Fig. 3
From: Retinal ganglion cell vulnerability to pathogenic tau in Alzheimer’s disease
Phosphorylated tau pathology in RGCs of MCI and AD patients. (a) Representative microscopic images of retinal cross-sections immunolabeled for paired helical filament of tau (PHF-1, red), RBPMS (green), and DAPI for nuclei (blue) in CN, MCI, and AD subjects. Scale bars: 10 μm. PHF-tau is usually not found within RGCs of MCI and AD patients, as shown in top and bottom left panels and indicated by white arrows. (b) Representative microscopic images of retinal cross-sections immunofluorescently co-labeled for PHF-tau (PHF-1, red) and hyperphosphorylated (p)tau at S396 epitope (pS396-tau, green), with nuclei (DAPI, blue) in MCI and AD donors. Scale bars: 10 μm. RGCs appear to stain for pS396-tau, and not PHF-tau, as shown by cell morphology and location in the GCL, while other layers (IPL, INL, OPL) show colocalization (yellow) of PHF-tau and pS396-tau. Scale bars: 10 μm. (c) Representative micrographs of retinal cross-sections immunolabeled for pS396-tau (red), RGCs (RBPMS, green), and nuclei (DAPI, blue) in CN, MCI, and AD donors. Scale bars: 20 μm. (d) Retinal micrographs immunolabeled for RBPMS RGCs (white), pS396-tau (red), amacrine and RGCs marker - parvalbumin (green), and nuclei (DAPI, blue) in CN, MCI, and AD subjects. Colocalization of pS396-tau in parvalbumin+ amacrine cells (yellow arrows, left panel) and RBPMS+ RGCs (white arrows, middle panel) are shown. Scale bars: 20 μm. (e) High-magnification microscopic images depicting pS396-tau (red) accumulation in hypertrophic RBPMS+ RGCs (green); white arrows indicate pS396-tau-laden RGCs in MCI and AD patients (yellow for colocalization). Scale bars: 20 μm (left) and 10 μm (right). Bottom panels show separate channels for each pS396-tau and RBPMS. (f) A representative microscopic image of peroxidase-based staining for pS396-tau isoforms (brown) within retinal layers, and specifically, in RGCs (by morphology and location, red arrows) of an MCI patient. Scale bar: 50 μm. (g) Quantification of pS396-tau percent area in the ST retina (n = 9 CN, n = 6 MCI, n = 10 AD). (h) Pearson’s correlation coefficient (rP) analysis between retinal pS396-tau and RBPMS+ RGCs. (i-j) Cell count of pS396-tau+ RGCs in (i) Central, Mid-, and Far-peripheral retinal subregions, and (j) total ST retina (n = 19–25). Individual data points and median, lower, and upper quartiles are shown in violin plots. *P < 0.05, **P < 0.01, ****P < 0.0001, by one-way or two-way ANOVA followed by Tukey’s post-hoc multiple comparison test. Fold changes are shown in red. F, female; M, male; Age (in years); Ethnicity: W, White; NFL, Nerve fiber layer, GCL, ganglion cell layer; IPL, Inner Plexiform Layer; INL, Inner Nuclear Layer, OPL, Outer Plexiform Layer; ONL, Outer Nuclear Layer; RGC, Retinal ganglion cells