Fig. 2

RGC hypertrophy and GVD-associated necroptosis in MCI and AD patients. (a) Representative microscopic images of retinal cross-section immunofluorescently labeled for RGC-specific marker RBPMS (green) and nuclei (DAPI blue), in the GCL, showing abnormal morphology of RGCs in MCI and AD retinas as compared with CN controls. Retinas from MCI and AD patients exhibit a hypertrophic cytoplasm (cell soma swelling), and abnormal morphology, including granulovacuolar vesicles degeneration (GVD)-like bodies and nucleus displacement. Red arrows point to nuclear displacement and white arrows indicate granulomatous cytoplasm. (b) Enlarged images of RBPMS⁺ RGCs (green channel only) in the respective diagnostic groups. Scale bars: 20 μm. (c) Quantitative analysis of RBPMS⁺ RGC soma cell size of the three largest cells per microscopic image, in patients with MCI and AD compared to CN controls. (d-g) Quantitative analysis of soma cell size for all RBPMS⁺ RGCs analyzed in total ST region (d), and in Cen (e) Mid-(f) and Far-(g) peripheral subregions, in n = 9 CN, n = 6 MCI, n = 10 AD; total number of cells that were analyzed are indicated below the graphs. (h) Representative microscopic images of retinal cross-section immunofluorescently labeled for the GVD marker, charged multivesicular body protein 2B (CHMP2B, red), RBPMS for RGCs (green), and DAPI for nuclei (blue) in CN, MCI, and AD donors. Colocalization of CHMP2B within RBPMS⁺ RGCs (yellow) is indicated by white arrows. Scale bars: 10 μm. (i) Representative microscopic images of retinal cross-section immunolabeled for the GVD-associated necroptotic marker, phosphorylated mixed lineage kinase domain-like (pMLKL, red), RBPMS (green), and DAPI (blue) in CN, MCI, and AD subjects. Colocalization of pMLKL within RBPMS (yellow) is indicated by white arrows. Scale bars: 10 μm. Individual data points and median, lower, and upper quartiles are shown in violin plots. *P < 0.05, ****P < 0.0001, by one-way ANOVA followed by Tukey’s post-hoc multiple comparison test. Fold changes are shown in red. GCL, Ganglion cell layer; NFL, Nerve fiber layer; IPL, Inner Plexiform Layer; INL, Inner Nuclear Layer; OPL, Outer Plexiform Layer; ONL, Outer Nuclear Layer