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

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 AppNL-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 AppNL-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 AppNL-F/NL-F knock-in mouse retina further showed that 50% of microglia in 3-month-old AppNL-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.