Age-Specific Retinal and Cerebral Immunodetection of Amyloid-β Plaques and Oligomers in a Rodent Model of Alzheimer’s Disease

Abstract

Background:

Amyloid-β soluble oligomers (Aβo) are believed to be the cause of the pathophysiology underlying Alzheimer’s disease (AD) and are normally detected some two decades before clinical onset of the disease. Retinal pathology associated with AD pathogenesis has previously been reported, including ganglion cell loss, accumulation of Aβ deposits in the retina, and reduction of nerve fiber layer thickness as well as abnormalities of the microvasculature.

Objective:

This study’s aim is to better understand the relationship between brain and retinal Aβo deposition and in particular to quantify levels of the toxic Aβo as a function of age in the retina of a rodent model of AD.

Methods:

Retinas and brain tissue from 5×FAD mice were stained with Congo red, Thioflavin-T (Th-T), and Aβ plaque-specific and Aβo-specific antibodies.

Results:

We show that retinas displayed an age-dependent increase of Th-T-specific amyloid fibrils. Staining with anti-Aβ antibody confirmed the presence of the Aβ plaques in all 5×FAD retinas tested. In contrast, staining with anti-Aβo antibody showed an age-dependent decrease of retinal Aβo. Of note, Aβo was observed mainly in the retinal nuclear layers. Finally, we confirmed the localization of Aβo to neurons, typically accumulating in late endosomes, indicating possible impairment of the endocytic pathway.

Conclusion:

Our results demonstrate the presence of intraneuronal Aβo in the retina and its accumulation inversely correlated with retinal Aβ plaque deposition, indicating an age-related conversion in this animal model. These results support the development of an early AD diagnostic test targeting Aβo in the eye.

Keywords

Anti-oligomer antibody Alzheimer’s disease amyloid-β oligomers retina retinal immunodetection 5×FAD mice

INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder associated with a gradual decline in cognitive function, memory loss, abnormal behavior, and reduction of brain volume [1 –4]. The neuropathological lesions observed in the brain of AD patients include extracellular deposition of amyloid-β (Aβ) plaques; intracellular deposition of hyperphosphorylated tau protein in the form of neurofibrillary tangles, ubiquitin, cerebral amyloid angiopathy, severe synaptic loss, and neuronal death [2 , 6]. Brain accumulation of misfolded/aggregated Aβ is believed to be one of the major pathological constituents for the development of the disease. Aβ peptide is derived from a larger protein, namely the amyloid-β protein precursor (AβPP) [5]. AβPP is enzymatically cleaved into amyloidogenic and non-amyloidogenic entities. In AD, the β- and γ-secretase sequentially cleave AβPP and produce the Aβ peptide fragments (36–43 amino acids) [7 –9], that aggregate and lead to accumulation of brain deposits or plaques [6 , 11]. Enzymatic cleavage leads to the formation of two major isoforms of Aβ; Aβ₄₀ (∼80–90%) and Aβ₄₂ (∼5–10%) [5 , 13]. In sporadic and familial AD, three major assemblies of Aβ have been reported [11 , 15], including monomeric Aβ composed of low molecular weight dimers and trimers; soluble oligomers, containing 12–24 monomers which become elongated to form protofibrils; and insoluble fibrils [16, 17]. Aβ soluble oligomers (Aβo) are neurotoxic and responsible for triggering the pathophysiology of AD [18 –21]. Experimental detection of Aβo in peripheral tissues and/or blood precedes its central accumulation in the brain by some two decades [22, 23], highlighting Aβo as a potential early diagnostic marker.

Various diagnostic approaches have been used for the detection of AD, which include neuro-clinical and neuropsychological examinations, blood and cerebrospinal fluid (CSF) screening, and brain imaging. However, most of these approaches are non-specific, and some are invasive, expensive, and time-consuming. Thus, there is an urgent need for a non-invasive and cost-effective diagnostic screen to identify AD-affected subjects in the preclinical or early clinical stages. Visual disturbances are often an early complaint reported by AD patients [24, 25], and have been linked to abnormalities of ocular physiology [26 –32]. Patients experience altered color vision [33, 34], peripheral vision loss [35 –37], and modified sensitivity to contrast and sometimes visual acuity [33, 38]. Alteration of retinal morphology has been reported in AD patients and include changes to the vasculature [26], optic nerve head [39], ganglion cell and axon loss [40, 41], and thinning of the retinal nerve fiber layer [28 , 40–50]. A study by O’Bryhim and colleagues using optical coherence tomography and angiography demonstrated that individuals with preclinical AD displayed early retinal architecture and vascular changes [51]. Another study demonstrated the feasibility to noninvasively detect and quantify, using a clinical scanning laser ophthalmoscope, amyloid deposits in the retina of human subjects given an oral solid lipid curcumin fluorochrome [47]. Koronyo et al. show in some cases that Aβ deposits in the retina was associated with blood vessels similar to cerebral vascular amyloid pathology [47] supporting other studies which indicate alteration to retinal vasculature [52 –54]. While Aβ deposits have been reported in the retina, how such levels change with age is not well documented. Furthermore, the relationship between retinal and brain soluble Aβo and insoluble oligomers is not well understood.

In this report, we assess in the 5×FAD mouse model age-related Aβo and Aβ plaque burden in the retina and brain of 6 to 17-month-old animals. Oakley and colleagues developed the 5×FAD, a transgenic rodent model that co-expresses five mutations [AβPP K670N/M671L (Swedish) + I716V (Florida) + V717I (London) and PS1 M146L+L286V] that displays intraneuronal accumulation of Aβ₄₂ before plaque formation [55]. We show that retinal accumulation of Aβo was similar to brain in the early stage of the disease and their levels were inversely proportional to the age-related increased in Aβ plaque increase.

We showed that Aβo was colocalized to late-endosomal compartments in retinal neurons, indicating impairment in their ability to process and degrade this oligomeric species. Our study highlights the possibility of targeting Aβo in the eye a preclinical diagnostic test for AD.

MATERIALS AND METHODS

Animals

The 5×FAD transgenic mice were made by co-injecting two vectors encoding AβPP (with Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations and PSEN1 (with M146L and L286V mutations), each driven by the mouse Thy1 promoter. This strain does not carry the retinal degeneration allele Pde6b^rd1. The 5×FAD mouse model rapidly develops severe amyloid pathology. Plaques spread throughout the hippocampus and cortex by six months of age. Synapse degeneration, neuronal loss and deficits in spatial learning are observed at approximately four months [55]. Age-matched wild type littermates were used as controls (Supplementary Table 1).

All procedures followed the requirements of the National Health and Medical Research Council of Australia statement for the use of animals in research, the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision, and were approved by the Howard Florey Animal Ethics Committee (13-068-UM). Mice were housed with free access to water and normal rodent chow (Barastoc, Melbourne, VIC, Australia) in the Melbourne Brain Centre (Parkville, VIC, Australia). Room temperature was maintained at 21°C, with animals exposed to a 12 h light/dark cycle (on at 7 AM, <50 lux inside the cage).

Tissue collection and histological assessment of brains and eyes

All mice (Supplementary Table 1) were perfused with saline and 10% neutral buffered formalin. Mouse brains and eyes were then fixed in 10% neutral buffered formalin, dehydrated using graded ethanol, washed with xylene, and finally embedded in paraffin. 3×6μm sections of each brain and retinal tissue were cut with a microtome (Thermofisher Scientific, Waltham, MA, USA) and then processed for routine hematoxylin and eosin (H & E), Congo red, and Thioflavin T (ThT) staining as well as immunohistochemistry and immunofluorescence. Sections were deparaffinized with xylene and rehydrated through graded alcohols and finally deionized water.

Congo red Aβ staining

Sections were placed in Congo red (Leica biosystems, Wetzlar, Germany) working solution for 20 min then rinsed in 5–8 changes of deionized water. This was followed by staining in Gill llHematoxylin (Leica biosystems) for 1–3 min and rinsing in three changes of deionized water. Sections were dehydrated in two changes of 95% alcohol and three changes of absolute alcohol for 1 min each. Finally, sections were cleared in two changes of xylene and mounted in a xylene miscible medium. Amyloid fibrils appeared as dull to brick red under light microscopy (Olympus CX 43, Shinjuku, Tokyo, Japan) and apple green birefringence under polarized light (Olympus CX 43).

Thioflavin-T staining of Aβ plaques

Following deparaffinization with xylene and ethanol, tissue sections were incubated in filtered 1% aqueous ThT (Sigma-Aldrich, St. Louis, MO, USA) for 8 min at room temperature. Sections were then rinsed in three changes of deionized water and mounted in aqueous mounting media (Agilent, Santa Clara, CA, USA). Finally, slides were sealed with clear nail polish and stored in a cold and dark place. Generally, ThT binds to the side chain channels along the long axis of amyloid fibrils. Upon binding to amyloid fibrils, ThT has a strong signal at excitation and emission maxima of 450 and 482 nm, respectively under fluorescence microscopy (Olympus VS 120).

Immunohistochemical assessment of Aβ plaques and soluble oligomers

Sections were pre-treated with antigen retrieval method (1× citrate buffer for 20 min in a water bath; pH 6) to expose the target antigen. Sections were then treated with 90% formic acid for 5 min at room temperature followed with cell membrane permeabilization which was achieved using 1% triton X for 1 min prior to addition of 0.3% H₂O₂ for 15 min to inactivate endogenous peroxidases. Sections were then blocked with Protein Block Serum-Free (Agilent, Santa Clara, CA, USA) for 15 min. Sections were then stained for 1 h with the following primary antibodies in PBS: mouse purified 4G8 anti-Aβ against 17–24 of Aβ peptide (1 : 500; Bio legend, San Diego, CA, USA) or A11 rabbit anti-Aβo Antibody (1 : 250; Merck Millipore, Burlington, MA, USA) respectively. Sections were also stained with IgG1 isotype control (BRIC 222 recognizing CD44 [56] or IgG2b isotype control (BRIC 126 recognizing CD47 [57] antibodies to confirm specificity and selectivity of both A11 and 4G8 antibodies. Next, sections were incubated for 1 h at room temperature with secondary antibodies in PBS: HRP-conjugated anti-mouse IgG(Sigma-Aldrich) or anti-rabbit IgG (Sigma-Aldrich) respectively. After washing three times with PBS, sections were covered with DAB solution and incubated for 5–10 min. Slides were then counterstained with hematoxylin for 1 min then imaged using the Olympus VS 120 Slide Scanner and were analyzed using ‘Olympus OlyVIA’ software.

Immunofluorescence co-localization studies

Double immuno-labelling was achieved by two different fluorescent labels, each having a separate emission wavelength. Sections were incubated overnight with both A11 and 4G8 at 4°C. Further, and in other experiments, sections were incubated with A11 and mouse Anti-NeuN mAb, clone A60 (Merck Millipore) to demonstrate neuronal homing of the oligomers. Additionally, sections were incubated with A11 and mouse anti-lysosomal-associated membrane protein 2 (LAMP2, Stressgen Bio reagents Corp, Victoria, British Columbia, Canada) antibody to assess whether Aβo localize to lysosomes/late endosomes. In both cases sections were incubated overnight at 4°C. Sections were then incubated with goat anti-rabbit IgG conjugated to FITC (Sigma-Aldrich) and donkey anti-mouse IgG conjugated to Texas red (Sigma-Aldrich) respectively for 2 h at 4°C. Sections were then mounted using fluorescence mounting media (Agilent, Santa Clara, CA, USA). Finally, the mounted sections were imaged using Olympus VS 120 Slide Scanner with a standard FITC/Texas Red double band-pass filter set.

Image quantification

Three sections from different 5×FAD and wild type mice were used for image quantification (Supplementary Figure 1). Three different areas of hippocampus, cerebral cortex and retina were analyzed. Immunofluorescence signal intensity was visualized by capturing red and green fluorescent field images using the Olympus VS 120 Slide Scanner. Images were analyzed using ‘Olympus OlyVIA’ software. Age-dependent accumulation of Aβ plaques and Aβo in 5×FAD was quantified using image processing software, cellSense (Olympus). The mean color threshold of fluorescent particles (red particles for plaques and green particles for oligomers) was calculated in several brain regions and eyes for each age group and the final result was presented as percentage fluorescence intensity and expressed as mean±S.E.M.

Statistical analysis

One-way ANOVA with Dunnett’s post-test was performed using GraphPad Prism version 7.00 for Windows (GraphPad, San Diego, CA, USA), for statistical analysis.

RESULTS

Histological assessment of retinal and cerebral lesions in 5×FAD mice

We first performed an initial assessment to confirm the presence of the typical neuropathological lesions associated with AD in the brain and retina of the 6–7-month-old 5×FAD mice (Fig. 1). H & E stain displayed widespread vacuolations, neuronal death, and presence of eosinophilic structures in the cortical and hippocampal region of the brain (Fig. 1D, E), in contrast with the retina which at the same age did not display the above structural changes (Fig. 1F).

Fig. 1

Photomicrographs of the microscopic lesions in the brains and retinas of six-month-old 5×FAD mice. A) Normal appearance of the cerebral cortex in a healthy wild type mouse following staining with H & E. B) Normal appearance of the hippocampus in a healthy wild type mouse following staining with H & E (M, molecular layer; CA4, Cornu Ammonis 4; DG, dentate gyrus). C) Normal appearance of the retina in a healthy wild type mouse following staining with H & E. The photomicrograph was derived from peripheral region of the retina, away from the optic disc. Widespread vacuolations, neuronal death, and presence of eosinophilic structures in a 6-month-old 5×FAD mouse brain. Vacuolations (yellow arrow), neuronal death (black arrow), and eosinophilic structures (red arrows) are observed in the D) cortical and E) hippocampal region of the brain following staining with H & E (DG, dentate gyrus). F) Normal appearance of the retina in a 6-month-old 5×FAD mouse brain following staining with H & E. The photomicrograph was derived from peripheral region of the retina, away from the optic disc. Representative of all affected mice in this age group.

Retinal and cerebral detection of Congophilic and ThT-specific Aβ fibrils in 5×FAD mice

One of the distinctive neuropathological features associated with human AD brains is the presence of extracellular Aβ plaques [4 , 58]. We initially used Congo red and ThT [59] to assess age-dependent accumulation of amyloid fibrils and compare amyloid burden in the retinas and brains derived from 6-, 7-, 12-, 14-, and 17-month-old 5×FAD and WT mice (Fig. 2). Here, we show the distinctive Congophilic red-brick coloration confirming the presence of amyloid fibrils in the brain and retina starting from 6 months (Fig. 2A, B) of age, respectively. This was confirmed by the presence of apple-green birefringence when examined under cross-polarized light (Fig. 2C, D). However, retinal apple-green birefringence was less intense compared to the brain. This pattern of amyloid fibrils distribution in retina and brain in these different age groups was confirmed with ThT staining; which displayed more pronounced staining in all age groups tested starting from 6 months onward (Fig. 2E, F). Congophilic- and ThT-specific retinal amyloid fibrils were clearly visible in the inner nuclear layer (INL), inner plexiform layer (IPL), and the ganglion cell layer (GCL) of all age groups (Fig. 2B, D, F). These results confirm that the retina of the 5×FAD mice show signs of AD pathogenesis [37]. Of note, Congophilic- and ThT-specific amyloid fibrils were not visible in the brains and retinas of the age-matched 6-month-oldwild type littermates (Supplementary Figure 2).

Fig. 2

Photomicrographs of Congo red- and Thioflavin T-specific amyloid-β fibrils in the brains and retinas of six-month-old 5×FAD mice. A) Distinctive red-brick staining of amyloid fibrils with Congo red in the brain and B) GCL and ONL of the retina of a 6-month-old 5×FAD mouse. The photomicrograph was derived from peripheral region of the retina, away from the optic disc. The presence of the amyloid fibrils was confirmed with the presence of apple-green birefringence in the C) brain and D) retina under polarized light. Thioflavin T staining displayed presence of amyloid fibrils in the E) hippocampus and in the F) INL, IPL, and GCL of the retina (blue arrows). Representative of all affected mice in this age group.

Retinal and cerebral immunodetection of Aβ plaques and Aβsoluble oligomers in 5×FAD mice

5×FAD mice are known to exhibit cerebral accumulation of intracellular Aβo and extracellular Aβ plaques [55]. Although, amyloid fibrils are a neuropathological hallmark of human AD, there is strong evidence that oligomers are the most toxic species and appear to be the main causative agent of neurological deficits [20, 60]. Moreover, it is now recognized that Aβo accumulation in serum of AD patients and experimental models can occur years before plaque build-up in the brain [61 –63]. We hypothesized that Aβo accumulation in the retina might also precede cerebral plaque build-up in 5×FAD mice in an age-dependent manner. We initially examined brains and retinas for the presence of Aβo and Aβ plaques by immunohistochemistry using A11 and 4G8, respectively (Figs. 3–5). 6-month-old 5×FAD mice displayed abundant intraneuronal Aβo deposits in the cerebral cortex, hippocampus, retinal nuclear layers (INL & ONL), and GCL (Fig. 3A–C) as well as few extracellular Aβ plaques (Fig. 3D–F). Interestingly, 12-month-old 5×FAD mice exhibited more pronounced extracellular cortical and hippocampal Aβ plaques but lower levels of intracellular Aβo compared to the 6-month-old age group (Fig. 4A–F). Nonetheless, levels of intracellular Aβo appeared lower in these brain structures compared to the 6-month-old age group (Fig. 4A, B). Similarly, retinal Aβo which could be seen in the GCL, INL, and ONL appeared less abundant in this age group compared to the 6-month-old mice (Fig. 4C). Occasional Aβ plaques were also evident in the retinal layers of 12-month-old 5×FAD mice, and their levels appeared lower than the 6-month-old age group (Fig. 4F). As the age of the 5×FAD mice progressed, fewer intracellular Aβo deposits were observed as opposed to the abundant accumulation of Aβ plaques in the cerebrum of 14 and 17-month-old mice (Fig. 5). Retinal Aβ plaques were also conspicuous in the ganglion layer but Aβo appeared to be absent in the 14- and 17-month-old 5×FAD mice (Fig. 5C, F). No Aβo and Aβ plaques deposits were seen in age-matched wild type littermates (data not shown). These results support previous findings that Aβ plaque burden increased over the course of the disease in both brain and retina, whereas Aβo levels appear to decrease in an age-dependent manner [64].

Fig. 3

Photomicrographs of the amyloid-β oligomers and plaques in the brain and retina of a six-month-old 5×FAD mouse. A) Immunohistochemical staining with A11 rabbit anti-Aβo polyclonal IgG antibody of a 6-month-old 5×FAD mouse which shows intraneuronal stained structures in the A) hippocampus, in the B) cerebral cortex and in the C) GCL, INL, and ONL of the retina. The photomicrograph was derived from peripheral region of the retina, away from the optic disc. D) Immunohistochemical staining with 4G8 murine anti-Aβ monoclonal IgG antibody of a 6-month-old 5×FAD mouse which shows characteristic extracellular AD Aβ plaques in the D) hippocampus, in the E) cerebral cortex and in the F) GCL and ONL of the retina. CA4, DG, and M refers to hippocampal Cornu Ammonis, Dentate gyrus, and the molecular layer, respectively. Representative of all affected mice in this age group.

Fig. 4

Photomicrographs of the amyloid-β oligomers and plaques in the brain and retina of a twelve-month-old 5×FAD mouse. A) Immunohistochemical staining with A11 rabbit anti-Aβo polyclonal IgG antibody of a 12-month-old 5×FAD mouse which shows less intense intraneuronal stained structures in the A) hippocampus, in the B) cerebral cortex and in the C) GCL of the retina (arrows). The photomicrograph was derived from peripheral region of the retina, away from the optic disc. Immunohistochemical staining with 4G8 murine anti-Aβ monoclonal IgG antibody of a 12-month-old 5×FAD mouse which shows widespread and intense staining of the extracellular AD Aβ plaques in the D) hippocampus, and in the E) cerebral cortex (arrows). F) Very few plaques were observed in the retina (arrows). CA3, DG, and M refers to hippocampal Cornu Ammonis, Dentate gyrus, and the molecular layer, respectively. Representative of all affected mice in this age group.

Fig. 5

Photomicrographs of the amyloid-β oligomers and plaques in the brain and retina of a seventeen-month-old 5×FAD mouse. A) Immunohistochemical staining with A11 rabbit anti-Aβo polyclonal IgG antibody of a 17-month-old 5×FAD mouse which shows scarce intraneuronal stained structures in the hippocampus, and in the B) cerebral cortex (arrows). C) Aβo were absent in the retina in this age group. The photomicrograph was derived from peripheral region of the retina, away from the optic disc. D) Immunohistochemical staining with 4G8 murine anti-Aβ monoclonal IgG antibody of a 17-month-old 5×FAD mouse which shows widespread and intense staining of the extracellular AD Aβ plaques in the hippocampus, and in the E) cerebral cortex (arrows). F) Plaques were observed in the GCL of the retina (arrows). CA1, DG, and M refers to hippocampal Cornu Ammonis, Dentate gyrus, and the molecular layer, respectively. Representative of all affected mice in this age group.

Aβ oligomers co-accumulate with Aβ plaques and co-localize with an endosomal marker in neurons of 5×FAD mice

Our immunostaining results confirmed that accumulation of intracellular Aβo and Aβ plaques in the retinal layers parallels their brain build-up. We then investigated whether Aβo and Aβ plaques co-localize/accumulate in different regions and structures of the retina and brain of the various age groups tested using A11 and 4G8 (Supplementary Figure 3). This immunofluorescence study confirmed the presence of intracellular Aβo and Aβ plaques in the retina of the 6-month-old 5×FAD mice (Supplementary Figure 3). Here, retinal Aβo was more prominent in the GCL and INL, and to a lesser extent in the IPL and ONL (Supplementary Figure 3). While present, retinal Aβ plaques were less prominent in this age group (Supplementary Figure 3). Remarkably, both Aβo and Aβ plaques were widespread in hippocampus and cerebral cortex of the 6-month-old 5×FAD mice (Supplementary Figure 4). At 12months of age, retinal oligomers and plaques co-localized in the GCL, INL, IPL, and ONL (Supplementary Figure 3) and co-accumulated and displayed very strong staining in the cerebral cortical region and hippocampus (Supplementary Figure 4). The immunofluorescence studies appeared to be more sensitive than immunohistochemistry as the latter allowed stronger detection of retinal Aβ plaques in the 12-month-old 5×FAD mice. Surprisingly, Aβo were detected in the retina and cerebrum of the 17-month-old 5×FAD mice (Supplementary Figures 3 and 4). Finally, and as expected, the 17-month-old 5×FAD mice displayed conspicuous widespread accumulation of cerebral Aβ plaques, which were also observed in the retinas of these animals (Supplementary Figures 3 and 4). No staining for both Aβo and Aβ plaques was seen in all brain and retinas of wild type age group (data not shown).

Immunofluorescence signal intensity of both Aβ plaques and oligomers were quantified by an image processing software, cellSense (Fig. 6) in the retina, hippocampus, and cortex of the 6-month (n = 5), 12-month (n = 5), and ≥14-month (n = 7) 5×FAD groups and compared with 6–7-month (n = 5), 12-month (n = 6), and 14-month (n = 5) wild type littermates. Of note, three different areas of each section were analyzed. Aβ plaque loads significantly increased from 6 months and onward (p < 0.001) in the retina (Fig.6G, I), hippocampus (Fig. 6D, F), and cortex (Fig. 6A, C) of the 6–7-, 12-, and ≥14-month-old age 5×FAD groups. In contrast, Aβ oligomers levels significantly decreased between 6-7 months and 12 months (p < 0.001) in the retina (Fig. 6H, I), hippocampus (Fig. 6E, F), and cortex (Fig. 6B, C) of the 6–7-month, 12-month, and ≥14-month-old age 5×FAD groups.

Fig. 6

Quantification of amyloid-β plaque burden and amyloid-β oligomers with CellSense image processing software. Total Aβ plaque burden (ABP) in the cerebral cortex (A, C, K), hippocampus (D, F, K), and retina (G, I, K) was quantified in 6–7-month-old (5×FAD = 5; wild type = 5); 12-month-old (5×FAD = 6; wild type = 6); and ≥14-month-old (5×FAD = 7; wild type = 5). Total Aβ oligomer load (Aβo) in the cerebral cortex (B, C, J), hippocampus (E, F, J), and retina (H, I, J) was quantified in 6-month-old (5×FAD = 5; wild type = 5); 12-month-old (5×FAD = 6; wild type = 6); and ≥14-month-old (5×FAD = 7; wild type = 5). Data represents mean±SEM.

Disturbances of the endosomal/lysosomal system is thought to be involved in neuronal toxicity and Aβ accumulation [65]. Inhibition of Aβ secretion can lead to intraneuronal Aβ accumulation in the endosomal/lysosomal compartment, which destabilizes its membrane leading to Aβ deposition in the cytosolic compartment [66 –68]. To verify the presence of Aβo deposits in the brain and retinal endosomal/lysosomal, we co-stained A11 and LAMP2, a marker against late endosome and lysosomes (Fig.7). Our data showed that Aβo co-localized with the lysosomal marker in the hippocampus, cortex and in the ONL, OPL, and INL of the retina in all age groups (Fig. 7A–F). Our results strongly indicate binding of A11 antibody in these organelles where clearance of Aβo is believed to occur. Finally, we confirmed that Aβo accumulation was in fact occurring in cerebral and retinal neurons as evident by co-staining with NeuN in all age groups (Fig. 7G, H). Moreover, Aβo deposits were more prominent in the retinal GCL and INL in the retina in the 6-month-old age group (Fig. 7H).

Fig. 7

Immunofluorescence co-localization of retinal and cerebral amyloid-β oligomers with lysosomal-associated membrane protein 2 (LAMP2) or neuron-specific nuclear protein (NeuN). Retinal and cerebral co-staining with A11 rabbit anti-Aβo polyclonal IgG antibody (green) and anti-mouse LAMP2 monoclonal IgG antibody (red) of a 6-, 12-, and 17-month-old 5×FAD mice or anti-mouse NeuN monoclonal IgG antibody (red) of a 6-month-old 5×FAD mouse. LAMP2 co-localized with Aβo in the (A, C, E) cerebral cortex and (B, D, F) in the GCL, IPL, INL, OPL, and ONL of the retina in all age groups (arrows). Aβo localized to NeuN in the (G) hippocampus, and in the (H) GCL, IPL, INL, and OPL of the retina in the 6-month-old age group (arrows). Representative of all affected mice in all age groups.

DISCUSSION

One of the principal neuropathological lesions associated with AD is the extracellular deposition of Aβ plaques [6]. Among the three major assemblies of Aβ, soluble oligomers are considered the most neurotoxic form and is the intermediary conformation recognized in early pathogenesis [11]. Soluble oligomers can lead to synaptic dysfunction, whereas large, insoluble deposits are believed to function as reservoirs of the bioactive oligomers [19]. In AD, Aβo are believed to form in the early phase of the disease and are present in blood and other tissues [69 –71]. Current strategies for AD detection include measurement of CSF-borne Aβ₄₂ levels and amyloid positron emission tomography (PET) imaging [70]. However, these approaches are considered to be invasive, display a high degree of multicenter variability, and are costly. A blood-based biomarker approach is gaining momentum as several groups have demonstrated its potential value, albeit work remains at experimental phase [72, 73]. Since Aβo is considered the toxic species and accumulates in preclinical stage of the disease [74 –77], several reports have demonstrated its potential as an early marker of the disease [78 –80]. A recent study by Nakamura and colleagues has identified high-performance plasma Aβ biomarkers using a combination of immunoprecipitation and mass spectrometry [81]. These authors measured and compared AβPP_669–711/Aβ_1–41 and Aβ_1–40/Aβ_1–41 ratios in order to predict Aβ-PET imaging status in cognitively normal, mild cognitive impaired, and AD individuals and shown that this test was highly predictive of brain Aβ burden.

In this study, we set out to provide proof-of-concept for early retinal detection of AD through identifying and quantifying levels of retinal Aβo in an AD mouse model. The eye is not considered as a hermetic anatomical structure and the barriers that separate the eye from the periphery are not completely sealed [82]. The blood-ocular barriers comprise the blood-retinal and blood-aqueous barrier; the latter is formed by tight junctions of the inner non-pigmented ciliary epithelium and the non-fenestrated endothelial cells of the iris blood vessels [82]. In contrast and because of the fenestrated structure of the ciliary body blood vessels, plasma proteins and molecules can enter the stroma as part of aqueous humor production [82]. Therefore, there is a distinct possibility that blood borne-Aβo might reach different structures of the eye, including the retina, and this provides a great potential to develop a non-invasive retinal eye test for AD. Of note, eye inflammation leads to blood-ocular barrier disruption, which also results in increased vascular permeability, potentially allowing higher levels of Aβo to spread in all structures of the eye.

Visual disturbances are part of early complaints reported by AD patients [83]. These disturbances include reduced blood vessel diameter and venous blood flow. Studies by Berisha and colleagues [26] and Feke and colleagues [52] suggested that alterations in retinal blood flow can distinguish mild cognitive impairment and AD. Furthermore, a study by Hadoux and colleagues demonstrated significance differences in the retinal reflectance spectra when comparing PET-specific Aβ burden in mild cognitively impaired individuals with age-matched PET-negative control individuals [84]. The authors show a direct correlation between retinal imaging scores and cerebral Aβ plaque burden [84].

In this study, we used 5×FAD mice brain and retinal tissues for pathological assessment and immune-detection of age-dependent Aβo accumulation. H & E staining revealed vacuolations, neuronal loss, and presence of eosinophilic aggregates in the neocortex and hippocampus of the brain but no obvious lesions were observed in the GCL and INL of the retina. We then used Congo red and ThT staining to demonstrate age-dependent accumulation of amyloid fibrils in the retina and brain of 5×FAD mice. We show that Congophilic-amyloid fibrils increased with age in the cortex and to a lesser extent in the retina. We wanted to verify whether the staining method was sensitive enough for the detection of retinal amyloid fibrils, hence we stained with ThT. This fluorescence reaction revealed staining of retinal amyloid fibrilsin the GCL and INLof 5×FAD mice. Amyloid fibrils were observed in the neocortex and the inferior layer of the hippocampus of 6-month-old 5×FAD mice. In older 5×FAD mice, amyloid fibrils were widely distributed throughout the cortex, hippocampus, brain stem, and cerebellum. Furthermore, we used lesion specific markers for immuno detection of Aβo and Aβ plaques. In young mice (6 months), we found high levels of Aβo in the hippocampus and neocortex and in retinal layers such as the GCL and INL, and to a lesser extent the IPL and ONL. Occasional Aβ plaques were also seen in the brain and retina of this age. The middle age group (12 months) displayed both Aβo and Aβ plaques in the brain. Retinal Aβo and to lesser extent Aβ plaques were observed in this age group, indicating that the retinal microenvironment is less efficient in sustaining plaque build-up. Finally, older 5×FAD mice (17 months) displayed widespread and extensive plaque burden in both brain and retina but only traces of Aβo. Taken together, these results strongly support the Aβo conversion to plaque paradigm in the brain. Studies by Kawarabayashiand colleagues [85] using the Tg2576 AD mouse model showed that full-length unmodified Aβ was present in the brain of young littermates which then turned into soluble Aβo at 6–10 months of age, followed by insoluble forms of Aβ which increased exponentially and converted into diffuse plaques from 12 to 23 months. Similar findings were reported for theAβPP^sw-tau^vlw mouse model which displayed an age-dependent exponential increase of Aβo deposition followed by plaque build-up [86].

We extend previous experimental studies to show that Aβo strongly co-localizes with Aβ plaques in both brain and retina of 5×FAD mice. A similar pattern of age-related changes in Aβo and Aβ plaques was evident between the brain and retina of 5×FAD mice. Specifically, both show an initial accumulation of the toxic soluble Aβo entities that are converted into Aβ plaques with age.

In the 6-month-old 5×FAD mice, Aβo localized predominantly to the retinal inner and middle nuclear layers. This is an important finding as it provides a rationale for the development of imaging approaches for detecting AD manifestation in the retina. Liu and colleagues have shown that the majority of Aβ plaques were present in the GCL and IPL with some plaques found in the ONL, photoreceptor outer segment, and optic nerve in 14-month-old Tg2576 mice [87]. That ours and other preclinical studies demonstrate a propensity for Aβo and Aβ plaques to localize to the inner retina is consistent with clinical observations of GCL and nerve fiber layer thinning and optic nerve degeneration in AD patients [88].

Koronyo-Hamaoui and colleagues [89] confirmed the presence of retinal Aβ plaques by systemic administration of curcumin in AβPP (SWE)/PS1 (ΔE9) mice. They confirmed presence of curcumin-positive retinal Aβ plaques in the retinal nerve fiber layer, GCL, IPL and OPL, and INL of the retina. The authors showed that plaques were detected as early as at 2.5 months of age which indicate that Aβ plaques in the retina precede brain plaques build-up. In our study, we detected both Aβo and Aβ plaques in the retinal layers of 6-month-old 5×FAD mice. Specifically, we found that Aβo was similar in the retinal layers when compared to levels in the brain of young mice. This suggest that detection of Aβo in the retina may be a sensitive marker for early diagnosis of AD. More work is required to understand the molecular ‘behavior’ of both Aβo and Aβ plaques in the retina.

We confirmed that Aβo was found in neurons as evident by co-staining with NeuN [90, 91]. It was previously reported that Aβ build-up is initiated in the intracellular compartments (reviewed by Bayer and colleagues [92]). This is consistent with other reports that Aβ₄₂ accumulates intraneuronally before being converted into extracellular plaques in human AD [90 , 94]. Our findings clearly show that Aβo localized to neurons in the hippocampus, neocortex and in various retinal layers, especially in the ONL, INL and GCL in 5×FAD mice across all ages. Aβ has been found in four intraneuronal compartments associated with the lysosomal system such as rab5-positive endosomes [66], autophagic vacuoles [95], lysosomes [96, 97], and multivesicular bodies [98]. Inhibition of Aβ secretion can lead to accumulation of intraneuronal Aβ in the lysosomal compartment and destabilizes its membrane and also deposits in the cytosolic compartment in the early AD pathogenesis [99]. In agreement, we show that Aβo co-localizes to late endosomes in retinal cells, indicating that disturbance of the endocytic pathway leads to its accumulation [100, 101].

Conclusion

Our study demonstrated an age-dependent inversely proportional accumulation of Aβo versus Aβ plaques in the retina of a transgenic AD animal model. The presence of these toxic Aβ soluble oligomers was detected as early as 6 months of age in this animal model, probably mimicking prodromal human AD pathogenesis. More work is needed in earlier age groups perhaps using a less ‘aggressive’ animal model in order to prove that the toxic assemblies can be detected in the retina before identification of cognitive deficiencies. Studies are currently underway to establish this before the useof fluorescence confocal scanning laser ophthalmoscopy for the non-invasive detection of retinal Aβo preceding their accumulation in the brain.

AVAILABILITY OF DATA AND MATERIALS

The datasets generated during this study are available from the corresponding author on request.

Footnotes

ACKNOWLEDGMENTS

We wish to thank Dr. Hong Yu at the West mead Institute for Medical Research, Sydney, Australia for help with image acquisition using the Olympus VS 120.

The Laboratory of Dr. MT received Institutional Start Up support and a Post-Graduate Scholarship for UH.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

References

Beyreuther

, Masters

(1991) Amyloid precursor protein (APP) and beta A4 amyloid in the etiology of Alzheimer’s disease: Precursor-product relationships in the derangement of neuronal function. Brain Pathol 1, 241–251.

Mott

, Hulette

(2005) Neuropathology of Alzheimer’s disease. Neuroimaging Clin N Am 15, 755–765, ix.

Perl

(2010) Neuropathology of Alzheimer’s disease. Mt Sinai J Med 77, 32–42.

Selkoe

, Hardy

(2016) The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol Med 8, 595–608.

Clark

, Karlawish

(2003) Alzheimer disease: Current concepts and emerging diagnostic and therapeutic strategies. Ann Intern Med 138, 400–410.

Serrano-Pozo

, Frosch

, Masliah

, Hyman

(2011) Neuropathological alterations in Alzheimer disease. Cold Spring Harb Perspect Med 1, a006189.

Cai

, Golde

, Younkin

(1993) Release of excess amyloid beta protein from a mutant amyloid beta protein precursor. Science 259, 514–516.

Kang

, Lemaire

, Unterbeck

, Salbaum

, Masters

, Grzeschik

, Multhaup

, Beyreuther

, Müller-Hill

(1987) The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature 325, 733–736.

Suzuki

, Cheung

, Cai

, Odaka

, Otvos

, Jr. , Eckman

, Golde

, Younkin

(1994) An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants. Science 264, 1336–1340.

10.

Chow

, Mattson

, Wong

, Gleichmann

(2010) An overview of APP processing enzymes and products. Neuromolecular Med 12, 1–12.

11.

Sengupta

, Nilson

, Kayed

(2016) The role of Amyloid-β oligomers in toxicity, propagation, and immunotherapy. EBioMedicine 6, 42–49.

12.

Alafuzoff

, Arzberger

, Al-Sarraj

, Bodi

, Bogdanovic

, Braak

, Bugiani

, Del-Tredici

, Ferrer

, Gelpi

, Giaccone

, Graeber

, Ince

, Kamphorst

, King

, Korkolopoulou

, Kovács

, Larionov

, Meyronet

, Monoranu

, Parchi

, Patsouris

, Roggendorf

, Seilhean

, Tagliavini

, Stadelmann

, Streichenberger

, Thal

, Wharton

, Kretzschmar

(2008) Staging of neurofibrillary pathology in Alzheimer’s disease: A study of the BrainNet Europe Consortium. Brain Pathol 18, 484–496.

13.

Murphy

, LeVine

, 3rd (2010) Alzheimer’s disease and the amyloid-beta peptide. J Alzheimers Dis 19, 311–323.

14.

Broersen

, Rousseau

, Schymkowitz

(2010) The culprit behind amyloid beta peptide related neurotoxicity in Alzheimer’s disease: Oligomer size or conformation? Alzheimers Res Ther 2, 12.

15.

Goure

, Krafft

, Jerecic

, Hefti

(2014) Targeting the proper amyloid-beta neuronal toxins: A path forward for Alzheimer’s disease immunotherapeutics. Alzheimers Res Ther 6, 42.

16.

Glabe

(2008) Structural classification of toxic amyloid oligomers. J Biol Chem 283, 29639–29643.

17.

Mroczko

, Groblewska

, Litman-Zawadzka

, Kornhuber

, Lewczuk

(2018) Amyloid β oligomers (AβOs) in Alzheimer’s disease. J Neural Transm (Vienna) 125, 177–191.

18.

Benilova

, Karran

, De

Strooper B

(2012) The toxic Aβ oligomer and Alzheimer’s disease: An emperor in need of clothes. Nat Neurosci 15, 349–357.

19.

Haass

, Selkoe

(2007) Soluble protein oligomers in neurodegeneration: Lessons from the Alzheimer’s amyloid beta-peptide. Nat Rev Mol Cell Biol 8, 101–112.

20.

Kayed

, Head

, Thompson

, McIntire

, Milton

, Cotman

, Glabe

(2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489.

21.

Zhao

, Long

, Mu

, Chew

(2012) The toxicity of amyloid β oligomers. Int J Mol Sci 13, 7303–7327.

22.

Frackowiak

, Zoltowska

, Wisniewski

(1994) Non-fibrillar beta-amyloid protein is associated with smooth muscle cells of vessel walls in Alzheimer disease. J Neuropathol Exp Neurol 53, 637–645.

23.

Viola

, Klein

(2015) Amyloid β oligomers in Alzheimer’s disease pathogenesis, treatment, and diagnosis. Acta Neuropathol 129, 183–206.

24.

Katz

, Rimmer

(1989) Ophthalmologic manifestations of Alzheimer’s disease. Surv Ophthalmol 34, 31–43.

25.

Sadun

, Borchert

, DeVita

, Hinton

, Bassi

(1987) Assessment of visual impairment in patients with Alzheimer’s disease. Am J Ophthalmol 104, 113–120.

26.

Berisha

, Feke

, Trempe

, McMeel

, Schepens

(2007) Retinal abnormalities in early Alzheimer’s disease. Invest Ophthalmol Vis Sci 48, 2285–2289.

27.

Frost

, Kanagasingam

, Sohrabi

, Vignarajan

, Bourgeat

, Salvado

, Villemagne

, Rowe

, Macaulay

, Szoeke

, Ellis

, Ames

, Masters

, Rainey-Smith

, Martins

, Group

(2013) Retinal vascular biomarkers for early detection and monitoring of Alzheimer’s disease. Transl Psychiatry 3, e233.

28.

Paquet

, Boissonnot

, Roger

, Dighiero

, Gil

, Hugon

(2007) Abnormal retinal thickness in patients with mild cognitive impairment and Alzheimer’s disease. Neurosci Lett 420, 97–99.

29.

Fotiou

, Brozou

, Haidich

, Tsiptsios

, Nakou

, Kabitsi

, Giantselidis

, Fotiou

(2007) Pupil reaction to light in Alzheimer’s disease: Evaluation of pupil size changes and mobility. Aging Clin Exp Res 19, 364–371.

30.

Frost

, Kanagasingam

, Sohrabi

, Bourgeat

, Villemagne

, Rowe

, Macaulay

, Szoeke

, Ellis

, Ames

, Masters

, Rainey-Smith

, Martins

(2013) Pupil response biomarkers for early detection and monitoring of Alzheimer’s disease. Curr Alzheimer Res 10, 931–939.

31.

Lim

, Li

, He

, Vingrys

, Wong

, Currier

, Mullen

, Bui

, Nguyen

(2016) The eye as a biomarker for Alzheimer’s disease. Front Neurosci 10, 536.

32.

Nguyen

CTO

, Hui

, Charng

, Velaedan

, van

Koeverden AK

, Lim

JKH

, He

, Wong

VHY

, Vingrys

, Bui

, Ivarsson

(2017) Retinal biomarkers provide “insight” into cortical pharmacology and disease. Pharmacol Ther 175, 151–177.

33.

Cronin-Golomb

, Corkin

, Rizzo

, Cohen

, Growdon

, Banks

(1991) Visual dysfunction in Alzheimer’s disease: Relation to normal aging. Ann Neurol 29, 41–52.

34.

Cronin-Golomb

, Sugiura

, Corkin

, Growdon

(1993) Incomplete achromatopsia in Alzheimer’s disease. Neurobiol Aging 14, 471–477.

35.

Armstrong

, Winsper

, Blair

(1996) Aluminium and Alzheimer’s disease: Review of possible pathogenic mechanisms. Dementia 7, 1–9.

36.

Trick

, Trick

, Morris

, Wolf

(1995) Visual field loss in senile dementia of the Alzheimer’s type. Neurology 45, 68–74.

37.

Valenti

(2013) Alzheimer’s disease: Screening biomarkers using frequency doubling technology visual field. ISRN Neurol 2013, 989583.

38.

Rizzo

, Anderson

, Dawson

, Nawrot

(2000) Vision and cognition in Alzheimer’s disease. Neuropsychologia 38, 1157–1169.

39.

Danesh-Meyer

, Birch

, Ku

, Carroll

, Gamble

(2006) Reduction of optic nerve fibers in patients with Alzheimer disease identified by laser imaging. Neurology 67, 1852–1854.

40.

Blanks

, Torigoe

, Hinton

, Blanks

(1996) Retinal pathology in Alzheimer’s disease. I. Ganglion cell loss in foveal/parafoveal retina. Neurobiol Aging 17, 377–384.

41.

Sadun

, Bassi

(1990) Optic nerve damage in Alzheimer’s disease. Ophthalmology 97, 9–17.

42.

Blanks

, Hinton

, Sadun

, Miller

(1989) Retinal ganglion cell degeneration in Alzheimer’s disease. Brain Res 501, 364–372.

43.

Blanks

, Schmidt

, Torigoe

, Porrello

, Hinton

, Blanks

(1996) Retinal pathology in Alzheimer’s disease. II. Regional neuron loss and glial changes in GCL. Neurobiol Aging 17, 385–395.

44.

Colligris

, Perez

de Lara MJ

, Colligris

, Pintor

(2018) Ocular manifestations of Alzheimer’s and other neurodegenerative diseases: The prospect of the eye as a tool for the early diagnosis of Alzheimer’s disease. J Ophthalmol 2018, 8538573.

45.

Dutescu

, Li

, Crowston

, Masters

, Baird

, Culvenor

(2009) Amyloid precursor protein processing and retinal pathology in mouse models of Alzheimer’s disease. Graefes Arch Clin Exp Ophthalmol 247, 1213–1221.

46.

Hinton

, Sadun

, Blanks

, Miller

(1986) Optic-nerve degeneration in Alzheimer’s disease. N Engl J Med 315, 485–487.

47.

Koronyo

, Biggs

, Barron

, Boyer

, Pearlman

, Au

, Kile

, Blanco

, Fuchs

, Ashfaq

, Frautschy

, Cole

, Miller

, Hinton

, Verdooner

, Black

, Koronyo-Hamaoui

(2017) Retinal amyloid pathology and proof-of-concept imaging trial in Alzheimer’s disease. JCI Insight 2, e93621.

48.

Morgia C

, Ross-Cisneros

, Koronyo

, Hannibal

, Gallassi

, Cantalupo

, Sambati

, Pan

, Tozer

, Barboni

, Provini

, Avanzini

, Carbonelli

, Pelosi

, Chui

, Liguori

, Baruzzi

, Koronyo-Hamaoui

, Sadun

, Carelli

(2016) Melanopsin retinal ganglion cell loss in Alzheimer disease. Ann Neurol 79, 90–109.

49.

, Li

, Zhang

, Ming

, Jia

, Wang

, Ma

(2010) Retinal nerve fiber layer structure abnormalities in early Alzheimer’s disease: Evidence in optical coherence tomography. Neurosci Lett 480, 69–72.

50.

Iseri

, Altinaş

, Tokay

, Yüksel

(2006) Relationship between cognitive impairment and retinal morphological and visual functional abnormalities in Alzheimer disease. J Neuroophthalmol 26, 18–24.

51.

O’Bryhim

, Apte

, Kung

, Coble

, Van

Stavern GP

(2018) Association of preclinical Alzheimer disease with optical coherence tomographic angiography findings. JAMA Ophthalmol 136, 1242–1248.

52.

Feke

, Hyman

, Stern

, Pasquale

(2015) Retinal blood flow in mild cognitive impairment and Alzheimer’s disease. Alzheimers Dement (Amst) 1, 144–151.

53.

Ratnayaka

, Serpell

, Lotery

(2015) Dementia of the eye: The role of amyloid beta in retinal degeneration. Eye (Lond) 29, 1013–1026.

54.

Williams

, McGowan

, Cardwell

, Cheung

, Craig

, Passmore

, Silvestri

, Maxwell

, McKay

(2015) Retinal microvascular network attenuation in Alzheimer’s disease. Alzheimers Dement (Amst) 1, 229–235.

55.

Oakley

, Cole

, Logan

, Maus

, Shao

, Craft

, Guillozet-Bongaarts

, Ohno

, Disterhoft

, Van

Eldik L

, Berry

, Vassar

(2006) Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: Potential factors in amyloid plaque formation. J Neurosci 26, 10129–10140.

56.

Anstee

, Gardner

, Spring

, Holmes

, Simpson

, Parsons

, Mallinson

, Yousaf

, Judson

(1991) New monoclonal antibodies in CD44 and CD58: Their use to quantify CD44 and CD58 on normal human erythrocytes and to compare the distribution of CD44 and CD58 in human tissues. Immunology 74, 197–205.

57.

Avent

, Ridgwell

, Mawby

, Tanner

, Anstee

, Kumpel

(1988) Protein-sequence studies on Rh-related polypeptides suggest the presence of at least two groups of proteins which associate in the human red-cell membrane. Biochem J 256, 1043–1046.

58.

Hardy

, Allsop

(1991) Amyloid deposition as the central event in the aetiology of Alzheimer’s disease. Trends Pharmacol Sci 12, 383–388.

59.

Teboul

, Feki

, Dubois

, Bozon

, Faure

, Hantraye

, Dhenain

, Delatour

, Delzescaux

(2007) A standardized method to automatically segment amyloid plaques in Congo Red stained sections from Alzheimer transgenic mice. Conf Proc IEEE Eng Med Biol Soc 2007, 5593–5596.

60.

Hardy

, Selkoe

(2002) The amyloid hypothesis of Alzheimer’s disease: Progress and problems on the road to therapeutics. Science 297, 353–356.

61.

Laske

, Sohrabi

, Frost

, López-de-Ipiña

, Garrard

, Buscema

, Dauwels

, Soekadar

, Mueller

, Linnemann

, Bridenbaugh

, Kanagasingam

, Martins

, O’Bryant

(2015) Innovative diagnostic tools for early detection of Alzheimer’s disease. Alzheimers Dement 11, 561–578.

62.

Schindler

, Bollinger

, Ovod

, Mawuenyega

, Li

, Gordon

, Holtzman

, Morris

, Benzinger

TLS

, Xiong

, Fagan

, Bateman

(2019) High-precision plasma β-amyloid 42/40 predicts current and future brain amyloidosis. Neurology 93, e1647–e1659.

63.

Wang

, Yi

, Han

, Park

, Jang

, Chun

, Kim

, Lee

, Kim

, Yu

, Lim

, Kang

, Park

, Youn

, An

SSA

, Kim

(2017) Oligomeric forms of amyloid-β protein in plasma as a potential blood-based biomarker for Alzheimer’s disease. Alzheimers Res Ther 9, 98.

64.

Criscuolo

, Cerri

, Fabiani

, Capsoni

, Cattaneo

, Domenici

(2018) The retina as a window to early dysfunctions of Alzheimer’s disease following studies with a 5xFAD mouse model. Neurobiol Aging 67, 181–188.

65.

Adamec

, Mohan

, Cataldo

, Vonsattel

, Nixon

(2000) Up-regulation of the lysosomal system in experimental models of neuronal injury: Implications for Alzheimer’s disease. Neuroscience 100, 663–675.

66.

Cataldo

, Petanceska

, Terio

, Peterhoff

, Durham

, Mercken

, Mehta

, Buxbaum

, Haroutunian

, Nixon

(2004) Abeta localization in abnormal endosomes: Association with earliest Abeta elevations in AD and Down syndrome. Neurobiol Aging 25, 1263–1272.

67.

Pasternak

, Callahan

, Mahuran

(2004) The role of the endosomal/lysosomal system in amyloid-beta production and the pathophysiology of Alzheimer’s disease: Reexamining the spatial paradox from a lysosomal perspective. J Alzheimers Dis 6, 53–65.

68.

Wolfe

, Lee

, Kumar

, Lee

, Orenstein

, Nixon

(2013) Autophagy failure in Alzheimer’s disease and the role of defective lysosomal acidification. Eur J Neurosci 37, 1949–1961.

69.

El-Agnaf

, Salem

, Paleologou

, Curran

, Gibson

, Court

, Schlossmacher

, Allsop

(2006) Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease. FASEB J 20, 419–425.

70.

McKhann

, Knopman

, Chertkow

, Hyman

, Jack

, Jr. , Kawas

, Klunk

, Koroshetz

, Manly

, Mayeux

, Mohs

, Morris

, Rossor

, Scheltens

, Carrillo

, Thies

, Weintraub

, Phelps

(2011) The diagnosis of dementia due to Alzheimer’s disease: Recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 7, 263–269.

71.

El-Agnaf

, Walsh

, Allsop

(2003) Soluble oligomers for the diagnosis of neurodegenerative diseases. Lancet Neurol 2, 461–462.

72.

Doecke

, Laws

, Faux

, Wilson

, Burnham

, Lam

, Mondal

, Bedo

, Bush

, Brown

, De

Ruyck K

, Ellis

, Fowler

, Gupta

, Head

, Macaulay

, Pertile

, Rowe

, Rembach

, Rodrigues

, Rumble

, Szoeke

, Taddei

, Trounson

, Ames

, Masters

, Martins

(2012) Blood-based protein biomarkers for diagnosis of Alzheimer disease. Arch Neurol 69, 1318–1325.

73.

Snyder

, Carrillo

, Grodstein

, Henriksen

, Jeromin

, Lovestone

, Mielke

, O’Bryant

, Sarasa

, Sjøgren

, Soares

, Teeling

, Trushina

, Ward

, West

, Bain

, Shineman

, Weiner

, Fillit

(2014) Developing novel blood-based biomarkers for Alzheimer’s disease. Alzheimers Dement 10, 109–114.

74.

Gong

, Chang

, Viola

, Lacor

, Lambert

, Finch

, Krafft

, Klein

(2003) Alzheimer’s disease-affected brain: Presence of oligomeric A beta ligands (ADDLs) suggests a molecular basis for reversible memory loss. Proc Natl Acad Sci U S A 100, 10417–10422.

75.

Larson

, Lesné

(2012) Soluble Aβ oligomer production and toxicity. J Neurochem 120 Suppl 1, 125–139.

76.

Lesné

, Sherman

, Grant

, Kuskowski

, Schneider

, Bennett

, Ashe

(2013) Brain amyloid-β oligomers in ageing and Alzheimer’s disease. Brain 136, 1383–1398.

77.

Sakono

, Zako

(2010) Amyloid oligomers: Formation and toxicity of Abeta oligomers. FEBS J 277, 1348–1358.

78.

Santos

, Simm

, Holthoff

, Boehm

(2008) A method for the detection of amyloid-beta1-40, amyloid-beta1-42 and amyloid-beta oligomers in blood using magnetic beads in combination with flow cytometry and its application in the diagnostics of Alzheimer’s disease. J Alzheimers Dis 14, 127–131.

79.

Xia

, Yang

, Shankar

, Smith

, Shen

, Walsh

, Selkoe

(2009) A specific enzyme-linked immunosorbent assay for measuring beta-amyloid protein oligomers in human plasma and brain tissue of patients with Alzheimer disease. Arch Neurol 66, 190–199.

80.

Zhou

, Chan

, Chu

, Kwan

, Song

, Chen

, Ho

, Cheng

, Ho

, Lam

(2012) Plasma amyloid-β oligomers level is a biomarker for Alzheimer’s disease diagnosis. Biochem Biophys Res Commun 423, 697–702.

81.

Nakamura

, Kaneko

, Villemagne

, Kato

, Doecke

, Doré

, Fowler

, Li

, Martins

, Rowe

, Tomita

, Matsuzaki

, Ishii

, Arahata

, Iwamoto

, Ito

, Tanaka

, Masters

, Yanagisawa

(2018) High performance plasma amyloid-β biomarkers for Alzheimer’s disease. Nature 554, 249–254.

82.

Zachary

, McGavin

(2016) Pathologic Basis of Veterinary Disease ExpertConsult-E-BOOK, Elsevier Health Sciences.

83.

Armstrong

(2009) Alzheimer’s disease and the eye. J Optom 2, 103–111.

84.

Hadoux

, Hui

, Lim

JKH

, Masters

, Pébay

, Chevalier

, Ha

, Loi

, Fowler

, Rowe

, Villemagne

, Taylor

, Fluke

, Soucy

, Lesage

, Sylvestre

, Rosa-Neto

, Mathotaarachchi

, Gauthier

, Nasreddine

, Arbour

, Rhéaume

, Beaulieu

, Dirani

, Nguyen

CTO

, Bui

, Williamson

, Crowston

, van

Wijngaarden P

(2019) Non-invasive in vivo hyperspectral imaging of the retina for potential biomarker use in Alzheimer’s disease. Nat Commun 10, 4227.

85.

Kawarabayashi

, Younkin

, Saido

, Shoji

, Ashe

, Younkin

(2001) Age-dependent changes in brain, CSF, and plasma amyloid (beta) protein in the Tg2576 transgenic mouse model of Alzheimer’s disease. J Neurosci 21, 372–381.

86.

DaRocha-Souto

, Scotton

, Coma

, Serrano-Pozo

, Hashimoto

, Serenó

, Rodríguez

, Sánchez

, Hyman

, Gómez-Isla

(2011) Brain oligomeric β-amyloid but not total amyloid plaque burden correlates with neuronal loss and astrocyte inflammatory response in amyloid precursor protein/tau transgenic mice. J Neuropathol Exp Neurol 70, 360–376.

87.

Liu

, Rasool

, Yang

, Glabe

, Schreiber

, Ge

, Tan

(2009) Amyloid-peptide vaccinations reduce {beta}-amyloid plaques but exacerbate vascular deposition and inflammation in the retina of Alzheimer’s transgenic mice. Am J Pathol 175, 2099–2110.

88.

Morin

, Abraham

, Amaratunga

, Johnson

, Huber

, Sandell

, Fine

(1993) Amyloid precursor protein is synthesized by retinal ganglion cells, rapidly transported to the optic nerve plasma membrane and nerve terminals, and metabolized. J Neurochem 61, 464–473.

89.

Koronyo-Hamaoui

, Koronyo

, Ljubimov

, Miller

, Ko

, Black

, Schwartz

, Farkas

(2011) Identification of amyloid plaques in retinas from Alzheimer’s patients and noninvasive in vivo optical imaging of retinal plaques in a mouse model. Neuroimage 54 Suppl 1, S204–217.

90.

Gouras

, Tsai

, Naslund

, Vincent

, Edgar

, Checler

, Greenfield

, Haroutunian

, Buxbaum

, Xu

, Greengard

, Relkin

(2000) Intraneuronal Abeta42 accumulation in human brain. Am J Pathol 156, 15–20.

91.

Umeda

, Tomiyama

, Sakama

, Tanaka

, Lambert

, Klein

, Mori

(2011) Intraneuronal amyloid β oligomers cause cell death via endoplasmic reticulum stress, endosomal/lysosomal leakage, and mitochondrial dysfunction in vivo. J Neurosci Res 89, 1031–1042.

92.

Bayer

, Wirths

, Majtényi

, Hartmann

, Multhaup

, Beyreuther

, Czech

(2001) Key factors in Alzheimer’s disease: Beta-amyloid precursor protein processing, metabolism and intraneuronal transport. Brain Pathol 11, 1–11.

93.

Iwatsubo

, Odaka

, Suzuki

, Mizusawa

, Nukina

, Ihara

(1994) Visualization of A beta 42(43) and A beta 40 in senile plaques with end-specific A beta monoclonals: Evidence that an initially deposited species is A beta 42(43). Neuron 13, 45–53.

94.

Wirths

, Multhaup

, Czech

, Blanchard

, Moussaoui

, Tremp

, Pradier

, Beyreuther

, Bayer

(2001) Intraneuronal Abeta accumulation precedes plaque formation in beta-amyloid precursor protein and presenilin-1 double-transgenic mice. Neurosci Lett 306, 116–120.

95.

, Cuervo

, Kumar

, Peterhoff

, Schmidt

, Lee

, Mohan

, Mercken

, Farmery

, Tjernberg

, Jiang

, Duff

, Uchiyama

, Näslund

, Mathews

, Cataldo

, Nixon

(2005) Macroautophagy–a novel Beta-amyloid peptide-generating pathway activated in Alzheimer’s disease. J Cell Biol 171, 87–98.

96.

Langui

, Girardot

, El

Hachimi KH

, Allinquant

, Blanchard

, Pradier

, Duyckaerts

(2004) Subcellular topography of neuronal Abeta peptide in APPxPS1 transgenic mice. Am J Pathol 165, 1465–1477.

97.

Zheng

, Kågedal

, Dehvari

, Benedikz

, Cowburn

, Marcusson

, Terman

(2009) Oxidative stress induces macroautophagy of amyloid beta-protein and ensuing apoptosis. Free Radic Biol Med 46, 422–429.

98.

Takahashi

, Milner

, Li

, Nam

, Edgar

, Yamaguchi

, Beal

, Xu

, Greengard

, Gouras

(2002) Intraneuronal Alzheimer abeta42 accumulates in multivesicular bodies and is associated with synaptic pathology. Am J Pathol 161, 1869–1879.

99.

Yang

, Chandswangbhuvana

, Margol

, Glabe

(1998) Loss of endosomal/lysosomal membrane impermeability is an early event in amyloid Abeta1-42 pathogenesis. J Neurosci Res 52, 691–698.

100.

, Nwabuisi-Heath

, Laxton

, Ladu

(2010) Endocytic pathways mediating oligomeric Abeta42 neurotoxicity. Mol Neurodegener 5, 19.

101.

Zheng

, Cedazo-Minguez

, Hallbeck

, Jerhammar

, Marcusson

, Terman

(2012) Intracellular distribution of amyloid beta peptide and its relationship to the lysosomal system. Transl Neurodegener 1, 19.