovascular integrity. Conclusion Recreated APOEε4mice recapitulate age‐dependent cerebrovascular deficits however running at a young timepoint will likely not affect cerebrovascular health. Longer term studies are underway to determine whether chronic running mitigates age‐dependent effects of APOEε4.

Background Myeloid cells have been strongly implicated in Alzheimer’s Disease and related dementias (ADRDs). Our previous study showed genetic diversity significantly modified brain myeloid cell responses and AD‐relevant phenotypes in mice. Therefore, incorporating natural genetic variation into mouse models is indispensable to understand the role of myeloid cells in AD. This study aims to characterize brain myeloid cells in our genetically diverse AD panel to determine their functions in ADRDs. Methods Brain myeloid cells were isolated from 8‐9 months‐old female mice from our genetically diverse AD mouse panel [C57BL/6J (B6), PWK/PhJ, WSB/EiJ and CAST/EiJ strains, each carrying APP/PS1] using magnetic activated cell sorting and were subjected to single‐cell RNA‐sequencing (scRNA‐seq). Data were processed using a customized pipeline allowing strain‐specific genome alignment followed by downstream analyses using Seurat and edgeR packages. Differences in myeloid cell responses between strains are being validated using functional assays in primary microglial cultures and immunofluorescence. To identify the putative genetic variations that drive and/or modify differences in myeloid cell responses, quantitative trait locus mapping of microglia number and morphology is being performed in brains from 120 Diversity Outbred (DO) mice, derived from eight mouse strains including those listed above. Results scRNA‐seq uncovered heterogeneous clusters of myeloid cells that vary significantly in cell abundance and gene expression between both wild‐type and APP/PS1 of B6, PWK, WSB and CAST mice. There was a significantly lower percentage of disease‐associated microglia in WSB.APP/PS1 mice and a significantly higher percentage of interferon‐responding microglia in PWK.APP/PS1 mice, compared to other AD strains. These data agreed with previously determined phenotype differences between strains. Differential gene expression analyses (all data compared to the B6 strain) revealed robust APP/PS1‐responding genes unique to strain background (strain by genotype effect) including Rpl29, Irf7, Cd36, H2‐Eb1, Lag3 and Cd34 in CAST; Ramp, Tmem176a, Tmem176b, Sepp1 and Inpp5d in PWK, and Ccl6, Slamf8, Nme1 and Pcna in WSB strain. Validation by primary culture and analyses of DO mice are underway. Conclusion As in humans, mouse genetic diversity significantly impacts the landscape and dynamics of brain myeloid cells in AD. These studies will improve the translatability of myeloid‐based therapies for ADRDs.