Detection of retinal and blood Aβ oligomers with nanobodies.

INTRODUCTION: Abnormal retinal changes are increasingly recognized as an early pathological change in Alzheimer's disease (AD). Although amyloid beta oligomers (Aβo) have been shown to accumulate in the blood and retina of AD patients and animals, it is not known whether the early Aβo deposition precedes their accumulation in brain. METHODS AND
RESULTS: Using nanobodies targeting Aβ1-40 and Aβ1-42 oligomers we were able to detect Aβ oligomers in the retina and blood but not in the brain of 3-month-old APP/PS1 mice. Furthermore, Aβ plaques were detected in the brain but not the retina of 3-month-old APP/PS1 mice.
CONCLUSION: These results suggest that retinal accumulation of Aβo originates from peripheral blood and precedes cognitive decline and Aβo deposition in the brain. This provides a very strong basis to develop and implement an "eye test" for early detection of AD using nanobodies targeting retinal Aβ.

INTRODUCTION

The importance of amyloid beta oligomer (Aβo) detection has gained momentum and experimental studies using human Alzheimer's disease (AD) samples have shown that this form can be detected as much as two decades before clinical onset of AD. , , , Aβo can potentially become a strong biomarker for early AD detection and could provide accurate biochemical information about various preclinical stages of AD. Several investigators have shown experimentally that blood‐borne Aβo is a viable biomarker for human AD. A study by Nakamura et al. has identified high‐performance plasma Aβ biomarkers using a combination of immunoprecipitation and mass spectrometry and suggested that plasma Aβ ratio can predict brain Aβ burden. Plasma Aβ precursor protein (APP)669–711/Aβ1–42 and Aβ1–40/Aβ1–42 were correlated with brain Aβ levels determined by Aβ positron emission tomography (PET) imaging.

Ocular disturbances are an early complaint in AD patients , , with reported changes in color vision, contrast sensitivity, visual memory and perception, , , nerve damage, and loss of nerve fibers. Ganglion cell loss and thinning of the retinal nerve fiber layer (RNFL) , have also been reported. A recent study by Coppola et al. reported that RNFL thinning was associated with neurodegenerative progressions in mild cognitive impaired (MCI) and AD patients compared to cognitively healthy individuals. Similar color vision and contrast sensitivity deficits were shown in a murine model of AD. In addition to neuronal changes in the retina, alteration of retinal blood flow and morphology has also been noted. Importantly, Aβ deposits in the retina of AD patients were identified by histology and in vivo imaging of MCI and AD patients. Subsequent studies have corroborated these findings and showed accumulation of Aβ and hyperphosphorylated tau (p‐tau) in the retina of AD patients , , and animal models.

Nanobodies are camelid‐derived antibody fragments with unique biological features, including lack of light chains, smaller size (more diffusible in tissues), hydrophilic (soluble in aqueous solution), highly stable, and more resistant against chemical denaturation. Previous studies reported that nanobodies targeting Aβ and neurofibrillary tangles in mice brain parenchyma are able to cross the blood‐brain barrier (BBB). Another study demonstrated that nanobodies specific for Aβ oligomers prevent neurotoxicity and fibril formation. Moreover, Vandesquille et al. showed that nanobodies were able to detect cerebral Aβ plaque deposits via magnetic resonance imaging (MRI) after intravenous injection.

In the current study, we used nanobody anti‐Aβ1‐40 (PrioAD12) and anti‐Aβ1‐42 (PrioAD13) oligomer antibodies to measure the levels of Aβo in the brain and retina of the APP/PS1 mice at 3 to 4 months of age with immunohistochemistry (IHC), before behavioral changes and appearance of cognitive deficits. We showed that retinal Aβ1‐40 and Aβ1‐42 oligomer levels were significantly higher in APP/PS1 mice compared to age‐matched WT controls. Furthermore, immunofluorescence (FL) analysis confirmed our IHC results and surprisingly resulted in the detection of large amounts of Aβo in the 18‐month‐old APP/PS1 age group. We also confirmed the localization of both Aβ1‐40 and Aβ1‐42 oligomers to neuronal late‐endosomal compartments in the retina and brain that was associated with activated astrocytes and microglia in APP/PS1 mice. Of importance, Aβ1‐40 and Aβ1‐42 levels in whole blood quantified by western blotting with the nanobodies were elevated in 3‐ and 18‐month‐old APP/PS1 mice compared to wild‐type (WT) controls. The observation that Aβo was detectable in the retina and blood but not in the brain of young APP/PS1 mice suggests that deposition of retinal Aβo might originate from the blood. Taken together, our results provide an important milestone in achieving an “eye” and/or blood‐based screening test for AD.

RESEARCH IN CONTEXT

Systematic review: Although several experimental studies have demonstrated the presence of blood‐borne amyloid beta oligomers (Aβo) decades before clinical Alzheimer's disease (AD) and neuropathology have ensued, few studies have focused on the early detection of Aβ oligomers in the retina and other eye structures.

Interpretation: The results of this study involving the double‐transgenic APP/PS1 AD mouse model showed that Aβo could be detected simultaneously in blood and retina before its deposition in the brain and appearance of cognitive decline.

Future directions: Future research in this field should aim to establish a routine clinical optical retinal and/or blood‐based test for the detection of human AD before cognitive decline and neuropathology have ensued. The ability to detect AD before clinical disease will potentially facilitate implementation of effective therapies.

METHODS

Animals and ethics statement

All procedures followed the requirements of the National Health and Medical Research Council of Australia statement for the use of animals in research and were approved by the Western Sydney University Animal Ethics Committee (ACEC # A12905). Mice were housed with free access to water and standard rodent chow (Gordon's Specialty Stock Feeds). APP/PS1 mice have the APP Swedish mutation K595N and M596L and PSEN1 with L166P mutation controlled by the Thy1 promoter (ww.alzforum.org). Cognitive impairment is usually observed after 7 months. , The APP/PS1 mouse model has high brain levels of Aβ1‐42 over Aβ1‐40, which increases with age. , Age‐matched WT littermates were used as a control.

Immunohistochemistry

APP/PS1 mice (n = 28) and WT littermates (n = 20) were first euthanized (Advanced Anesthesia Specialists, DarvallVet) before perfusion with saline followed with 10% neutral buffered formalin. Formalin fixed paraffin embedded blocks (FFPE) were prepared using 10% neutral buffered formalin as a fixative followed by graded ethanol and xylene. 6μm thick brain and eye tissue sections were cut using a microtome (Thermo Fisher Scientific). Sections were then deparaffinized with xylene and rehydrated through graded alcohols and finally washed with deionized water.

Sections were pretreated using the 2100 antigen retriever (Aptum Biologics Ltd) to expose the target epitopes. Sections were then treated with 90% formic acid for 5 minutes at room temperature followed by cell membrane permeabilization, which was achieved by using 1% triton X for 1 minute prior to addition of 0.3% H2O2 for 15 minutes to inactivate endogenous peroxidases. Sections were then blocked with Protein Block Serum‐Free (Agilent) for 15 minutes. Sections were then stained for 1 hour with the following primary antibodies in phosphate‐buffered saline (PBS): anti‐Aβ1‐40 (PrioAD12), anti‐Aβ1‐42 (PrioAD13) antibodies (1:500), or mouse anti‐Aβ purified 4G8 antibody (1:500; BioLegend). After washing with PBS, sections were incubated for 1 hour at room temperature with secondary antibodies in PBS:horseradish peroxidase (HRP)‐conjugated anti‐llama immunoglobulin G (IgG; Bethyl Laboratories) or anti‐mouse IgG (Sigma‐Aldrich). Sections were then washed in PBS (x3) before addition of DAB substrate chromogen system and incubated for 5 to 10 minutes. Slides were then counterstained with hematoxylin for 1 minutes. The Olympus VS 120 Slide Scanner was used to visualize images and the Olympus OlyVIA and Olympus cellSens imaging software were used for analysis.

Immunofluorescence co‐localization studies

Double immuno‐labeling was achieved by two different fluorescent labels, each having a separate emission wavelength. Sections were incubated overnight with anti‐Aβ1‐40 (PrioAD12), anti‐Aβ1‐42 (PrioAD13) or 4G8 antibody at 4°C. Sections were also incubated with camelid antibodies and mouse anti‐lysosomal‐associated membrane protein 2 (LAMP2, Stressgen Bioreagents Corp) antibody to assess whether Aβo localizes to lysosomes/late endosomes. Sections derived from the 3‐ to 4‐month‐old group were incubated with camelid antibodies and GFAP or Iba1 (Thermo Fisher Scientific). Finally, sections were incubated with camelid‐derived antibodies and anti‐NeuN mAb, clone A60 (MilliporeSigma) to confirm the intra‐neuronal localization of the Aβos. All the sections were incubated overnight at 4°C. After washing with PBS, sections were then incubated with goat anti‐llama IgG conjugated to fluorescein isothiocyanate (FITC; Bethyl Laboratories, Inc) and donkey‐anti‐mouse IgG conjugated to Texas Red (Sigma‐Aldrich) for 2 hours at 4°C. Sections were then mounted using fluorescence mounting media (Agilent) then visualized using an Olympus VS 120 slide scanner with a standard FITC/Texas Red double band‐pass filter set.

Image quantification

For the quantification of the age‐dependent accumulation of Aβ plaque (Aβp) and Aβo, we used three sections derived from the 3‐ to 4‐month‐old APP/PS1 (n = 8) and WT (n = 8) mice as well as the 17‐ to 18‐month‐old APP/PS1 (n = 8) and WT (n = 8) mice. Three different hippocampus, cerebral cortex, and retinal sections were analyzed. Immunohistochemical signal intensity was visualized by capturing bright field images using the Olympus VS 120 slide scanner. Images were analyzed using OlyVIA software. , Age‐dependent accumulation of Aβp and Aβo in APP/PS1 mice was quantified using cellSens image processing software. The mean intensity of particles was calculated in several brain and retinal regions from each age group and the result was presented as percentage intensity and expressed as mean ± standard error of the mean.

Immunoprecipitation and western blot analysis of Aβo in whole blood

To measure blood levels of Aβo in APP/PS1 mice, we performed immunoprecipitation to enrich/isolate Aβ from 3 to 4‐ and 17 to 18‐month‐old mice as described. Samples were loaded on precast gels (Bio‐Rad) and electrophoresed and 1 μg/mL of nanobody Aβ1‐40 (PrioAD12), Aβ1‐42 (PrioAD13) anti‐oligomer antibodies or A11 rabbit‐anti‐Aβo antibody (MilliporeSigma) was added followed by anti‐llama (Bethyl Laboratories) or anti‐rabbit IgG (Sigma‐Aldrich) HRP conjugated antibody. The resulting digital images were analyzed with ImageJ processing program for the densitometry analysis and values between the transgenic APP/PS1 and WT controls were compared.

Statistical analyses

Statistical analyses were performed using SAS Enterprise Guide version 8.2. A natural logarithm transformation was applied to the measurements. Shapiro‐Wilk test was used to determine normality. Group differences were analyzed by Wilcoxon‐Mann‐Whitney test due to non‐normality. The blood‐borne Aβo performance at 3 to 4 months when predicting Aβp at 17 to 18 months in both brain and eye was not performed because these data were not measured on the same animal over time. There were 36 comparisons overall so a Bonferroni correction of 0.05/36 = 0.0014 was applied, meaning P‐values less than this were considered statistically significant.

RESULTS

Immunodetection of Aβ plaques and oligomers in the brain and retina of APP/PS1 mice using single‐domain antibodies

In this study, we wanted to test the hypothesis that retinal Aβo accumulation precedes neurobehavioral deficits but also predates brain deposition of both Aβo and Aβp in young APP/PS1 mice using single‐domain camelid‐derived anti‐Aβo antibody fragments and the 4G8 anti‐Aβp antibody (Table 1 and Figures 1, 2, 3, 4). Of note, the single‐domain antibody fragments, called PrioAD12 and PrioAD13, were previously shown to bind to Aβ1‐40 and Aβ1‐42, respectively. Here, we showed widespread intra‐neuronal Aβ1‐40 and Aβ1‐42 oligomers in the retinal inner nuclear layer (INL), outer nuclear layer (ONL), and ganglion cell layer (GCL) of the 3‐month‐old APP/PS1 mice (Figure 1C & F); in contrast, no Aβo depositions were seen in the brains at the same age (Figure 1A, B, D, E), indicating that retinal Aβo accumulation precedes its appearance in the brain. Furthermore, no 4G8‐specific Aβp was found in the brain and retina of the 3‐month‐old APP/PS1 mice (Figure 1G, H, I). Interestingly, both Aβ1‐40 and Aβ1‐42 were detected at 8 months of age in the cerebral cortex and hippocampus of APP/PS1 mice (Figure 2A, B, D, E) as well as in the retina similar to 3‐month‐old APP/PS1 mice (Figure 2C, F). 4G8‐specific Aβp was also observed in the cerebral cortex, hippocampus, and INL of the retina at 8 months (Figure 2G, H, I). Both Aβo and Aβp were detectable in the cerebral cortex, hippocampus, and retinal layers of 11‐month‐old APP/PS1 mice (Figure 3A‐I). Finally, no Aβo were detected in 18‐month‐old APP/PS1 mice (Figure 4A, B, C, D, E, F), whereas extensive, widespread, and conspicuous Aβp was observed in the cerebral cortex, hippocampus (Figure 4 G, H), and retinal INL (Figure 4 I). No Aβo or Aβp deposits were seen in age‐matched WT littermates (data not shown). Overall our data confirm that Aβo deposits first appear in the retina months before they are detectable in the brain and support the proposition that the retinal oligomers probably originate from the blood. These results also validate our previous findings that showed Aβp burden increased over the course of the disease in both brain and retina, whereas Aβo levels appeared to decrease in an age‐dependent manner.

TABLE 1

Age‐dependent accumulation of Aβ oligomers and plaques in the blood, retina and brain of APP/PS1 mice

Age groups (months)	PRIOAD12 (Aβ1‐40) or PRIOAD13 (Aβ1‐42)				4G8 (Aβ plaques)
	Blood	Retinal layers	Brain		Retinal layers	Brain
			Cerebral cortex	Hippocampus		Cerebral cortex	Hippocampus
3–4 (n = 16)	Present	Present	Absent	Absent	Absent	Absent	Absent
8–11 (n = 16)	Nd*	Present	Present	Present	Present	Present	Present
17–18 (n = 16)	Present	Absent	Absent	Absent	Present	Present	Present

Abbreviations: Aβ, amyloid beta; Nd, Not determined.

FIGURE 1

Immunohistochemical staining of amyloid beta (Aβ) in the brain and retina of 3‐month‐old APP/PS1 mice. Immunohistochemical staining with anti‐Aβ1‐40 and anti‐Aβ1‐42 oligomer nanobodies and 4G8 anti‐Aβ plaque antibody of 3‐month‐old APP/PS1 mice. Immunohistochemical staining with anti‐Aβ1‐40 (PrioAD12) and anti‐Aβ1‐42 (PrioAD13) nanobodies of a 3‐month‐old APP/PS1 mice did not demonstrate Aβ1‐40 depositions in the (A) cerebral cortex and (B) hippocampus as well as Aβ1‐42 depositions in the (D) cerebral cortex and (E) hippocampus. Aβ1‐40 and Aβ1‐42 depositions were observed in the (C, F) ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) of the retina. The photomicrograph was derived from peripheral region of the retina—away from the optic disc. Immunohistochemical staining with 4G8 antibody of 3‐month‐old APP/PS1 mice did not display characteristic extracellular Aβ plaques in the (G) hippocampus, (H) cerebral cortex, and (I) retina. Representative of all affected mice in this age group

FIGURE 2

Immunohistochemical staining of amyloid beta (Aβ) in the brain and retina of 8‐month‐old APP/PS1 mice. Immunohistochemical staining with anti‐Aβ1‐40 and anti‐Aβ1‐42 oligomer nanobodies and 4G8 anti‐Aβ plaque antibody of 8‐month‐old APP/PS1 mice. Immunohistochemical staining with anti‐Aβ1‐40 (PrioAD12) and anti‐Aβ1‐42 (PrioAD13) nanobodies of 8‐month‐old APP/PS1 mice showed presence of Aβ1‐40 depositions in the (A) cerebral cortex and (B) hippocampus as well as Aβ1‐42 depositions in the (D) cerebral cortex and (E) hippocampus. Aβ1‐40 and Aβ1‐42 depositions were observed in the (C, F) ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) of the retina. The photomicrograph was derived from peripheral region of the retina—away from the optic disc. Immunohistochemical staining with 4G8 antibody of 8‐month‐old APP/PS1 mice displayed extensive extracellular Aβ plaque staining in the (G) hippocampus, (H) cerebral cortex, and (I) retina. Representative of all affected mice in this age group

FIGURE 3

Immunohistochemical staining of amyloid beta (Aβ) in the brain and retina of 11‐month‐old APP/PS1 mice. Immunohistochemical staining with anti‐Aβ1‐40 and anti‐Aβ1‐42 oligomer nanobodies and 4G8 anti‐Aβ plaque antibody of 11‐month‐old APP/PS1 mice. Immunohistochemical staining with anti‐Aβ1‐40 (PrioAD12) and anti‐Aβ1‐42 (PrioAD13) nanobodies of 11‐month‐old APP/PS1 mice showed presence of Aβ1‐40 oligomer depositions in the (A) cerebral cortex and (B) hippocampus as well as Aβ1‐42 oligomer depositions in the (D) cerebral cortex and (E) hippocampus. Aβ1‐40 and Aβ1‐42 depositions were observed in the (C, F) ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) of the retina. The photomicrograph was derived from peripheral region of the retina—away from the optic disc. Immunohistochemical staining with 4G8 antibody of 11‐month‐old APP/PS1 mice displayed extensive extracellular Aβ plaque staining in the (G) hippocampus, (H) cerebral cortex, and (I) retina. Representative of all affected mice in this age group

FIGURE 4

Immunohistochemical staining of amyloid beta (Aβ) in the brain and retina of 18‐month‐old APP/PS1 mice. Immunohistochemical staining with anti‐Aβ1‐40 and anti‐Aβ1‐42 oligomer nanobodies and 4G8 anti‐Aβ plaque antibody of 18‐month‐old APP/PS1 mice. Immunohistochemical staining with anti‐Aβ1‐40 (PrioAD12) and anti‐Aβ1‐42 (PrioAD13) nanobodies of 18‐month‐old APP/PS1 mice did not show presence of Aβ1‐40 depositions in the (A) cerebral cortex and (B) hippocampus as well as Aβ1‐42 in the (D) cerebral cortex and (E) hippocampus. Aβ1‐40 and Aβ1‐42 depositions were not observed (C, F) in the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) of the retina. The photomicrograph was derived from peripheral region of the retina—away from the optic disc. Immunohistochemical staining with 4G8 antibody of 18‐month‐old APP/PS1 mice displayed extensive extracellular Aβ plaque staining in the (G) hippocampus and (H) cerebral cortex and (I) plaques were observed in the retina (white arrows). Representative of all affected mice in this age group

Quantitative analysis of Aβ plaques and oligomers in the brain, retina, and whole blood of APP/PS1 mice using single‐domain antibodies

Age‐dependent retinal and brain accumulation of Aβp and Aβo in the 3‐ to 4‐month‐old APP/PS1 mice (n = 8) and WT littermates (n = 8) was quantified and compared to Aβp and Aβo levels in the 17‐ to 18‐month‐old APP/PS1 mice (n = 8) and WT littermates (n = 8; Tables 2 and 3; Figure 5). Three different areas of retina, hippocampus, and cerebral cortex of each section (32 x 3 sections) were analyzed. Figure 5 shows the normalized intensity of retinal and brain Aβ as measured by the cellSens image processing software and the values for Aβ1‐40 (PRIOAD12 antibody), Aβ1‐42 (PRIOAD13 antibody), and total Aβ (4G8 antibody). We found that the normalized intensity of retinal Aβ1‐40 and Aβ1‐42 oligomers were significantly higher in 3‐ to 4‐month‐old compared to the 17‐ to 18‐month‐old APP/PS1 mice (P = .0002; Tables 2 and 3), whereas normalized intensity of brain Aβp was significantly higher in the retina and brain of 17‐ to 18‐month‐old compared to the 3‐ to 4‐month‐old APP/PS1 mice (P = .0002; Tables 2 and 3; Figure 5).

TABLE 2

Statistical analysis of the age‐dependent retinal, brain, and blood accumulation of Aβp and Aβo in the 3‐ to 4‐month‐ and 17‐ to 18‐month‐old APP/PS1 mice and compared to wild‐type littermates. Bonferroni correction of 0.05/36 = 0.0014 was applied, meaning P‐values less than this value were considered statistically significant

Antibody	Age	Predictor	APP/PS1					Wild type					Difference
			N	Median	Lower quartile	Upper quartile	IQR	N	Median	Lower quartile	Upper quartile	IQR	z	P
PRIOAD12	3–4 months	logeye	8	8.67	8.53	8.84	0.32	8	3.28	2.51	4.04	1.53	‐3.3082	.0002
PRIOAD12		logbrain	8	4.00	3.82	4.04	0.22	8	2.55	2.43	2.63	0.20	‐3.3082	.0002
PRIOAD12		logblood	8	3.94	3.78	4.28	0.51	8	2.30	2.30	2.56	0.25	‐2.2185	.0286
PRIOAD13		logeye	8	8.34	7.99	8.57	0.58	8	3.01	2.43	3.97	1.53	‐3.3106	.0002
PRIOAD13		logbrain	8	3.83	3.40	4.11	0.71	8	2.50	2.33	2.88	0.55	‐3.3106	.0002
PRIOAD13		logblood	8	4.08	3.96	4.12	0.16	8	2.30	2.30	2.30	0.00	‐2.3067	.0286
4G8		logeye	8	2.30	2.30	2.30	0.00	8	2.30	2.30	2.30	0.00	0	1
4G8		logbrain	8	2.30	2.30	2.30	0.00	8	2.30	2.30	2.30	0.00	0	1
PRIOAD12	17–18 months	logeye	8	2.93	2.33	3.77	1.43	8	2.30	2.30	2.30	0.00	2.8359	.007
PRIOAD12		logbrain	8	2.57	2.38	2.80	0.42	8	2.30	2.30	2.30	0.00	3.1854	.0014
PRIOAD12		logblood	8	4.43	4.28	4.47	0.19	8	2.30	2.30	2.30	0.00	2.3067	.0286
PRIOAD13		logeye	8	2.74	2.35	2.93	0.58	8	2.30	2.30	2.30	0.00	2.8359	.007
PRIOAD13		logbrain	8	2.76	2.57	2.98	0.41	8	2.30	2.30	2.30	0.00	3.1825	.0014
PRIOAD13		logblood	8	2.83	2.72	3.22	0.50	8	2.30	2.30	2.30	0.00	2.3067	.0286
4G8		logeye	8	4.35	4.06	4.66	0.60	8	2.30	2.30	2.30	0.00	3.5336	.0002
4G8		logbrain	8	7.73	7.68	7.77	0.09	8	2.30	2.30	2.30	0.00	3.5366	.0002

TABLE 3

Age‐dependent retinal and brain accumulation of Aβp and Aβo in the 3‐ to 4‐month‐old APP/PS1 mice were quantified and compared to Aβp and Aβo levels in the 17‐ to 18‐month‐old APP/PS1 mice. Bonferroni correction of 0.05/36 = 0.0014 was applied, meaning P‐values less than this value were considered statistically significant

Antibody	Group	Log	N	Age (month)	Difference
					z	P
PRIOAD12	APP/PS1	EYE	8	3–4 vs. 17–18	–3.3106	.0002
PRIOAD13		EYE			–3.3106
4G8		EYE			3.5336
4G8		BRAIN			3.5366

Abbreviations: Aβ, amyloid beta; Aβo, amyloid beta oligomers; Aβp, amyloid beta plaques.

FIGURE 5

Age‐dependent accumulation of amyloid beta (Aβ) oligomers and Aβ plaques. Quantification of the age dependent accumulation of cerebral and retinal Aβ oligomers and Aβ plaques with nanobodies: Immunodetection and quantification of retinal and cerebral Aβ1‐40 and Aβ1‐42 with PrioAD12 and PrioAD13 nanobodies in the cerebral cortex and hippocampus and retina of 3‐ to 4‐month‐old (n = 8) and 17‐ to 18‐month‐old (n = 8) APP/PS1 mice using cellSens software image analysis after immunohistochemical staining. Total Aβ plaque burden (Aβp) was quantified in the cerebral cortex and hippocampus and retina of 3‐ to 4‐month‐old (n = 8) and 17‐ to 18‐month‐old (n = 8) APP/PS1 mice. Wilcoxon‐Mann‐Whitney test was performed and normalized intensity of both Aβ1‐40 and Aβ1‐42 oligomers were significantly higher in the retina of 3‐ to 4‐month‐old compared to the 17‐ to 18‐month‐old age group APP/PS1 mice (P = .0002) whereas Aβp load was significantly higher in brain and retina of the 17‐ to 18‐month‐old age group compared to the 3‐ to 4‐month‐old age group (P = .0002). Error bars represent interquartile range

Several studies have demonstrated the presence of Aβo in plasma of patients with MCI and AD; , , plasma levels may also predict the brain Aβ burden. In this study, we hypothesized that Aβo accumulation in blood might also precede retinal accumulation or at least occur simultaneously with retinal accumulation. Here, we used fresh whole blood in blood lysis buffer to enrich Aβo. Initially and after lysing whole blood derived from APP/PS1 mice, anti‐oligomer‐A11‐coated immunomagnetic microbeads were used to isolate Aβ from APP/PS1 mice and WT littermates (APP/PS1, n = 16, and WT, n = 16). After western blotting, anti‐Aβ1‐40 (PrioAD12) or anti‐Aβ1‐42 (PrioAD13) oligomer single‐domain antibodies were used to immunodetect Aβo isoforms in 3‐ and 18‐month‐old APP/PS1 and WT mice. Anti‐Aβ1‐40 PrioAD12 displayed a two‐band pattern ranging between 10 and 15 kDa in the 3‐month‐old APP/PS1 mice, whereas only one band at 10 to 15 kDa was seen in the 18‐month‐old APP/PS1 age group (Figure S1A, B in supporting information). In contrast, anti‐Aβ1‐42 PrioAD13 showed only one band at 10 to 15 KDa in both the 3‐ and 18‐month‐old APP/PS1 age groups (Figure S1A, B). Furthermore, we also used A11 rabbit anti‐Aβo antibody to immuno‐compare Aβ1‐40 and Aβ1‐42 levels detected with PrioAD12 and PrioAD13. In both age groups A11 displayed a different band pattern compared to the single‐domain antibodies and ranged between 70 and 80 kDa (Figure S1A, B). We then performed densitometric analysis of scanned western blot membranes. Table 2 shows the normalized intensity of whole‐blood Aβ as measured by ImageJ software and the values for Aβ1‐40 (PRIOAD12 antibody) and Aβ1‐42 (PRIOAD13 antibody). Levels of both Aβ1–40 and Aβ1–42 oligomers were not significantly higher compared to the WT levels in the 3‐month age group (P = .0286) when a Bonferroni correction was applied. However, when the statistical analysis was performed using paired t‐tests (P‐values below .05 were deemed significant in this case) to compare levels of both Aβ1–40 and Aβ1–42 oligomers in APP/PS1 versus WT, these were significant (P < .05; Figure 6). Levels of Aβ1‐40 increased significantly from 3 to 18 months while Aβ1‐42 decreased by at least three‐fold in the 18‐month‐old APP/PS1 mice (Figure 6). These results also highlight the high binding affinity of the camelid‐derived single domain antibodies for detection of Aβ1‐40 and Aβ1‐42 oligomers in whole blood.

FIGURE 6

Quantification of amyloid beta (Aβ) oligomers in blood. Quantification of the age‐dependent accumulation of blood‐borne Aβ oligomers with nanobodies. Immunodetection and quantification of blood‐borne Aβ1‐40 and Aβ1‐42 with PrioAD12 and PrioAD13 nanobodies in whole blood of 3‐ to 4‐month‐old (n = 8) and 17‐ to 18‐month‐old (n = 8) APP/PS1 mice using ImageJ software analysis after western blotting. The normalized intensity was calculated in three independent experiments for each age group and the final result was presented as median intensity. Paired t‐tests were performed and P‐values below .05 were considered significant. Levels of both Aβ1–40 and Aβ1–42 oligomers were significantly higher in the 3‐ to 4‐month age group. Aβ1‐40 oligomer level increased significantly from 3 to 4 to 17 to 18 months whereas Aβ1‐42 oligomer level decreased by at least three‐fold in the 17‐ to 18‐month‐old APP/PS1 mice. Error bars represent interquartile range

Co‐localization of Aβ oligomers and plaques in the retina and brain of APP/PS1 mice

To determine whether Aβ1‐40 or Aβ1‐42 oligomers co‐localized with Aβp in different anatomical regions and structures of the retina and brain, we co‐stained brain and retinal sections with 4G8 antibody with either anti‐Aβ1‐40 (PrioAD12) or anti‐Aβ1‐42 (PrioAD13) oligomer single‐domain antibodies (Figure 7). We did not observe any co‐localization in the brain and retina of the 3‐month‐old APP/PS1 mice (Figure 7A‐F) but confirmed the presence of Aβ1‐40 or Aβ1‐42 oligomer in the retinal layers (Figure 7C, F). However, both retinal Aβp and Aβ1‐40 or Aβp and Aβ1‐42 were shown to co‐localize in the GCL, IPL, and INL of the 8‐month‐old APP/PS1 age group (Figure 7I, L). Furthermore, co‐accumulation of Aβp and Aβ1‐40 or Aβp and Aβ1‐42 was also seen in the cerebral cortex and hippocampus (Figure 7G, H, J, K), noticeably higher levels of Aβo in this age group compared to plaques. Aβp in 11‐month‐old APP/PS1 mice was markedly increased while Aβ1‐40 and Aβ1‐42 oligomers decreased in the brain (Figure 7M, N, P, Q). High levels of Aβ1‐40 and Aβ1‐42 oligomers were consistently found in the retina (Figure 7O, R). Finally, 18‐month‐old APP/PS1 mice showed that both Aβ1‐40 and Aβ1‐42 co‐localized with Aβp in the cerebral cortex and hippocampus and in the retinal GCL, INL, and ONL (Figure 7S‐X). Surprisingly, high levels of Aβ1‐40 and Aβ1‐42 oligomers were observed in the retina and brain of 18‐month‐old APP/PS1 mice (Figure 7S‐X), perhaps confirming the hypothesis that plaques act as a reservoir for the toxic Aβo. WT age‐matched littermates did not show any co‐localization of Aβp with Aβ1‐40 or Aβ1‐42 (data not shown).

FIGURE 7

Co‐localization of amyloid beta (Aβ) oligomers and plaques. Immunofluorescence co‐localization of cerebral and retinal Aβ oligomers and Aβ plaques in different APP/PS1 age groups. Cerebral and retinal co‐staining with anti‐Aβ1‐40 (PrioAD12) and anti‐Aβ1‐42 (PrioAD13) nanobodies (GREEN) and 4G8 antibody (RED) of 3‐ (A‐F), 8‐ (G‐L), 11‐ (M‐R), and 18‐month‐old (S‐X) APP/PS1 mice. No distinctive oligomers co‐localized with plaques in the brain cortical region (A, D) and in the hippocampus (B, E). Large number of oligomers found in the (C, F) retinal ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) but no co‐localization observed in the 3‐month‐old mice (white arrows). Then Aβ1‐40 (G, H) and Aβ1‐42 (J, K) oligomers co‐localized with plaques in the brain cortical region and hippocampus, respectively, and in the retinal GCL and INL (I, L) of the 8‐month‐old mice (white arrows), respectively. With age progression, Aβ1‐40 (M, N) and Aβ1‐42 (P, Q) oligomers co‐localized with plaques in the brain cortical region and hippocampus, respectively, and in the retinal INL (O, R) of the 11‐month‐old mice (white arrows). Finally, in 18‐month‐old APP/PS1 mice, Aβ oligomers co‐localized with plaques in the brain cortical region (S, V) and in the hippocampus (T, W) and also in the retinal GCL, INL, and ONL (U, X) of the 18‐month‐old mice, respectively (white arrows). Representative of all affected mice in all age groups

DISCUSSION

Behavioral assessment of the APP/PS1 AD mouse model demonstrated that memory decline and cognitive deficits start after 7 months of age. Although our study did not include behavioral assessments, mice appeared healthy until 10 months of age. Of importance, we show that increased Aβo in blood and retinal accumulation of Aβo was observed at 3 months in APP/PS1 mice in the absence of Aβp accumulation in the retina and before appearance of both Aβo and Aβp in brain. The accumulation of blood and retinal Aβo occur at a very early age, likely months before the expected memory and cognitive deficits in APP/PS1 mice. These data indicate that these assemblies are likely to be responsible for the toxic effects associated with AD, , can be detected before AD onset, and might originate from the blood. Several diagnostic strategies have been developed for early AD detection, including systems for the detection of Aβo in plasma and in the cerebrospinal fluid (CSF). The experimental value of detecting blood‐borne Aβ biomarkers has gained considerable momentum; , however, a decade of research efforts in this area has not yet led to a clinical diagnostic due to the complexity and lack of reproducibility of these approaches. Nonetheless, pursuing a blood‐detection approach might have great diagnostic value. A recent study combining immunoprecipitation and mass spectrometry led to the identification of high‐performance blood‐borne Aβs derived from human MCI and AD. Similarly, using our unique camelid single‐domain anti‐Aβ1‐40 or anti‐Aβ1‐42 oligomer antibody, we were able to detect both Aβ1‐40 and Aβ1‐42 oligomer in whole blood derived from 3‐ to 18‐month‐old APP/PS1 mice via immunoprecipitation followed by western blotting. Aβ1‐40 and Aβ1‐42 oligomer levels were significantly higher compared to WT mice in the 3‐month‐old age group, while their levels were elevated for Aβ1‐40 oligomers and reduced for Aβ1‐42 oligomers in the 18‐month‐old age group compared to the 3‐month‐old age group. The reduction of Aβ1‐42 in the older age group mirrors the biological behavior of this assembly in human AD in which plasma Aβ1‐42 or total Aβ1‐42/ Aβ1‐40 ratio is used as a strong predictor of amyloid‐PET status. , Although the levels of blood‐borne Aβ levels were significantly higher in APP/PS1 compared to the levels in the WT littermates, the western blot technique has its limitations and might generally lead to false positives.

We have previously shown a strong inverse correlation between retinal Aβo and brain Aβp deposition. This previous study provided the rationale for assessing and comparing age‐dependent accumulation of Aβo in the retina, whole blood, and brain. In this current study, the 3‐ to 4‐month‐old APP/PS1 age group displayed extensive accumulation of Aβo deposits in the ONL, INL, and GCL of the retina, whereas the brain remained free of Aβo deposition. In addition, Aβ plaques were completely absent in both brain and retina in this age group. Retinal Aβo deposition was lower with age, and was no longer detected in the 17‐ to 18‐month‐old age group using IHC. In contrast, cerebral Aβo was first detected at 8 months of age in our APP/PS1 mice and remained unchanged in 11‐month‐old mice, but was undetectable in 18‐month‐old APP/PS1 mice. Consistent with our previous study, retinal Aβp was first detected in 8‐ to 11‐month‐old APP/PS1 and increased in the 18‐month age group. The lack of detection of Aβo in older animals supports the hypothesis of Aβo conversion to plaques as the disease progresses. This is highly speculative as this “conversion” from oligomers to plaques has not been demonstrated at a molecular level; however, in our study, the mere fact that Aβo are present in the retina and not in the brain provides momentum to pursue this diagnostic strategy in vivo. Taken together, and acknowledging the limitations of the study in relation to the lack of data related to animal behavior in our current study, these results suggest that retinal Aβo accumulation precedes its cerebral deposition and that its simultaneous presence in the blood at high levels strongly suggests that retinal Aβo originate from the blood, , albeit a lymphatic and/or a CSF origin cannot be ruled out. Of note, fluorescence assessment also showed presence of cerebral Aβo depositions forming the dense core of the Aβp plaques in the 18‐month age group. This data strengthens the hypothesis of Haass et al. suggesting that Aβ plaques might act as a reservoir for Aβ oligomers.

In this study, we established that Aβo could be detected simultaneously in the blood and retina of APP/PS1 mice before their appearance in the brain. Aβo neuroinvasion appears to originate from blood before reaching the retina probably via “leaky” blood–ocular barriers. A study by Morin et al. reported that APP is synthesized in retinal ganglion cells and transported to the optic nerve in small transport vesicles. It can be speculated that blood‐borne Aβo deposition in the retina might initiate a seeding reaction leading to aggregation and spread to the brain. The ability to detect Aβo concurrently in the blood and retina using nanobodies that specifically bind Aβ1‐40 and Aβ1‐42 oligomers before cognitive decline and neuropathology are evident offers a real possibility to establish a screening platform (retinal imaging of Aβo) and a reference diagnostic testing platform (blood testing of Aβo).

CONFLICTS OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial and non‐financial competing interests that could be construed as a potential conflict of interest.

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