Retinal Aβ Pathology in Alzheimer’s Patients
One decade ago, and more than a century following the identification of Aβ plaques in the postmortem brain of the first person diagnosed with AD, Auguste Deter, AD-specific pathological hallmarks were shown for the first time in the human retina (Koronyo-Hamaoui et al., 2011). In this original study, Aβ plaques were identified in all flatmount retinas isolated from 13 cases with definite and probable AD, as confirmed by both brain pathology and clinical reports (Figures 2A–C’). Retinal Aβ-plaque pathology in these patients was in stark contrast to minimal to no pathology found in the retina of age- and gender-matched cognitively normal individuals (Figures 2B–C’) (Koronyo-Hamaoui et al., 2011; La Morgia et al., 2016; Koronyo et al., 2017).
FIGURE 2  Increased retinal Aβ42 deposition correlates with cerebral amyloid plaque burden in Alzheimer’s patients. (A) Representative micrographs from an AD brain and (B) flat-mount retinas from a cognitively normal (CN) subject and (C) AD patient stained with anti-Aβ42 mAb (12F4). Although smaller in size, retinal Aβ plaques are similar in morphology to brain plaques. Scale bar: 20 μm. (C) High-magnification images reveal diffuse, compact, and “classical” mature plaque morphology of retinal Aβ aggregates. Scale bar: 10 μm. (D) Quantitation of retinal Aβ42 plaque burden, measured by 12F4 immunoreactive area, in AD patients (n = 8) and sex-/age-matched CN control subjects (n = 7). Data shown as group mean ± SEM. **P < 0.01, unpaired 2-tailed Student’s t-test. (E,F) Pearson’s correlation coefficient tests between retinal Aβ42 plaque load (12F4-immunoreactivity) and mean cerebral neuritic plaque burden (e; r = 0.87, P = 0.0048, n = 8; severity score of Gallyas silver staining) or regional plaque burden either in the entorhinal (F; black symbols; r = 0.84, P = 0.0092, n = 8) or primary visual cortex (F; orange symbols; r = 0.84, P = 0.0097, n = 8). Reproduced from Koronyo et al. (2017) with permission of ASCI via Copyright Clearance Center. This and two subsequent studies on human cohorts of over 50 patients and control donor eyes, examining retinal flatmounts and cross-sections with Aβ-specific monoclonal antibodies (12F4, 11A5-B10, 6E10, 4G8), anti-Aβ dyes (i.e., Curcumin, Thioflavin-S, Congo-Red) and Gallyas silver stain, showed that all neuropathologically confirmed AD patients exhibited Aβ deposits in the retina (Koronyo-Hamaoui et al., 2011; La Morgia et al., 2016; Koronyo et al., 2017). Interestingly, through scanning of retinal flatmounts, the team discovered a non-uniform manifestation of Aβ deposits across the human retina. Plaques were more often detected in peripheral regions, especially in the superior and inferior quadrants (Koronyo et al., 2017).
A quantitative histological analysis of whole-mount retinas in a subset of confirmed AD patients compared to age- and sex-matched cognitively normal controls revealed a significant 4.7-fold increase in Aβ42-containing retinal plaque burden in patients (Figure 2D; Koronyo et al., 2017). Another group, using a sample of retinas isolated from 10 neuropathologically and clinically confirmed AD and 10 control patients, demonstrated a significant 2.7-fold increase in the number of retinal Aβ42 plaques that were also found to be larger in volume relative to deposits detected in normal control tissue (Grimaldi et al., 2019). Importantly, although retinal Aβ plaques are typically smaller in size compared to brain plaques, their burden in the retina significantly correlated with severity of plaque pathology in the brain (Figures 2E,F; Koronyo et al., 2017). In particular, retinal amyloid deposits were more strongly correlated with plaque burden in the primary visual cortex and the entorhinal cortex (Figure 2F; Koronyo et al., 2017).
Transmission electron microscopy (TEM) analysis of (12F4+)Aβ42-positive immunoreactivity in retinal tissues from AD patients revealed the ultrastructure of Aβ in plaques, fibrils, protofibrils and annular oligomer-like forms (Figures 3A–B’; Koronyo et al., 2017). Gallyas silver stain further exposed the existence of retinal neuritic-like plaques. While marked increases in retinal Aβ pathology were noted in AD patients as compared with age-matched cognitively normal individuals, retinal plaques in patients frequently appeared in clusters and preferentially in the mid- and far-peripheral regions (La Morgia et al., 2016; Koronyo et al., 2017). These findings suggest that regional and geometric differences in plaque density should be considered when examining retinal tissue from patients. Moreover, the use of traditional histological techniques in retinal cross sections of limited regions could account for the few studies unable to consistently detect Aβ in the AD retina (Schön et al., 2012; Ho et al., 2014). This challenge emphasizes the necessity to standardize approaches for analyzing AD-related pathology across diverse topographical regions of the human retina. Indeed, following untraditional histological protocols developed by Koronyo and colleagues, three additional independent groups were able to detect Aβ deposits in the retina of confirmed AD patients (Tsai et al., 2014; den Haan et al., 2018; Grimaldi et al., 2019).
FIGURE 3  Ultrastructure of Aβ deposits in AD retina identified by transmission electron microscopy (TEM). (A–B’) Representative TEM images of retinal cross-sections from definite AD patients showing (A) ultrastructure of Aβ plaque (pl), fibrils (fib) and protofibrils (pfib) near a blood vessel (bv). Scale bar: 1 μm. (A’) High-magnification image showing Aβ fibrils, protofibrils and Aβ deposits (abd). Scale bar: 50 nm. (B) Aβ plaque-like deposits (pl; demarcated by red line), near basement membrane (bm) of a blood vessel (bv). Scale bar: 0.5 μm. (B) High magnification image of region marked by red asterisk in (B) showing dense Aβ deposition with structural similarity to annular oligomers (red arrowhead). Scale bar: 40 nm. All sections were prestained with anti-Aβ42 mAb (12F4) and peroxidase-based system and DAB substrate chromogen. Reproduced from Koronyo et al. (2017) with permission of ASCI via Copyright Clearance Center. Prior examinations of normal aged eyes demonstrated Aβ immunoreactivity in the sub-retinal pigment epithelium (RPE) (Loffler et al., 1995) as well as toxic Aβ oligomers in drusen in the macular RPE of aged and AMD patients (Johnson et al., 2002; Anderson et al., 2004; Luibl et al., 2006). As it relates to AD, an early biochemical evaluation of Aβ40 and Aβ42 alloforms in retinal tissues of patients showed their existence in the human AD retina, albeit without comparing to levels in control retinas nor assessing correlation with respective brain levels (Alexandrov et al., 2011). Importantly, a recent study corroborated these findings of amyloidogenic Aβ40 and Aβ42 alloforms in the retina of AD patients (Schultz et al., 2020). The study measured Aβ40 and Aβ42 levels in retinal and hippocampal tissues of human cohorts with neurodegenerative diseases and compared between ApoE ε4 carriers and non-carriers (Schultz et al., 2020). Results from this study showed higher levels of retinal and hippocampal Aβ40 and Aβ42 in individuals with AD-related pathological changes and ApoE ε4 carriers. Further, levels of both alloforms in the retina correlated with their counterparts in the hippocampus, as well as with NFT and Aβ plaque burden severity (Schultz et al., 2020).
A recent histological study confirmed the presence of retinal plaques in 6 AD patients and 6 healthy controls, including finding similar sized 12F4+Aβ42-containing deposits, and gave additional insight into the spatial distribution and subtypes of Aβ aggregates in the human retina (den Haan et al., 2018). Aβ-positive immunoreactivity and deposits were found in various cell layers in postmortem retinas of AD patients, particularly the INL; deposits were found within horizontal, amacrine, and Müller cells (den Haan et al., 2018). Analyses of retinal pathology in AD donors indicated that Aβ deposits were more abundant in the inner retinal layers, concentrating in the NFL and GCL (Koronyo et al., 2017). Aβ42 was present in both fibrillar and proto-fibrillar forms, confirmed with TEM and Birefringence (apple-green) of Congo red-stained retinas under polarized light (Koronyo et al., 2017). More recently, Aβ40 was quantified and mapped in a larger cohort of postmortem human AD retinas (n = 47), showing significant increases in both retinal vascular and abluminal Aβ40 in AD patients as compared with matched controls (Shi et al., 2020). Retinal Aβ40 was especially abundant in the inner retinal layers of the central retina. Increased Aβ40 in the retina of AD patients as compared with cognitively normal individuals was further validated by biochemical ELISA analysis (Shi et al., 2020).
Overall, these growing studies confirm the presence of disease-associated Aβ species in the human retina and highlight the striking similarities between retinal and cerebral vulnerability to hallmark AD pathologies.