Endothelial Cells Are Heterogeneous in Different Brain Regions and Are Dramatically Altered in Alzheimer's Disease.

Vascular endothelial cells play an important role in maintaining brain health, but their contribution to Alzheimer's disease (AD) is obscured by limited understanding of the cellular heterogeneity in normal aged brain and in disease. To address this, we performed single nucleus RNAseq on tissue from 32 human AD and non-AD donors (19 female, 13 male) each with five cortical regions: entorhinal cortex, inferior temporal gyrus, prefrontal cortex, visual association cortex, and primary visual cortex. Analysis of 51,586 endothelial cells revealed unique gene expression patterns across the five regions in non-AD donors. Alzheimer's brain endothelial cells were characterized by upregulated protein folding genes and distinct transcriptomic differences in response to amyloid ß plaques and cerebral amyloid angiopathy. This dataset demonstrates previously unrecognized regional heterogeneity in the endothelial cell transcriptome in both aged non-AD and AD brain.SIGNIFICANCE STATEMENT In this work, we show that vascular endothelial cells collected from five different brain regions display surprising variability in gene expression. In the presence of Alzheimer's disease pathology, endothelial cell gene expression is dramatically altered with clear differences in regional and temporal changes. These findings help explain why certain brain regions appear to differ in susceptibility to disease-related vascular remodeling events that may impact blood flow.

Tau induces blood vessel abnormalities and angiogenesis-related gene expression in P301L transgenic mice and human Alzheimer's disease.

Mixed pathology, with both Alzheimer's disease and vascular abnormalities, is the most common cause of clinical dementia in the elderly. While usually thought to be concurrent diseases, the fact that changes in cerebral blood flow are a prominent early and persistent alteration in Alzheimer's disease raises the possibility that vascular alterations and Alzheimer pathology are more directly linked. Here, we report that aged tau-overexpressing mice develop changes to blood vessels including abnormal, spiraling morphologies; reduced blood vessel diameters; and increased overall blood vessel density in cortex. Blood flow in these vessels was altered, with periods of obstructed flow rarely observed in normal capillaries. These changes were accompanied by cortical atrophy as well as increased expression of angiogenesis-related genes such as Vegfa, Serpine1, and Plau in CD31-positive endothelial cells. Interestingly, mice overexpressing nonmutant forms of tau in the absence of frank neurodegeneration also demonstrated similar changes. Furthermore, many of the genes we observe in mice are also altered in human RNA datasets from Alzheimer patients, particularly in brain regions classically associated with tau pathology such as the temporal lobe and limbic system regions. Together these data indicate that tau pathological changes in neurons can impact brain endothelial cell biology, altering the integrity of the brain's microvasculature.

Partial reduction of microglia does not affect tau pathology in aged mice.

BACKGROUND: Activation of inflammation pathways in the brain occurs in Alzheimer's disease and may contribute to the accumulation and spread of pathological proteins including tau. The goal of this study was to identify how changes in microglia, a key inflammatory cell type, may contribute to tau protein accumulation and pathology-associated changes in immune and non-immune cell processes such as neuronal degeneration, astrocyte physiology, cytokine expression, and blood vessel morphology. METHODS: We used PLX3397 (290 mg/kg), a colony-stimulating factor receptor 1 (CSF1R) inhibitor, to reduce the number of microglia in the brains of a tau-overexpressing mouse model. Mice were fed PLX3397 in chow or a control diet for 3 months beginning at 12 months of age and then were subsequently analyzed for changes in blood vessel morphology by in vivo two-photon microscopy and tissues were collected for biochemistry and histology. RESULTS: PLX3397 reduced microglial numbers by 30% regardless of genotype compared to control diet-treated mice. No change in tau burden, cortical atrophy, blood vessels, or astrocyte activation was detected. All Tg4510 mice were observed to have an increased in "disease-associated" microglial gene expression, but PLX3397 treatment did not reduce expression of these genes. Surprisingly, PLX3397 treatment resulted in upregulation of CD68 and Tgf1ß. CONCLUSIONS: Manipulating microglial activity may not be an effective strategy to combat tau pathological lesions. Higher doses of PLX3397 may be required or earlier intervention in the disease course. Overall, this indicates a need for a better understanding of specific microglial changes and their relation to the disease process.

Endothelial Cells are Heterogeneous in Different Brain Regions and are Dramatically Altered in Alzheimer's Disease.

Vascular endothelial cells play an important role in maintaining brain health, but their contribution to Alzheimer's disease (AD) is obscured by limited understanding of the cellular heterogeneity in normal aged brain and in disease. To address this, we performed single nucleus RNAseq on tissue from 32 AD and non-AD donors each with five cortical regions: entorhinal cortex, inferior temporal gyrus, prefrontal cortex, visual association cortex and primary visual cortex. Analysis of 51,586 endothelial cells revealed unique gene expression patterns across the five regions in non-AD donors. Alzheimer's brain endothelial cells were characterized by upregulated protein folding genes and distinct transcriptomic differences in response to amyloid beta plaques and cerebral amyloid angiopathy (CAA). This dataset demonstrates previously unrecognized regional heterogeneity in the endothelial cell transcriptome in both aged non-AD and AD brain. Significance Statement: In this work, we show that vascular endothelial cells collected from five different brain regions display surprising variability in gene expression. In the presence of Alzheimer's disease pathology, endothelial cell gene expression is dramatically altered with clear differences in regional and temporal changes. These findings help explain why certain brain regions appear to differ in susceptibility to disease-related vascular remodeling events that may impact blood flow.

Tau reduction in aged mice does not impact Microangiopathy.

Microangiopathy, including proliferation of small diameter capillaries, increasing vessel tortuosity, and increased capillary blockage by leukocytes, was previously observed in the aged rTg4510 mouse model. Similar gene expression changes related to angiogenesis were observed in both rTg4510 and Alzheimer's disease (AD). It is uncertain if tau is directly responsible for these vascular changes by interacting directly with microvessels, and/or if it contributes indirectly via neurodegeneration and concurrent neuronal loss and inflammation. To better understand the nature of tau-related microangiopathy in human AD and in tau mice, we isolated capillaries and observed that bioactive soluble tau protein could be readily detected in association with vasculature. To examine whether this soluble tau is directly responsible for the microangiopathic changes, we made use of the tetracycline-repressible gene expression cassette in the rTg4510 mouse model and measured vascular pathology following tau reduction. These data suggest that reduction of tau is insufficient to alter established microvascular complications including morphological alterations, enhanced expression of inflammatory genes involved in leukocyte adherence, and blood brain barrier compromise. These data imply that 1) soluble bioactive tau surprisingly accumulates at the blood brain barrier in human brain and in mouse models, and 2) the morphological and molecular phenotype of microvascular disturbance does not resolve with reduction of whole brain soluble tau. Additional consideration of vascular-directed therapies and strategies that target tau in the vascular space may be required to restore normal function in neurodegenerative disease.

Cerebrovascular Senescence Is Associated With Tau Pathology in Alzheimer's Disease.

Alzheimer's Disease (AD) is associated with neuropathological changes, including aggregation of tau neurofibrillary tangles (NFTs) and amyloid-beta plaques. Mounting evidence indicates that vascular dysfunction also plays a key role in the pathogenesis and progression of AD, in part through endothelial dysfunction. Based on findings in animal models that tau pathology induces vascular abnormalities and cellular senescence, we hypothesized that tau pathology in the human AD brain leads to vascular senescence. To explore this hypothesis, we isolated intact microvessels from the dorsolateral prefrontal cortex (PFC, BA9) from 16 subjects with advanced Braak stages (Braak V/VI, B3) and 12 control subjects (Braak 0/I/II, B1), and quantified expression of 42 genes associated with senescence, cell adhesion, and various endothelial cell functions. Genes associated with endothelial senescence and leukocyte adhesion, including SERPINE1 (PAI-1), CXCL8 (IL8), CXCL1, CXCL2, ICAM-2, and TIE1, were significantly upregulated in B3 microvessels after adjusting for sex and cerebrovascular pathology. In particular, the senescence-associated secretory phenotype genes SERPINE1 and CXCL8 were upregulated by more than 2-fold in B3 microvessels after adjusting for sex, cerebrovascular pathology, and age at death. Protein quantification data from longitudinal plasma samples for a subset of 13 (n = 9 B3, n = 4 B1) subjects showed no significant differences in plasma senescence or adhesion-associated protein levels, suggesting that these changes were not associated with systemic vascular alterations. Future investigations of senescence biomarkers in both the peripheral and cortical vasculature could further elucidate links between tau pathology and vascular changes in human AD.

Experimental evidence for the age dependence of tau protein spread in the brain.

The incidence of Alzheimer's disease (AD), which is characterized by progressive cognitive decline that correlates with the spread of tau protein aggregation in the cortical mantle, is strongly age-related. It could be that age predisposes the brain for tau misfolding and supports the propagation of tau pathology. We tested this hypothesis using an experimental setup that allowed for exploration of age-related factors of tau spread and regional vulnerability. We virally expressed human tau locally in entorhinal cortex (EC) neurons of young or old mice and monitored the cell-to-cell tau protein spread by immunolabeling. Old animals showed more tau spreading in the hippocampus and adjacent cortical areas and accumulated more misfolded tau in EC neurons. No misfolding, at any age, was observed in the striatum, a brain region mostly unaffected by tangles. Age and brain region dependent tau spreading and misfolding likely contribute to the profound age-related risk for sporadic AD.