Despite the indispensable role that astrocytes play in the neurovascular unit, few studies have investigated the functional impact of astrocyte signaling in cognitive decline and dementia related to vascular pathology. Diet-mediated induction of hyperhomocysteinemia (HHcy) recapitulates numerous features of vascular contributions to cognitive impairment and dementia (VCID). Here, we used astrocyte targeting approaches to evaluate astrocyte Ca2+dysregulation and the impact of aberrant astrocyte signaling on cerebrovascular dysfunction and synapse impairment in male and female HHcy diet mice. Two-photon imaging carried out in fully awake mice revealed activity-dependent Ca2+dysregulation in barrel cortex astrocytes under HHcy. Stimulation of contralateral whiskers elicited larger Ca2+transients in individual astrocytes of HHcy diet mice compared to control diet mice. However, evoked Ca2+signaling across astrocyte networks was impaired in HHcy mice. HHcy also was associated with increased activation of the Ca2+/calcineurin (CN) dependent transcription factor NFAT4, which has been linked previously to the reactive astrocyte phenotype and synapse dysfunction in amyloid and brain injury models. Targeting the NFAT inhibitor VIVIT to astrocytes, using adeno-associated virus (AAV) vectors, led to reduced GFAP promoter activity in HHcy diet mice, and improved functional hyperemia in arterioles and capillaries. VIVIT expression in astrocytes also preserved CA1 synaptic function and improved spontaneous alternation performance on the Y maze. Together, the results demonstrate that aberrant astrocyte signaling can impair the major functional properties of the neurovascular unit (i.e.cerebral vessel regulation and synaptic regulation) and may therefore represent a promising drug target for treating VCID and possibly other ADRDs.

Introduction Vascular contributions to cognitive impairment and dementia (VCID) are a leading cause of dementia. An underappreciated, modifiable risk factor for VCID is hyperhomocysteinemia (HHcy), defined by elevated levels of plasma homocysteine, most often due to impaired B vitamin absorption in aged persons. Studies aimed at identifying neuropathologic features and gene expression profiles associated with HHcy have been lacking. Methods A subset of research volunteers from the University of Kentucky Alzheimer's Disease Research Center longitudinal cohort came to autopsy and had ante mortem plasma homocysteine levels available. Brain tissue and blood plasma drawn closest to death were used to measure homocysteine and related metabolites in the current pilot study. Genetic expression profiles of inflammatory markers were evaluated using the Human Neuroinflammation NanoString panel. Further analyses included an evaluation of plasma homocysteine effects on amyloid beta, tau, ionized calcium‐binding adaptor molecule 1, and glial fibrillary acidic protein immunohistochemistry in the frontal and occipital cortices. Analytes and other study outcomes were evaluated in relation to ante mortem HHcy status: We identified 13 persons with normal ante mortem plasma homocysteine levels (<14 µmol/L) and 18 who had high plasma homocysteine levels (≥14 µmol/L). Results Participants with HHcy demonstrated increased levels of several plasma homocysteine cycle metabolites such as total cysteine, S‐adenosyl‐homocysteine, cystathionine, and choline. Inflammatory gene expression profiles showed a general downregulation in the setting of elevated plasma homocysteine. HHcy was associated with more and longer microglial processes, but smaller and fewer astrocytes, especially in participants of older age at death. HHcy in older participants was also associated with occipital cortex microhemorrhages and increased severity of atherosclerosis throughout the cerebral vasculature. Conclusions Increased plasma homocysteine and older age were associated with the downregulation of inflammatory gene expression markers in association with significant glial and vascular pathology changes. Impaired immune function is a plausible mechanism by which HHcy increases cerebrovascular damage leading to impaired cognitive function.

APOE is the largest genetic risk factor for late-onset Alzheimer disease (AD) with E4 conferring an increased risk for AD compared to E3. The ApoE protein can impact diverse pathways in the brain including neuroinflammation but the precise impact of ApoE isoforms on inflammation remains unknown. As microglia are a primary source of neuroinflammation, this study determined whether ApoE isoforms have an impact on microglial morphology and activation using immunohistochemistry and digital analyses. Analysis of ionized calcium-binding adaptor molecule 1 (Iba1) immunoreactivity indicated greater microglial activation in both the hippocampus and superior and middle temporal gyrus (SMTG) in dementia participants versus non-demented controls. Further, only an increase in activation was seen in E3-Dementia participants in the entire SMTG, whereas in the grey matter of the SMTG, only a diagnosis of dementia impacted activation. Specific microglial morphologies showed a reduction in ramified microglia in the dementia group. For rod microglia, a reduction was seen in E4-Control patients in the hippocampus whereas in the SMTG an increase was seen in E4-Dementia patients. These findings suggest an association between ApoE isoforms and microglial morphologies and highlight the importance of considering ApoE isoforms in studies of AD pathology.

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BackgroundVascular contributions to cognitive impairment and dementia (VCID) are one of the leading causes of dementia; VCID affects roughly 10‐40% of all dementia patients. A major, yet underrecognized, modifiable risk factor for VCID is hyperhomocysteinemia (HHcy). Defined as elevated levels of plasma homocysteine (a non‐protein‐forming amino acid), most late‐life HHcy is caused by impaired B vitamin absorption. Although HHcy has been recognized as a risk factor for VCID, studies aimed at identifying pathologies associated with HHcy have been lacking.MethodTo determine pathologies associated with HHcy, we identified 31 autopsied research volunteers with antemortem homocysteine levels; 13 cases had normal plasma homocysteine levels (>14μmol/L) and 18 had high plasma homocysteine levels (<14μmol/L). We then measured levels of homocysteine and related metabolites in both plasma samples taken closest to autopsy and frontal cortex. Next, we determined whether the level of plasma homocysteine was associated with several markers identified via immunohistochemistry, including Aβ, PHF‐1, IBA‐1 and GFAP. Plasma and brain protein markers for inflammation and angiogenesis were also measured to determine associations with plasma homocysteine. Finally, we used the Human Neuroinflammation NanoString panel to determine gene expression changes of inflammatory markers associated with high homocysteine levels in the frontal cortex and occipital lobe.ResultPlasma metabolite analysis showed patients who had elevated levels of homocysteine also had increased levels of several homocysteine cycle metabolites such as cysteine, S‐adenosyl‐homocysteine, cystathionine and choline. Flt1, an angiogenic marker, and IL5, an inflammatory marker, had a positive correlation with increased plasma homocysteine. No correlation between IBA‐1 or GFAP immunohistochemistry with plasma homocysteine was found. Gene expression showed that most genes were downregulated in the presence of high plasma homocysteine, including many significant genes involved in apoptosis, growth factor and cytokine signaling, and the innate and adaptive immune response.ConclusionThese preliminary data show that increased plasma homocysteine correlates with protein inflammatory and angiogenic markers and with a significant downregulation of inflammation‐related gene expression markers in the brain. Overall, this could reflect impaired normal immune function, providing possible mechanisms by which hyperhomocysteinemia induces cognitive deficits and cerebrovascular damage.