Retinal amyloid pathology and proof-of-concept imaging trial in Alzheimer's disease

Abstract

Background: Noninvasive detection of Alzheimer's disease (AD) with high specificity and sensitivity can greatly facilitate identification of at-risk populations for earlier, more effective intervention. AD patients exhibit a myriad of retinal pathologies, including hallmark amyloid β-protein (Aβ) deposits.

Methods: Burden, distribution, cellular layer, and structure of retinal Aβ plaques were analyzed in flat mounts and cross sections of definite AD patients and controls (n = 37). In a proof-of-concept retinal imaging trial (n = 16), amyloid probe curcumin formulation was determined and protocol was established for retinal amyloid imaging in live patients.

Results: Histological examination uncovered classical and neuritic-like Aβ deposits with increased retinal Aβ42 plaques (4.7-fold; P = 0.0063) and neuronal loss (P = 0.0023) in AD patients versus matched controls. Retinal Aβ plaque mirrored brain pathology, especially in the primary visual cortex (P = 0.0097 to P = 0.0018; Pearson's r = 0.84-0.91). Retinal deposits often associated with blood vessels and occurred in hot spot peripheral regions of the superior quadrant and innermost retinal layers. Transmission electron microscopy revealed retinal Aβ assembled into protofibrils and fibrils. Moreover, the ability to image retinal amyloid deposits with solid-lipid curcumin and a modified scanning laser ophthalmoscope was demonstrated in live patients. A fully automated calculation of the retinal amyloid index (RAI), a quantitative measure of increased curcumin fluorescence, was constructed. Analysis of RAI scores showed a 2.1-fold increase in AD patients versus controls (P = 0.0031).

Conclusion: The geometric distribution and increased burden of retinal amyloid pathology in AD, together with the feasibility to noninvasively detect discrete retinal amyloid deposits in living patients, may lead to a practical approach for large-scale AD diagnosis and monitoring.

Funding: National Institute on Aging award (AG044897) and The Saban and The Marciano Family Foundations.

Keywords: Neuroscience; Ophthalmology.

Conflict of interest statement

Conflict of interest: Y. Koronyo, K.L. Black, S.R. Verdooner, and M. Koronyo-Hamaoui are founding members of NeuroVision Imaging. S. Frautschy and G.M. Cole are founding members of Optimized Curcumin Longvida.

Figures

Figure 1. Schematic diagram of donor eye dissection, retinal isolation, and tissue processing for histological analysis.

(A) Anterior chamber extraction from human donor eyeballs, isolation of whole retinas, and vitreous body removal (n = 37 AD patients and controls, Supplemental Table 1). (B) Preparation of retinal flat mounts and sectioning into 12 geometric regions. (C) Representative segmentation and imaging of regional flat mount stained for Aβ (top right). Region dissection of Aβ-positive areas and preparation of horizontal (en face) and vertical (cross) sections for transmission electron microscopy (TEM) analysis (top left). Microscopic image shows a vertical TEM image with ultrastructure of retinal Aβ plaque in a confirmed AD patient (bottom left). Illustration of paraffin-embedded retinal cross section prepared from Aβ-immunoreactive areas, showing cellular layers (bottom right). P. Pole, posterior pole; O.D., optic disc; M, macula; F, fovea.

Figure 2. Burden and distribution of Aβ deposits in postmortem retinas of AD patients.

(A) Representative micrographs from flat-mount retinas of definite AD patients (n = 8) and age- and sex-matched non-AD controls (CTRLs; n = 7) at varying ages, stained with anti-Aβ42 mAb (12F4) and the standard peroxidase technique (plaques visible as dark brown spots). Scale bar: 20 μm. Human donor number, sex (male [M] or female [F]), and age in years (y) are shown (for additional details on donor eyes and brains, see Supplemental Table 1). High-magnification images display diverse morphology of Aβ aggregates, including diffuse, compact, and “classical” mature plaques. Scale bar: 10 μm. (B) AD patients brain sections and their respective retinal flat mounts stained with 12F4 mAb. Retinal plaques are generally smaller but similar in morphology to brain plaques (arrowheads). Retinal Aβ deposits are apparent inside blood vessels (bv), perivascular, and along the vessel walls (purple arrowheads). Scale bar: 20 μm. (C) Correlation analyses using Pearson’s correlation coefficient (r) test between retinal 12F4+-plaque burden and cerebral plaques (Gallyas silver or thioflavin-S staining), including mean plaque burden from 7 brain regions (see Methods), primary visual (PV) cortex (Ctx.) only, and entorhinal Ctx. only, in a subset of AD patients and CTRLs (n = 8). (D) Anti-Aβ antibody and Longvida curcumin (Cur) fluorescent staining of Aβ deposits in cortical and retinal tissues from the same patient subset. Sudan black B (SBB) was applied to quench autofluorescence. Retinal Aβ plaques positive for 6E10 single staining. Colocalization of curcumin and 6E10 in cortical and retinal Aβ plaques, showing patterns unique to each staining method. Scale bar: 10 μm (left retina and brain); 5 μm (right retina). (B and D) Representative paired retinal and brain samples from n = 5 AD patients and n = 2 controls. (E) Distribution of plaques in large retinal regions (n = 16 AD patients and controls), using fluorescent-based (top; scale bar: 5 μm) and peroxidase-based (bottom; scale bar: 20 μm) staining. (F) Qualitative geometric hot spot regions for retinal Aβ deposits found in AD patients (S, superior; T, temporal; I, inferior; N, nasal; cumulative data from multiple experiments). (G) Quantitative 12F4-immunoreactive (IR) area of Aβ42-containing plaques (geometric regions ST1-3) in a subset of definite AD patients (n = 8) and age- and sex-matched controls (n = 7). Group mean and SEM are shown. **P < 0.01, unpaired 2-tailed Student’s t test.

Figure 3. Congo red–positive amyloid fibrils and Gallyas silver–stained neuritic-like plaques found in retinas of AD patients.

(A) Amyloid deposits stained with Congo red detected in flat-mount retinas of AD patients (top left, n = 3). Birefringence (apple-green) of Congo red–stained retinas under polarized light indicates presence of amyloid fibrils (bottom left). Scale bar: 25 μm. Congo red–positive amyloid plaques in AD patient retinas (right). Scale bar: 100 μm. Two intersecting blood vessels surrounded (along blood vessels and perivascular) by extensive Congo red–positive staining; matched control retina was negative (data not shown). Background blue counterstain is Toluidine blue. (B–E) Gallyas silver stain in Paraffin-embedded retinal cross sections from AD patients (n = 12) and matched CTRLs (n = 8). Scale bar: 20 μm, unless indicated otherwise. (B) Age-matched controls exhibited intact retinal tissue that lacked major protein aggregates. (C) AD-associated neuropathologies in the retina (red arrowheads), notably observed in the ganglion cell layer (GCL). A compact plaque near a blood vessel (bv; left). A classic Aβ plaque and compact deposits (middle); a higher-magnification image is shown in the inset. Neuritic components of senile plaques in GCL (right); a higher-magnification image is shown below (scale bar: 10 μm). (D) Retinal deposits in GCL near and surrounding a blood vessel. (E) Intracellular/soma-positive silver stain aggregates and nuclear-dominant silver stain (red arrowheads) are observed in GCL and INL (scale bar: 5 μm).

Figure 4. Aβ deposits associated with neuronal loss are detected in the retinas of AD patients.

(A and B) Paraffin-embedded retinal cross sections from superior quadrants of AD patients (n = 12) and matched CTRLs (n = 8) stained with anti-Aβ42 mAbs (12F4) and peroxidase-based labeling (brown). Hematoxylin counter stain for nuclei (violet). (A) Retinas of controls were clear of Aβ immunoreactivity. (B) Retinas of AD patients contained a multitude of Aβ deposits (arrowheads), especially in the ganglion cell layer (GCL). Marked loss of retinal cells apparent in the GCL, inner nuclear layer (INL), and outer nuclear layer (ONL); areas of nuclei loss are indicated by asterisks. Scale bar: 20 μm. Higher-magnification images are shown. Intracellular cytoplasmic Aβ (top; arrow). Aβ deposits near and inside blood vessel walls (middle; bv; arrowheads); these vascular and perivascular deposits are frequent in GCL. Scale bar: 10 μm. A compact multicore Aβ deposit found in GCL (bottom; scale bar: 5 μm). (C) Nissl staining of retinal cross sections from a definite AD patient and matched CTRLs (n = 17 subjects; experiment repeated 3 times). Altered Nissl neuronal staining is observed in AD patients; changes in cytoplasmic staining patterns (chromatolysis) that could associate with neuronal loss are observed in retinal GCL, INL, and ONL. Scale bar: 20 μm. (D) Quantitative Nissl neuronal count and total area in a subset of AD patients (n = 9) and age- and sex-matched CTRLs (n = 8). Percentage change compared with CTRLs is in red. Group mean and SEM are shown. *P < 0.05, **P < 0.01, unpaired 2-tailed Student’s t test. (E–G) Representative images from n = 8 AD patients and n = 8 matched CTRLs; fluorescent images from AD patient retinas showing curcumin-positive Aβ deposits colocalized with various anti-Aβ mAbs (4G8, 6E10, 12F4), recognizing diverse N′- and C′-terminus epitopes within the Aβ sequence (arrowheads). (E) Aβ deposits detected in the ONL, above the outer limiting membrane (OLM), and in the GCL near and inside blood vessel walls (left, arrowheads; scale bar: 20 μm). Colocalization of curcumin and 4G8 in a retinal Aβ plaque near DAPI nuclear staining demonstrates each unique staining pattern (right; scale bar: 5 μm). (F) Intracellular/somatic Aβ immunoreactivity (arrow) and colocalization of 6E10 with curcumin (arrowheads; left; scale bar: 10 μm). Compact multicore Aβ deposits (right; scale bar: 5 μm. (G) Curcumin-positive Aβ deposits colocalized with the anti-Aβ mAbs (12F4) in the GCL/IPL (arrowheads). Scale bar: 10 μm.

Figure 5. Ultrastructures of Aβ deposits in human AD retina.

Transmission electron microscopy (TEM) analyses of vertical (cross; A–C) and en face (horizontal; D) retinal sections from definite AD patients (n = 3; experiments repeated 3 times). Retinas were prestained with anti-Aβ42 mAb (12F4) and a high-sensitivity immunoperoxidase-based system and DAB substrate chromogen. (A) TEM image showing ultrastructure of Aβ plaque (pl), fibrils (fib), and protofibrils (pfib) near a blood vessel (bv) in vertical sections. Scale bar: 1 μm. Higher-magnification image (right), indicating presence of 10- to 150-nm-wide Aβ fibrils as well as protofibrils and Aβ deposits (abd; scale bar: 50 nm). (B) Fibrillar Aβ ultrastructure surrounded by multiple Aβ deposits. Scale bar: 200 nm. (C) Aβ plaque-like structure, marked by an asterisk and bordering red line, in close proximity to basement membrane (bm) of a blood vessel (scale bar: 0.5 μm). Higher-magnification image showing Aβ plaque-like area, with structures resembling paranuclei containing annular oligomers (arrowhead; scale bar: 40nm). (D) TEM images of en face sections demonstrating retinal Aβ plaque ultrastructures, with radial fibrillar arms emanating from a central dense core found in the innermost retinal layers. Darker black signal represents condensed Aβ aggregate core. Scale bar: 0.5 μm.

Figure 6. Development of a noninvasive retinal amyloid imaging method using curcumin labeling in live human patients.

(A) Curcumin regimen and retinal imaging protocols in living human subjects (n = 16 AD and CTRLs and n = 2 AMD patients). Oral Longvida curcumin administration for 2 or 10 days. Subjects’ retinas were imaged with modified ophthalmoscopes prior to (day 0, baseline image) and after curcumin intake, per the regimen. (B) Repeated regional fundus imaging in a mild AD patient receiving curcumin for 10 days. Increase of curcumin fluorescent intensity due to retinal amyloid deposits (white spots) is observed from baseline (day 0) through days 1–10. Decreased curcumin signal is observed at day 29 (washout). (C) Spot line profile analysis of curcumin fluorescence from an individual plaque (marked in B by red arrows), showing increased signal at days 1 and 10 versus baseline levels. At day 29, fluorescent signal decays to baseline levels (washout). Representative spot line profile from n = 3 patients. (D) Representative retinal fundus images from a moderate AD patient (top) and their pseudocolor images (bottom); arrowheads mark individual plaques, and circles demarcate a cluster of deposits. Increased spot number and area from baseline image to day 2 and 10 images were quantified by ImageJ analysis (bottom). (B–D) Representative images from all human subjects (n = 16; Supplemental Table 2). (E) Longvida curcumin pharmacokinetic (PK) analyses in living healthy controls receiving curcumin for 10 days (n = 6 subjects; all or a subset analyzed for each time point, as shown). Tissue curcumin levels in red blood cells (RBCs) and free curcumin in plasma peaked at day 10 (P < 0.0001). Group mean and SEM are shown. *P < 0.05, ****P < 0.0001 for comparison between day 0 and days 3, 10, or 30, by 1-way ANOVA and Tukey’s post test. Logarithmic transformation analysis of covariance (subject × day) demonstrated that curcumin levels increased with duration of treatment in RBCs (R2 = 0.654; day: P = 0.001; subject: P = 0.024) and plasma (R2 = 0.559; day: P = 0.003; subject: P = 0.09). (F) A quantitative longitudinal retinal curcumin imaging in a representative healthy control and mild AD patient, both receiving 2-day curcumin regimen. Exponential decay of integrated fluorescent intensity occurred after day 2. Decay rate = 10.4% per day, half-life = 6.3 days, offset IFI = 21.5. Scale bar: 200 μm.

Figure 7. Noninvasive detection of retinal amyloid deposits in live AD patients.

(A) Multistep manual postacquisition image processing and analysis to detect and quantify spots with increased curcumin fluorescence signal in the retina (superior quadrant; representative images of n = 16 human subjects). Scale bar: 800 μm. (B) Representative images demonstrate detection of retinal curcumin spots in two live AD patients and contrasting minimal spots in a healthy control (Normal) and patient suffering from vascular dementia (Non-AD Dementia). Curcumin fluorescence fundography of superior temporal region (ST) reveals amyloid deposits often in retinal peripheries. Amyloid deposit detection in inferior temporal (IT) region of another AD patient. Regions of interest are indicated by white squares. Scale bar: 400 μm. (C) Higher-magnification image of the above regions of interest. Red circles in the top images highlight retinal spots of curcumin-increased fluorescence. Scale bar: 400 μm. Bottom images display postprocessing spot number and fluorescent area (μm2). (D and E) Representative optical coherence tomography (OCT) of a selected curcumin-positive region in an AD patient with no maculopathy (repeated experiments in n = 3 patients). Scale bar: 200 μm. (D) Curcumin fluorescence fundography indicating certain retinal amyloid plaque with red arrows. Green lines delineate region of OCT segmentation. (E) Retinal cross section by OCT reveals amyloid plaque in outer retinal layers. (F) High-magnification OCT image displays curcumin-positive deposit located above retinal pigment epithelium (RPE), along with intact RPE and Bruch’s membrane.

Figure 8. Increased retinal amyloid index in AD patients — a proof-of-concept human trial.

(A–D) Representative automated image-processing sequence after repeated fundus image acquisition at day 0 (Baseline) and day 2, showing retinal superior temporal region in an AD patient. (A) Collection of Z-stack scanning laser ophthalmoscope (SLO) fundus images. Each, at baseline and on day 2, underwent illumination correction, alignment of baseline to day 2, and contrast enhancement. (B) Pseudocolor image for visualization. (C and D) Multistep automated postacquisition image processing. (E–G) Threshold defining increased curcumin fluorescent signal in left eye superior hemisphere (LS) and calculation of retinal amyloid index (RAI) scores in a mild AD patient and age-matched CTRL. (E) Blue lines are 1:1 reference, and green lines represent the threshold level, determined at 500 counts and above (red spots are above the threshold). (F) Color-coded spot overlay images. Red spots are above threshold and considered curcumin-positive amyloid deposits, green spots exceed 1:1 reference but not threshold, and blue spots fall below reference. (G) Heatmap images with red spot centroids (left). Day 2 combined overlay images show heatmaps with regions of interest (right). The same automated image processing and analysis was applied on all human subjects. (H) Significant Pearson’s r correlation between RAI scores and retinal spot number in all data set (n = 16 living subjects, see Supplemental Table 2). (I) RAI scores in comparison between AD patients (n = 10) and non-age-matched CTRLs (n = 6) (left) and a comparison of RAI scores in an age-matched subgroup of AD patients (n = 6) and CTRLs (n = 5, see Table 1) (right). Group mean and SEM are shown. **P < 0.005, ***P < 0.0005, unpaired 2-tailed Student’s t test. (J) Pearson’s r correlation analysis of RAI scores and age. (K) RAI and MMSE cognitive scores in AD patient group. Scale bar: 800 μm (A–D); 400 μm (F and G).