The fluorescent ligand bTVBT2 reveals increased p-tau uptake by retinal microglia in Alzheimer's disease patients and AppNL-F/NL-F mice

Abstract

Background: Amyloid beta (Aβ) deposits and hyperphosphorylated tau (p-tau) accumulation have been identified in the retina of Alzheimer's disease (AD) patients and transgenic AD mice. Previous studies have shown that retinal microglia engulf Aβ, but this property decreases in AD patients. Whether retinal microglia also take up p-tau and if this event is affected in AD is yet not described. In the current study, we use the p-tau-specific thiophene-based ligand bTVBT2 to investigate the relationship between disease progression and p-tau uptake by microglia in the retina of AD patients and App^NL-F/NL-F knock-in mice, an AD mouse model known to demonstrate extracellular Aβ plaques and dystrophic neurites in the brain from 6 months of age.

Methods: Evaluation of bTVBT2 specificity and its presence within microglia was assessed by immunofluorescent staining of hippocampal sections and flat-mount retina samples from non-demented controls, AD patients, 3-, 9-, and 12-month-old App^NL-F/NL-F knock-in mice and 12- and 18-month-old wild type (WT) mice. We used ImageJ to analyze the amount of bTVBT2 inside Iba1-positive microglia. Co-localization between the ligand and p-tau variant Ser396/Ser404 (PHF-1), Aβ, phosphorylated TAR DNA binding protein 43 (pTDP-43), and islet amyloid polypeptide (IAPP) in the brain and retina was analyzed using confocal imaging.

Results: Confocal imaging analysis showed that bTVBT2 binds to PHF-1- and AT8-positive aggregates inside retinal microglia, and not to Aβ, pTDP-43, or IAPP. The density of bTVBT2-positive microglia was higher in cases with a high Aβ load compared to those with a low Aβ load. This density correlated with the neurofibrillary tangle load in the brain, but not with retinal levels of high molecular weight (aggregated) Aβ40 or Aβ42. Analysis of App^NL-F/NL-F knock-in mouse retina further showed that 50% of microglia in 3-month-old App^NL-F/NL-F knock-in mice contained bTVBT2. The percentage significantly increased in 9- and 12-month-old mice.

Conclusion: Our study suggests that the microglial capability to uptake p-tau in the retina persists and intensifies with AD progression. These results also highlight bTVBT2 as a ligand of interest in future monitoring of retinal AD pathology.

Keywords: Alzheimer’s disease; Retina; Tauopathy; Thiophene-based ligands.

Fig. 1

bTVBT2 inside microglia in the human retina. A representation of the human retina showing the region used in this study (inferior-nasal, green field) is seen in A. The molecular structure of bTVBT2 is shown in B. Confocal image of the orthogonal view of a bTVBT2-positive signal inside an Iba1-positive microglia is shown in C. Graph in D shows a higher number of bTVBT2-positive microglia density in the retina of Aβ_high cases compared to Aβ_low cases. Data was analyzed using the Mann–Whitney U test and is presented as a median with a 95% confidence interval. Significant difference at * = p < 0.05. A case with low amyloid-beta load (Aβ_low) showing no bTVBT2 inside retinal microglia is seen in E, and a case with high amyloid-beta load (Aβ_high) with bTVBT2-positive deposits inside retinal microglia (white arrows) is shown in F. Scale bar in B represent 20 µm and scale bar in D and E represent 20 µm. Scatter plot in G shows the correlation between bTVBT2-positive microglia density in the retina and NFT staging in the brain. Data was analyzed using Spearman’s rank correlation coefficient

Fig. 2

bTVBT2 co-localization with tau in human hippocampus. Cornu ammonis 1 (CA1) from an AD case stained against tau (A), bTVBT2 (B), and merged image (C), showing little (long arrows) or no (short arrows) co-localization of bTVBT2 with PHF-1-positive NFTs. Hippocampal sections stained with bTVBT2 together with tau (D), p-tau181 (E), AT8 (F), and PHF-1 (G) showing co-localization of bTVBT2 with tau-positive dystrophic neurites. Scale bars in A–C and D–G represent 10 µm and 20 µm, respectively

Fig. 3

bTVBT2 co-localization with AT8 and PHF-1 but not p-tau181 in human retina. Confocal image of the orthogonal view of a flat-mount retina sample from an AD stained against p-tau181 (A) (purple indicated with an arrow), PHF-1 (B) (purple indicated with arrow), and AT8 (C) (purple) together with bTVBT2 (red in A–C) and Iba-1 (green in A–C). The three p-tau stainings are merged with a staining against DAPI (purple, red, green, and blue in A–C). Scale bars in (A–C), represent 5 µm

Fig. 4

bTVBT2 co-localization with tau in mouse retina. Image in A shows a confocal orthogonal view of a bTVBT2-positive signal inside an Iba1-positive retinal microglia in a 3-month-old App^{NL−F/NL−F} knock-in mouse. The graph in B shows that the proportion of bTVBT2-positive microglia in the retina increases significantly in 9–12-month-old App^{NL−F/NL−F} knock-in mice compared to 3-month-old App^{NL−F/NL−F} knock-in mice, whereas 12- to 18-month-old WT mice have similar levels as 3-month-old App^{NL−F/NL−F} knock-in mice. Data is presented as mean ± SD and was analyzed using one-way ANOVA, followed by the Tukey test with (n = 3) comparisons. Each point represents the mean of (n = 50) microglia in each group (n = 6). Significant difference at ** = p < 0.01. Scale bar in A represents 5 µm