This study demonstrates the feasibility to noninvasively detect and quantify amyloid deposits in the retinas of live human subjects by using a solid lipid curcumin fluorochrome and a modified point SLO. In a proof-of-concept trial, the mean RAI score in AD patients was elevated compared with that of the healthy controls. A histological examination of retinas from neuropathologically confirmed AD patients revealed certain layer and geographical distribution of Aβ deposits, especially in previously disregarded retinal regions. These results expand past identification of retinal Aβ plaques and Aβ 42 peptides in the retinas of AD patients (26, 39, 40, 42) and further demonstrate the existence of classical retinal deposits and morphologies relevant to AD neuropathology, including neuritic-like plaques and NFT-like structures. Retinal deposits frequently appeared in clusters and, although smaller, mirrored Aβ plaque morphology and vascular pathology in the brain. Retinal plaques were also associated with retinal neuronal loss in these patients. Electron microscopy analysis and Congo red staining verified the existence of retinal Aβ fibrils and protofibrils and the potential existence of Aβ oligomers. Importantly, AD patients showed a substantial increase in Aβ 42 -containing deposits as compared with age- and sex-matched healthy controls. Collectively, these findings suggest the utility of noninvasive retinal amyloid plaque imaging as a surrogate biomarker of AD.

Retinal Aβ deposits in AD patients were frequently concentrated in the mid- and far-periphery of the superior quadrants, matching the pattern we detected in living patients via retinal curcumin imaging. These findings, together with superior retina neuronal loss may elucidate previous reports of axonal loss and NFL thinning in these patients (24–26, 29, 76–78), detected mostly in the same retinal regions that amyloid pathology was evident. Similarly, Blanks and colleagues described an extensive loss of neurons, typically in the superior quadrant and the mid- and far-peripheries (16). The significant NFL reduction in the superior quadrant is consistent with recent findings of mRGC loss and Aβ accumulation in the same retinal regions (26). This increase in Aβ pathology in the superior quadrant, along with severe thinning of the NFL and loss of mRGCs, also known as intrinsically photosensitive RGCs containing melanopsin and implicated in regulation of circadian rhythm (26), may help explain sleep disturbances and visual field dysfunctions in AD, even at early stages (26, 32, 44, 79). Furthermore, the existence of Aβ deposits in regions in which rod cells are abundant could contribute to the impairments in visual motion and low-contrast sensitivity documented in AD patients (80, 81). In addition, the reason for the geographical predilection of Aβ accumulation and associated pathology in AD for the superior quadrant is unknown; we postulate that unique tissue properties and structural parameters of this retinal region perhaps deem it more susceptible to AD pathology. In the brain, Aβ plaques are also detected earlier and more frequently in certain brain regions; cerebral Aβ accumulation is a complex multifactorial process caused by increased production/aggregation or decreased clearance of Aβ (reviewed in Zuroff et al., ref. 82). It is possible that differences in retinal blood flow, vessel diameter, choroid thickness, tissue permeability, and light stimulation in this retinal region are some of the factors that may adversely influence Aβ aggregation or clearance.

Histological assessment of Aβ pathology in the human retina, including plaques containing Aβ 40 and Aβ 42 isoforms, was achieved by applying certain isolation techniques and tissue preparation and biochemical labeling protocols as well as screening of large retinal areas (Figure 1). A couple of studies that were unable to detect Aβ in the human AD retina analyzed horizontal cross sections spanning a narrow strip from the nasal to temporal quadrants (41, 43), rather than screening large regions in flat mounts, thus limiting their analysis to regions that are largely spared of amyloid pathology in AD patients. Therefore, while Ho and colleagues (43) were unable to detect Aβ, p-tau, or α-synuclein in any ocular tissue of AD or Parkinson’s disease (PD) patients, considerably more independent studies in fact reported the presence of Aβ deposits in the retina and lenses (11, 39, 40, 42, 51, 83) as well as identified p-tau (41) and α-synuclein (84) in the retinas of AD and PD patients, respectively. Future studies should apply suitable techniques and comprehensive examination of flat-mount retinas to study additional pathological hallmarks of AD in the retina.

Analysis of retinal cross sections derived from the superior quadrants indicated accumulation of Aβ deposits mostly in the innermost retinal layers (GCL, IPL, and INL). Gallyas silver stain evidenced classical plaques, neuritic (senile-like) plaques, and NFT-like structures in the GCL, although their exact identity requires further study employing specific antibodies against pTau and NFT. In addition, neuronal reduction observed in INL, ONL, and especially GCL of AD patients, quantified by Nissl stain, was accompanied by retinal Aβ pathology. Indeed, the abundance of deposits in the GCL may explain the ganglion cell degeneration and abnormal electroretinogram patterns reported in AD patients (26, 85, 86) and animal models of AD (22, 54, 57, 87). The finding of intracellular Aβ accumulation in AD retinas, either somatic or perinuclear, suggests an alternate mode of Aβ-mediated toxicity. Recent studies have demonstrated the detrimental effects of intracellular Aβ, by way of oligomerization into pathological forms within the endoplasmic reticulum (88), as well as the formation of neuritic plaques from degenerating neurons containing intracellular Aβ (89). Ultrastructure examination of Aβ 42 -containing deposits by TEM revealed the existence of retinal Aβ 42 fibrils, protofibrils, and other intermediates in the pathway of Aβ deposition. We detected assemblies with morphology resembling paranuclei containing annular oligomers (72, 73), the building blocks of Aβ protofibrils and fibrils. The concurrent existence of various Aβ structures in the retina of the same patient may suggest that Aβ accumulates in different disease stages. This supports the hypothesis that different types of Aβ deposits develop independently rather than sequentially from the same plaque type (90). It is likely that multiple factors contribute to the formation of each type of retinal deposit, but the underlying mechanisms of plaque and nonfibrillar oligomer formation in the retina are not yet understood. This uncertainty merits future investigation of the various plaque phenotypes and distribution of each type in the human retina during normal aging and disease progression (AD).

In this study, we frequently found retinal Aβ deposits in association with blood vessels similar to cerebral vascular amyloid pathology (91). As retinal and cerebral microvasculature share many morphological and physiological properties (34), these vascular amyloid findings may explain the well-documented narrowing of venous blood column diameter and reduced blood flow in both the retinas (19, 20, 33, 76, 92) and brains (93–95) of AD patients. Recently, a mechanism for such phenomena was proposed in a study using murine models of AD demonstrating that vascular amyloid could harden blood vessel walls and decrease blood flow (96). Still, larger studies are required to confirm the association between cerebral amyloid angiopathy and retinal vascular amyloid pathology.

To detect amyloid deposits in retinas of living patients, we translated our retinal curcumin optical imaging approach, previously developed in rodent models of AD (40, 51). Using a modified ophthalmic device, we analyzed single, cluster, and large-area deposits with increased curcumin fluorescent intensity in human subjects. Retinal amyloid deposits at baseline (before curcumin intake) generally exhibited faint autofluorescence. Previous studies also report weak autofluorescence near plaques in the brains of ADtg mice that had never received curcumin (40, 97). The low-grade autofluorescence of plaques in the absence of curcumin may result from oxidative damage-induced lipid peroxidation production of aldehydes that create autofluorescent Schiff’s bases in and near aggregated amyloids (98). In addition, pigmentation (i.e., lipofuscin accumulation) often occurs in the retinas of elderly individuals and can also generate autofluorescent spots (99, 100). Therefore, measuring increased fluorescence of curcumin spots allows for specific detection of Aβ-containing deposits in vivo. Importantly, we previously confirmed the identity of retinal amyloid plaques that were imaged in vivo with curcumin in ADtg mouse models by subsequent ex vivo immunolabeling, demonstrating specific anti-Aβ labeling in the same plaques (40). The future availability of postmortem retinal tissues from previously in vivo–imaged human subjects will warrant investigation in order to determine the correlation between RAI scores and retinal histopathology of Aβ deposits.

Curcumin has been reported to have high affinity and specificity for the β-pleated sheets of Aβ and moderately stains NFTs that are positive for PHF-tau. Moreover, curcumin has high affinity to Aβ 42 , oligomers, and fibrils, which are tightly linked to AD (62–68). Curcumin intensely stains the central dense amyloid core in Aβ fibrils and plaques, as was also previously demonstrated in the brain of murine models of AD (101). Further, it labels the nonfibrillar forms of Aβ 40 and Aβ 42 based on its specific binding to the Aβ-hexapeptide 16KLVFFA21 amino acid sequence domain (64, 102). Previous reports showed that curcumin readily crosses the blood-retinal and -brain barriers in murine models and specifically labels Aβ aggregates (40, 51, 103). We found here that Longvida oral curcumin formulation consistently colabeled Aβ plaques with various sequence-specific mAbs in postmortem human retinas and brains, including 12F4, which recognizes the C′-terminus Aβ 42 alloforms and not APP. Curcumin showed a distinct labeling pattern that was similar to the amyloid spots observed in living patients. Curcumin is considered as generally recognized as safe by the FDA based on previous clinical trials (74, 75, 104), and the Longvida curcumin used in this study caused minimal to no adverse effects. Longvida’s safety has been demonstrated in pharmacokinetic studies in humans (105) and extensive toxicity testing in animals (106), and its efficacy has been demonstrated in two clinical trials (74, 75). The choice of curcumin as an amyloid contrast agent in living patients was further supported by the experiments presented here (Figure 6 and Supplemental Figures 4–5 and 7), previously in vivo and ex vivo in ADtg mice (40, 51), and in a study using OCT imaging in MCI patients (107). The high bioavailability of optimized curcumin was established by increased tissue absorption of free rather than glucuronidated or other curcumin metabolites from the gastrointestinal system to the blood stream.

An OCT examination in a small subset of patients showed increased curcumin fluorescent spots indicative of amyloid deposits in AD patients, located in peripheral regions above an intact RPE layer and Bruch’s membrane and in the absence of maculopathy. These findings are distinct from drusen described in AMD patients (108–110). AMD is a common ocular disease described by sub-RPE drusen deposits, Bruch’s membrane thickening, and outer retina degeneration of the RPE and photoreceptors within the macula centralis (111). Aβ immunoreactivity was detected in 30%–40% of soft drusen in AMD patients, often occurring in the posterior pole below a disrupted RPE and Bruch’s membrane and along with maculopathy (108–110). Our preliminary data on two AMD patients, using the same retinal curcumin imaging protocol, showed deposits with increased curcumin intensity within large diffuse lesions at the posterior pole/central retina, rather than plaques in the peripheral retina, as detected in AD patients. Further, drusen are often seen as hypofluorescent spots (108–110). Yet, this study cannot rule out the existence of pseudodrusen, also known as subretinal drusenoid deposits, in the retinas of AD patients, although these are mostly located in the macula in AMD and differ from our in vivo and histological observations in the AD retina. Future studies should determine through large sample sizes whether the location of retinal amyloid pathology in AMD and AD is different, which could help with differential diagnosis.

Glaucoma is another common ocular disease that was previously implicated as sharing commonalities with AD (reviewed in Hart et al., ref. 11). While retinal pathology found in AD has revealed NFL and GCL loss in addition to hallmark Aβ plaque and tauopathy, predominantly in the innermost retinal layers of the peripheral superior quadrant, glaucoma pathology indicated a particular pattern of central retina neuronal loss in the GCL along with optic disc cupping. This and other pathological evidence relating the two diseases remains quite controversial. Thus, further investigation is needed before positing claims of shared abnormalities.

Within our small AD patient group, we did not obtain a correlation between MMSE scores and RAI scores. Although analyses performed on a limited sample size are challenging to interpret, this result is comparable to amyloid burden in the brain. Cerebral amyloid-PET imaging studies predominantly indicate that amyloid burden plateaus early in the course of AD (112). Here, in a subset of patients, for which retinal and cerebral amyloid burden was available, we found a significant correlation between superior temporal Aβ 42 deposits and Gallyas silver– and thioflavin-S–positive plaques in the brain, which was seemingly stronger for the primary visual cortex. The apparent correlation between retinal and brain plaques calls for further study in larger groups of AD patients to determine their relationship during disease progression as well as to analyze the association with specific brain regions.

While the timing of Aβ plaque appearance in the retinas of AD patients is unknown, our previous histological analysis revealed its existence in early-stage cases (e.g., probable and possible AD) (40). In addition, we detected retinal Aβ plaques in flat mounts from ADtg mice at least 2 months prior to their presence in the respective hippocampi and cortices (40). These data may suggest that Aβ accumulation in the retina is an early event in AD pathogenesis, yet large longitudinal studies in presymptomatic AD and MCI patients are required to confirm retinal Aβ plaque as an early biomarker. Further, the capability of the RAI score to detect AD with high sensitivity and specificity should be evaluated in follow-up studies with a greater number of cases compared with age- and sex-matched controls. Such studies could provide a better understanding of AD manifestation in the retina and further validate our methodology by determining whether retinal amyloid burden is a reliable biomarker of AD.

In summary, we present here a quantitative and detailed histological report of retinal Aβ deposits and the pathological hallmarks of AD, including their distribution and ultrastructure in AD patients, together with the demonstrated feasibility to noninvasively image and quantify retinal amyloid deposits in living patients. Such retinal amyloid imaging technology, capable of detecting discrete deposits at high resolution in the CNS, may present a sensitive yet inexpensive tool for screening populations at risk for AD, assessing disease progression, and monitoring response to therapy.