Brain Vasculature Accumulates Tau and Is Spatially Related to Tau Tangle Pathology in Alzheimer's Disease

Abstract

Insoluble pathogenic proteins accumulate along blood vessels in conditions of cerebral amyloid angiopathy (CAA), exerting a toxic effect on vascular cells and impacting cerebral homeostasis. In this work we provide new evidence from three-dimensional human brain histology that tau protein, the main component of neurofibrillary tangles, can similarly accumulate along brain vascular segments. We quantitatively assessed n=6 Alzheimer's disease (AD), and n=6 normal aging control brains and saw that tau-positive blood vessel segments were present in all AD cases. Tau-positive vessels are enriched for tau at levels higher than the surrounding tissue and appear to affect arterioles across cortical layers (I-V). Further, vessels isolated from these AD tissues were enriched for N-terminal tau and tau phosphorylated at T181 and T217. Importantly, tau-positive vessels are associated with local areas of increased tau neurofibrillary tangles. This suggests that accumulation of tau around blood vessels may reflect a local clearance failure. In sum, these data indicate tau, like amyloid beta, accumulates along blood vessels and may exert a significant influence on vasculature in the setting of AD.

Keywords: Alzheimer’s disease; blood vessels; cerebral amyloid angiopathy; cerebral vasculature; neurofibrillary tangles; tau.

Figure 1. Three-dimensional histology reveals the presence of vascular-associated tau.

Confocal fluorescence microscopy images showing raw data from the inferior temporal gyrus of an AD (A) and a control (B) donor. Images are immunolabeled for vasculature (GLUT1, green), neurons (HuD, magenta), nuclei (DAPI, blue), and AT8 tau (white). (C–E) Vascular tau accumulation in blood vessels AD donors compared with a (F) control donor. Images show staining for vasculature (green) and AT8 tau (white) in a 40 μm thick z-slice.

Figure 2. Examination of tau along individual blood vessels throughout the cortex.

(A) An overall schema of the method used to quantify vascular tau. Blood vessels are first traced in virtual reality (magenta) and are shown overlying the original GLUT1-positive blood vessel imaging data (green). Tracing allows for the isolation of individual blood vessels and their surround, including tau pathology (white) ≤ 100 μm from the blood vessel surface (example is from AD 5 vessel 5). Subsequently, quantification of tau intensity along and away from the blood vessel surface was conducted. (B) Measures of the average tau intensity at the vessel surface (within 3 microns) per donor and cortical layer. (C) The average AT8-positive tangle density and (D) HuD-positive neuron density per cubic mm was also measured for each cortical layer. (E) The average diameter of vessels measured per donor. Dots represent individuals, bars represent means +/− SEM. (F) Separate tissue was labeled with antibodies to GLUT1, SMA, and tau show areas of tau accumulation on blood vessels that are also SMA-positive (indicated by asterisks).

Figure 3.. Mapping tau accumulation on blood vessels.

(A) Heatmaps showing log normalized tau intensity within 3 microns from the surface of each segmented blood vessel (n=107 AD, n=127 control). Rows are individual vessels and columns are tau intensity measures along the vessel length. (B) Data is binned every 10 microns along the blood vessel’s surface and shows the mean intensity of each bin, normalized to the mean tau intensity of the whole image. Red asterisks highlight example vessels shown in panels C–H. (B) A schematic showing how data binning and surface tau measures were acquired. Each bin was then percentile ranked by AT8 tau staining intensity. (C, D, E) Example of isolated blood vessel (green) and tau labeling (white). (F, G, H) Corresponding maps of tau intensity along the vessel surface. Color corresponds to percentiles (deciles).

Figure 4.. Tau intensity is related to distance from the blood vessel surface.

Blood vessel segments were grouped according to surface tau intensity by percentiles (deciles) and the amount of tau immunolabeling is plotted by distance from blood vessel surface. Values for each donor (colored lines) are normalized to the average tau value in the whole image (background) such that a 0% change (grey dashed line) means the tau labeling intensity is no different than the average level of tau in the whole image.

Figure 5.. Post-translationally modified tau is enriched in AD blood vessels.

(A) Isolation of blood vessels from the inf. temp. gyrus of n=3 control and n=5 AD brains shows that tau is enriched in vasculature and can be visualized with multiple antibodies including total tau = DAKO rabbit polyclonal. Quantification of total signal per lane for (B) total tau, (C) Tau13 n-terminal antibody, (D) Tau46 c-terminal antibody, (E) phospho-T181 tau, (F) phospho-S202 tau, (G) phospho-T217 tau and (H) phospho-T231 tau. All values normalized to the average of controls. One-tailed t-test *p<0.05, **p<0.01. Error bars = means +/− SEM.

Figure 6.. NFT and neuron density analysis.

Examples of blood vessels showing NFT accumulation near the blood vessel surface in samples (A) AD 2, (B) AD 1, (C) AD6, and (D) control 3. (E) Isolated blood vessel with surrounding tau pathology (AT8, white) and surrounding neurons (HuD, magenta). (F) NFTs and neurons were identified and segmented using Ilastik. Visualization of segmentation masks were generated using Imaris and with a value of 1 μm to smooth the surfaces. (G) A custom MATLAB script was developed to calculate the distance of each segmented tangle and neuron from the surface of each blood vessel. Plots show the blood vessel (blue), its calculated centerline for object distance calculations (red), and objects colored according to their distance along the blood vessel. (H) Plot shows the percent of neurons with NFT in regions near (0–30 microns) blood vessels with varying amounts of surface tau. Repeated measures ANOVA P value = 0.037, R²= 0.47. Dots represent individuals, bars represent means +/− SEM.