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

Abstract

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.

Keywords: Alzheimer’s disease; angiogenesis; blood vessels; brain microvessels; tau.

Fig. 1.

Altered blood vessel morphology, number, and density in aged Tg4510 mice. In vivo two-photon microscopy of 15-mo-old wild-type control (A) and Tg4510 (B) mice injected with i.v. fluorescein–dextran revealed abnormal spiral morphologies in Tg4510 mice (B, Inset, asterisks denote spirals). [Scale bar, (A and B) 50 μm and (B, Inset) 20 μm.] Images were captured from multiple fields of view per animal, and the average number of blood vessels per cubic millimeters was calculated (C) in addition to the average diameter of vessels per mouse (D). (E) A histogram of average diameter measures in each genotype shows a shift toward smaller-diameter vessels in Tg4510 mice. Individual diameter measures per mouse can be found in Fig. S1. (F) The percentage of blood vessels without red blood cell flow is indicated. (G) Representative images of small-diameter vessels with rhodamine-6G–labeled adherent leukocytes appearing red against green fluorescein–dextran-labeled sera. (Scale bar, 10 μm.) n = 3 mice per genotype. *P < 0.05, **P < 0.01.

Fig. 2.

Time course of cortical blood vessel changes. (A) Separate groups of wild-type control and Tg4510 mice were imaged at 2, 9, 12, 15, or 18 mo of age (n = 3–5 mice per group). (Scale bar, 20 μm.) Blood vessel (B) density, (C) length, and (D) capillary diameters were compared at each age. (E) Cresyl violet-stained sections were used to measure cortical atrophy. (Scale bar, 400 μm.) (F) Nonnormalized blood vessel density plotted against cortical thickness highlights the relationship between these variables in Tg4510. The blood vessel density and cortical thickness ±2 SDs in control animals are indicated by shaded regions for a reference. *P < 0.05, **P < 0.01.

Fig. 3.

Altered hypoxia and angiogenesis genes in Tg4510 mice. Quantitative PCR arrays examining 84 genes related to hypoxia and angiogenesis were assessed using samples from n = 3 Tg4510 and n = 3 wild-type control mice. The results of total cortex expression changes in Tg4510 mice are plotted (A), where significant P values appear above the dotted line and fold changes >2 appear to the left or right of the dotted line. (B) In two separate cohorts of mice, whole hemispheres were dissociated into a cell suspension that was further separated into either microglial cells or astrocytes and then endothelial cells. The purity of each cell population was assessed by cell type-specific qPCR (C, E, and G) before running on the hypoxia and angiogenesis gene array (D, F, and H).

Fig. 4.

PAI-1 expression in tau-overexpressing mice. (A) PAI-1, the protein product of Serpine1, was detected in 2-, 9-, 12-, and 15-mo cortical homogenates of Tg4510 mice by Western blotting (n = 3–4 mice per genotype). The 2- and 9-mo samples were run on one blot and 12- and 15-mo samples on a second. (B) PAI-1 bands were quantified by densitometry and normalized to actin loading control. (C) Coronal sections from 15-mo-old mice were probed for microglia (Iba-1), tau (MC1), and PAI-1. In Tg4510 mice, colocalization of PAI-1 was most prominent in microglial cells and cells containing misfolded MC1-positive tau aggregates. (Scale bar, 20 μm.) (D) Additional labeling for both microglia (m) and blood vessels (b.v.) using tomato-lectin indicates PAI-1 is also localized to blood vessels in 15-mo Tg4510 mice. DAPI was used to label nuclei. (Scale bar, 10 μm.) Error bars represent ±SDs. *P < 0.05, **P < 0.01. A.U., arbitrary unit.

Fig. 5.

Effects of amyloid-beta on blood vessels. (A) We imaged 15-mo-old mice from the APP/PS1-Tg4510 line and the related Tg21221 line (from APP/PS1-Tg21221 litters). (Scale bar, 20 μm.). n = 3–4 mice per genotype. (B) Cresyl violet-stained sections were used to measure cortical atrophy, which was observed in Tg4510 and APP/PS1-Tg4510 mice but not APP/PS1, Tg21221, or wild-type controls. (Scale bar, 200 μm.) (C) Blood vessel density was assessed from two-photon images and normalized to cortical thickness for each mouse. (D and E) PAI-1 was increased in mice carrying the Tg4510 (P301L tau) or Tg21221 (wild-type tau) transgene but not in APP/PS1-only mice. Beta-actin is shown as a protein loading control, and NeuN indicates neuronal protein. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

Bubble chart analysis of gene expression changes in human brain with high neurofibrillary tangle load. (A) RNA expression datasets from the AMP-AD knowledge portal were assessed for differential expression of the following 14 genes, comparing Braak stages V/VI (B3) to Braak stages 0/I/II (B1): Egr1, Hmox, Lax, Lgals3, Met, Plau, Pgf, Atr, Eif4ebp, Hif1an, Mmp9, Serpine1, Slc16a3, and Vegfa. Differentially expressed genes were identified at an FDR of 25% and are listed in Dataset S2. Data are plotted by brain region. The size of each bubble is determined by the −log10(P value) such that highly significant changes are represented by larger bubbles. The smallest correspond to a P value of 0.051 and the largest 2.9 × 10E-8. The color of each bubble indicates the direction and magnitude of gene expression fold change. Genes from each dataset are shown as separate bubbles—in regions with RNA-seq and microarray data (dorsolateral prefrontal cortex, frontal pole, inferior frontal gyrus, PHG, and superior temporal gyrus), multiple bubbles per gene indicate changes in multiple datasets. NA, nucleus accumbens; OVC, occipital visual cortex; SPL, superior parietal lobe; TP, temporal pole. (B) Western blot of cortical homogenates from cases with low neurofibrillary tangle load (Control) versus high neurofibrillary tangle load (AD) confirmed an increase of the Serpine1 protein product PAI-1. Beta-actin is shown for loading control. **P < 0.01. A.U., arbitrary unit.