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Aims: We sought to identify and optimise a universally available histological marker for pericytes in the human brain. Such a marker could be a useful tool for researchers. Further, identifying a gene expressed relatively specifically in human pericytes could provide new insights into the biological functions of this fascinating cell type. Methods: We analysed single‐cell RNA expression profiles derived from different human and mouse brain regions using a high‐throughput and low‐cost single‐cell transcriptome sequencing method called EasySci. Through this analysis, we were able to identify specific gene markers for pericytes, some of which had not been previously characterised. We then used commercially (and therefore universally) available antibodies to immunolabel the pericyte‐specific gene products in formalin‐fixed paraffin‐embedded (FFPE) human brains and also performed immunoblots to determine whether appropriately sized proteins were recognised. Results: In the EasySci data sets, highly pericyte‐enriched expression was notable for SLC6A12 and SLC19A1. Antibodies against these proteins recognised bands of approximately the correct size in immunoblots of human brain extracts. Following optimisation of the immunohistochemical technique, staining for both antibodies was strongly positive in small blood vessels and was far more effective than a PDGFRB antibody at staining pericyte‐like cells in FFPE human brain sections. In an exploratory sample of other human organs (kidney, lung, liver, muscle), immunohistochemistry did not show the same pericyte‐like pattern of staining. Conclusions: The SLC6A12 antibody was well suited for labelling pericytes in human FFPE brain sections, based on the combined results of single‐cell RNA‐seq analyses, immunoblots and immunohistochemical studies. [ABSTRACT FROM AUTHOR]

The p38 alpha mitogen-activated protein kinase (p38α) is linked to both innate and adaptive immune responses and is under investigation as a target for drug development in the context of Alzheimer's disease (AD) and other conditions with neuroinflammatory dysfunction. While preclinical data has shown that p38α inhibition can protect against AD-associated neuropathology, the underlying mechanisms are not fully elucidated. Inhibitors of p38α may provide benefit via modulation of microglial-associated neuroinflammatory responses that contribute to AD pathology. The present study tests this hypothesis by knocking out microglial p38α and assessing early-stage pathological changes. Conditional knockout of microglial p38α was accomplished in 5-month-old C57BL/6J wild-type and amyloidogenic AD model (APPswe/PS1dE9) mice using a tamoxifen-inducible Cre/loxP system under control of the Cx3cr1 promoter. Beginning at 7.5 months of age, animals underwent behavioral assessment on the open field, followed by a later radial arm water maze test and collection of cortical and hippocampal tissues at 11 months. Additional endpoint measures included quantification of proinflammatory cytokines, assessment of amyloid burden and plaque deposition, and characterization of microglia-plaque dynamics. Loss of microglial p38α did not alter behavioral outcomes, proinflammatory cytokine levels, or overall amyloid plaque burden. However, this manipulation did significantly increase hippocampal levels of soluble Aβ42 and reduce colocalization of Iba1 and 6E10 in a subset of microglia in close proximity to plaques. The data presented here suggest that rather than reducing inflammation per se, the net effect of microglial p38α inhibition in the context of early AD-type amyloid pathology is a subtle alteration of microglia-plaque interactions. Encouragingly from a therapeutic standpoint, these data suggest no detrimental effect of even substantial decreases in microglial p38α in this context. Additionally, these results support future investigations of microglial p38α signaling at different stages of disease, as well as its relationship to phagocytic processes in this particular cell-type. [ABSTRACT FROM AUTHOR]

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 Ca21 dysregulation and the impact of aberrant astrocyte signaling on cerebrovascular dysfunction and synapse impairment in male and female HHcy diet mice. Two-photon imaging conducted in fully awake mice revealed activity-dependent Ca21 dysregulation in barrel cortex astrocytes under HHcy. Stimulation of contralateral whiskers elicited larger Ca21 transients in individual astrocytes of HHcy diet mice compared with control diet mice. However, evoked Ca21 signaling across astrocyte networks was impaired in HHcy mice. HHcy also was associated with increased activation of the Ca21/calcineurin-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 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 Alzheimer's disease and other related dementias. [ABSTRACT FROM AUTHOR]

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:Asubset 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 deathwere used tomeasure homocysteine and relatedmetabolites in the current pilot study. Genetic expression profiles of inflammatory markers were evaluated using the HumanNeuroinflammation 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. [ABSTRACT FROM AUTHOR]

Background: The p38 alpha mitogen‐activated protein kinase (p38α) pathway is linked to both innate and adaptive immune responses, and is currently under investigation as a target for drug development in the context of Alzheimer's disease (AD) and other conditions with neuroinflammatory dysfunction (Asih et al., 2020). Preclinical data indicates that p38α inhibition can protect against AD‐associated neuropathology, although the cellular mechanisms and signaling pathways that underlie these effects have not been fully elucidated. Evidence suggests that inhibition of p38α may provide benefit by modulating microglial‐associated neuroinflammatory processes that contribute to the development of AD pathology. The present study tests this hypothesis by assessing early‐stage pathological changes in AD model mice following microglial‐specific knock out (KO) of p38α. Methods: KO of p38α in microglia was accomplished in 5‐month‐old C57BL/6J wild‐type and amyloidogenic AD model (APPswe/PS1dE9) mice using a tamoxifen‐inducible Cre/loxP system under control of the Cx3Cr1 promoter. Animals underwent behavioral assessment on the open field test at 7.5 months of age and on the radial arm water maze test at 11 months of age, followed by collection of cortical and hippocampal tissues. Other endpoint measures included quantification of proinflammatory cytokines, assessment of amyloid burden and plaque deposition, and characterization of microglia‐plaque dynamics using a combination of ELISA, immunohistochemical, and immunofluorescent techniques. Results: Suppression of microglial p38α did not alter behavioral outcomes, proinflammatory cytokine levels, or overall amyloid plaque burden, but did significantly increase hippocampal levels of soluble Aβ42 and reduced hippocampal colocalization of Iba1 and 6E10 in a subset of microglia in close proximity to plaques. Conclusion: This work suggests that rather than reducing inflammation per se, microglial p38α inhibition in the context of early AD‐type amyloid pathology may alter hippocampal microglia‐plaque interactions. Additionally, these results support future investigations of microglial p38α signaling at different stages of disease, as well as the potential relationship between p38α and phagocytic processes in this particular cell‐type. Funding: This work is supported by the National Institutes of Health (RF1 AG064859; F32 AG058456; T32 AG057461). [ABSTRACT FROM AUTHOR]

Background: With the aged population estimated to double by 2050 and the rate of dementia expected to triple, it is critical to identify treatments for dementia. One of the leading causes of dementia is vascular contributions to cognitive impairment and dementia (VCID), and is frequently found co‐morbidly with Alzheimer's disease, the other leading cause of dementia. A modifiable risk factor for VCID is hyperhomocysteinemia (HHcy), which is defined as elevated levels of plasma homocysteine (a non‐protein‐forming amino acid), and most late‐life HHcy is caused by impaired B vitamin absorption. Although HHcy is a risk factor for VCID, the mechanism through which it induces cognitive impairment remains unclear. Method: We used human and mouse brain tissue to determine glial and vascular changes associated with HHcy. Wild‐type (C57Bl6J) mice were placed on a HHcy or control diet for 6, 10, 14, or 18 weeks. Using qPCR, we investigated pro‐ and anti‐inflammatory cytokines. We also determined changes in microglia (IBA‐1) and identified the number of microhemorrhages and cognitive changes. In humans, 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 determined whether the level of plasma homocysteine was associated with microglia (IBA‐1) and astrocytes (GFAP) as well as microhemorrhages and atherosclerosis severity in the frontal and occipital cortices. Both gene expression and protein inflammatory markers were measured to determine associations with plasma homocysteine. Result: In our mouse model of HHcy, we identified a time course of glial changes that first begins with an increase in pro‐inflammatory cytokines and increases in microglial staining at 6 weeks on diet. This is followed by cognitive changes at 10 weeks on diet. Finally, at 14 weeks on diet, we see significant cognitive deficits due to HHcy. In human tissue, HHcy was associated with more microglia but fewer astrocytes, a significant decrease in inflammation and increased microhemorrhages in the frontal cortex. Conclusion: Taken together, this data shows that in both mouse and human tissue, HHcy induces significant glial and vascular pathology that could be driven by inflammatory effects. [ABSTRACT FROM AUTHOR]

Background: Cerebrovascular pathology, often found in autopsy confirmed Alzheimer's disease (AD), is widely believed to exacerbate AD pathophysiology and complicate anti‐AD treatment strategies. Perivascular cells such as astrocytes maintain the microenvironment for proper brain cell function and vascular integrity. Early occurrence of cerebral small vessel disease and astrocyte reactivity could lower the threshold of neuronal disorders and AD. Studies of astrocyte and microvascular pathology and function in the brain have been limited and reaction of astrocytes to vascular damage has yet to be fully understood. Method: AAV‐Gfa‐EGFP or AAV‐Gfa‐GCaMP6 was injected into the barrel cortex of wild type mice followed by glass window installation. After 3 weeks recovery, rhodamine dextran was IV‐injected and intravital brain imaging through two‐photon microscopy was performed at pre‐ and post‐photoinduction. Photoactivation of IV‐injected Rose Bengal dye (RB) technique was used to create vascular pathology. ImageJ was used for data analysis and GraphPad prism was used for statistical analysis. Result: After photoinduction, green fluorescence intensity was increased in blood stream of both EGFP and GCaMP expressing brains. This result indicates a leakage of brain parenchyma component to blood stream. Astrocytic EGFP fluorescence intensity and astrocyte processes complexity were reduced after photoinduction in a time‐dependent manner. Interestingly, astrocyte calcium signaling was strikingly increased after photoinduction and returned back to the baseline at ∼1,000s post‐induction. The highest peak of astrocyte calcium signaling was located near a site of vascular damage, indicated by an increased in rhodamine fluorescence intensity in the brain parenchyma area. Astrocytic GCaMP intensity at the astrocyte endfeet surrounding small vasculature was increased, on the other hand, GCaMP intensity at the astrocyte processes are decreased in a time‐dependent manner. Consistently, we observed a reduction of vascular diameter after photoinduction in both EGFP and GCaMP expressing brains. Conclusion: Acute vascular injury induced by rose bengal photoinduction altered astrocyte integrity and cellular signaling. This injury model resembles vascular constriction and hemorrhages found in human brain that could accelerate neurodegeneration and AD progression. [ABSTRACT FROM AUTHOR]

Background: Tumor necrosis factor alpha (TNFα) is a prominent pleiotropic cytokine with dichotomous effects. Primarily produced in the brain by glial cells, TNFα interacts with two subtypes of TNF receptors (TNFRs) that dictate the cellular response, be that pro‐ or anti‐inflammator. The diverse cellular responses include cell survival, cell death, proliferation, and angiogenesis. Due to the critical role inflammation plays in the pathogenesis of Alzheimer's disease (AD) and vascular contributions to cognitive impairment and dementia (VCID), understanding the specific roles of pleiotropic cytokines like TNFa in dementia‐causing diseases will provide mechanistic and therapeutic insights. Both inhibition of soluble TNFα (sTNFα) and deletion of pro‐inflammatory TNF receptor 1 (TNFR1) have been shown to decrease severity of amyloid deposition in AD models. However, similar studies have not been conducted regarding VCID outcomes. Method: This study sought to examine gene and protein expression of TNFα and the two receptors, TNFR1 and TNFR2, within two mouse models of VCID; the hyperhomocysteinemia (HHcy)‐induced VCID co‐morbid with amyloid deposition and aged Tg2576 with severe cerebral amyloid angiopathy (CAA)‐associated VCID. Measures were taken via qPCR for mRNA assessment and Meso Scale Discovery assays (MSD) for protein quantification. Result: In HHcy‐induced VCID, TNFα mRNA levels were significantly increased regardless of the presence of amyloid deposition. Similarly, protein levels of TNFα trended higher in HHcy groups compared to their controls regardless of amyloid presence. Levels of TNFR1 mRNA trended lower in the HHcy model but was significantly increased in HHcy‐amyloid co‐morbid mice. When investigated in aged Tg2576 mice with CAA, TNFR1 protein expression increased at older timepoints compared to younger, and against wild type controls. Anti‐inflammatory and innate immune‐associated TNFR2 was increased at 20 months in CAA mice when compared to age‐matched controls. Conclusion: These findings display differences in gene and protein expression of TNFa and its receptors in two models of VCID. The unique expression in these models merits further research into the differences in TNF‐mediated inflammation in VCID. Further research is also necessary to determine potential therapeutic advantages of drugs such as TNFα inhibitors in cases of VCID with and without amyloid deposition. [ABSTRACT FROM AUTHOR]

Background: Vascular 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. Method: To 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. Result: Plasma 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. Conclusion: These 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. [ABSTRACT FROM AUTHOR]

Abstract: Superoxide dismutase (SOD) is major cytosolic antioxidant enzyme responsible for dismutation of superoxide anion (). Alterations in SOD expression and activity are associated with various neurological disorders. In the present study, we utilized neuronal HT-22 cells to investigate heat-stressed induced cytotoxicity. Heat stress at 43°C for 30min caused a decrease in SOD-1 mRNA levels, cytoplasmic SOD protein and enzyme activity and a corresponding decline in cell number during a 48h recovery at 37°C. During the recovery phase, there was an increase in reactive oxygen species generation and an increase in NADPH oxidase activity with a corresponding increase in DNA fragmentation and release of cytochrome c from the mitochondria. The increase in ROS accumulation and cell death was abolished by pretreatment with the SOD mimetics EUK-134 and Mn(III)TBAP and the NADPH oxidase inhibitor apocynin. These data suggest that hyperthermia increases ROS generation by increasing NADPH activity and decreasing SOD activity leading to cytotoxicity in HT-22 cells. [ABSTRACT FROM AUTHOR]

This study was conducted to determine the capacity of the asialoglycoprotein receptor on the hepatocyte-derived cell line HepG2 to exhibit an anomeric preference with respect to the D-galacto moiety on cationic liposome membrane-anchored cholesteryl-α-D-galactopyranoside (Choαgal) and cholesteryl-β-Dgalactopyranoside (Cholβgal) in cationic liposome/pGL3 plasmid DNA complexes constructed for non-viral, hepatocyte-directed gene transfer. In addition, cholesteryl-α-D-glucopyranoside (Choαglu) and cholesteryl-β-D-glucopyranoside (Cholβglu) were separately formulated into cationic liposomes at the same mole ratio (11%) to examine the C-4 epimeric selectivity of the asialoglycoprotein lectin for the glucopyranosides displayed in derived lipoplexes. Lipoplex formation was examined by gel retardation, ethidium displacement assays, and transmission electron microscopy. Plasmid DNA was shown to be fully liposome associated and maximally compacted at a liposome:DNA ratio of 6:1 (weight ratio), corresponding to a +/- charge ratio of 1.3 with complexes falling in the 80–200 nm size range, whereas at a 5:1 w/wratio [1.1 (+/-) charge ratio] lipoplexes were somewhat smaller (50–100 nm) but promoted higher transgene activity in HepG2 cells than 6:1 (w/w) lipoplexes, in the following order: Cholβgal > Cholαgal > Cholβglu = Cholαglu. Transgene activity levels in HeLa cells which lack the asialoglycoprotein receptor were approximately 10% of those achieved in HepG2 cells. Moreover, transgene activity in HepG2 cells was reduced by approximately 90% in the presence of excess asialofetuin, a ligand for the asialoglycoprotein lectin. [ABSTRACT FROM AUTHOR]