Amyloid-beta and phosphorylated tau in post-mortem Alzheimer's disease retinas

Abstract

In-vivo labeling of retinal amyloid-beta(Aβ) and tau has potential as non-invasive biomarker for Alzheimer's disease (AD). However, literature on the presence of Aβ and phosphorylated tau (pTau) in AD retinas is inconclusive. We therefore assessed the presence of Aβ and pTau in post-mortem retinas in 6 AD and 6 control cases who donated brains and eyes to the Netherlands Brain Bank. Neuropathological diagnosis of AD was made according to NIA-AA criteria. Formalin fixed retinas were dissected in quadrants and cross-sections of medial and superior retinas were made. Immuno-histochemical stainings were performed for Aβ, amyloid precursor protein (APP) and pTau. To assess translation to an in-vivo set up using curcumin as labelling fluorophore, co-stainings with curcumin were performed. No typical Aβ-plaques and neurofibrillary tangles, like in the cerebral cortex, were observed in AD retinas. A diffuse immunoreactive signal for pTau was increased in the inner and outer plexiform layers of the retina in AD cases compared to control cases with absence of cerebral amyloid pathology. Immunostaining with anti-Aβ and anti-APP antibodies yielded signal in ganglion cells, amacrine cells, horizontal cells and Müller cells in both control and AD cases. We observed small extracellular deposits positive for anti-Aβ antibodies 12F4 and 6E10 and negative for 4G8 and curcumin. A subset of these deposits could be characterized as corpora amylacea. In conclusion we found that retinal manifestations of AD pathology appear to be different compared to cerebral AD pathology. Using a qualitative cross-sectional approach, we did not find Aβ/APP related differences in the retina between AD and control subjects. In contrast, tau related changes were found to be present in cases with cerebral AD pathology, suggesting retinal tau as a potential biomarker for AD.

Keywords: Alzheimer’s disease; Amyloid; Neurodegeneration; Post-mortem; Retina; Tau.

Fig. 1

Processing of post-mortem eyes. Anterior parts of eyes were removed (a). Formalin fixed eyes were dissected through the horizontal (b) and vertical meridian (c). Superior (red, arrows) and nasal (green, arrows) parts were cut in 10 μm sections from anterior to posterior. As a result, sections contained all retinal layers from ora serrata to the posterior pole: retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptors (PR), retinal pigment epithelium (RPE), choroid and sclera

Fig. 2

Overview of different structures positive for different anti-Aβ antibodies. Overview of structures that were stained with anti-Aβ antibodies 6E10,12F4 and 4G8. Distribution of these structures over retinal layers is shown on the left (a-h). Abbreviations: RNFL = retinal nerve fiber layer, GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, PR = photo receptors, RPE = retinal pigment epithelium, LPR = liquid permanent red. Scale bars 50 μm

Fig. 3

APP/Aβ in the medial and superior retina. a Representative stainings for APP/Aβ antibodies and amyloid (Thioflavin-S) in AD hippocampus and AD and control retinas showing different staining patterns for different antibodies (superior and medial). b Kluver-PAS staining of corpora amylacea in hippocampus and retina compared to 6E10 and 12F4 stainings. c IHC co-stainings with PAS and 6E10. d Curcumin and 6E10 co-staining in AD hippocampus and in retinas showing a positive ganglion cell and extracellular deposit. All size bars 50 μm

Fig. 4

(Phosphorylated) tau in AD retinas. Representative stainings of different tau antibodies in AD hippocampus and AD and control retinas (superior and medial). Antibodies are associated with total tau (HT-7), phosphorylated tau (AT-270, AT-8, AT-100), tau fibrils (anti-pS422) and paired helical filaments (MC-1). Size bars: 50 μm. Hipp. = hippocampus

Fig. 5

Phosphorylated tau in AD and control retinas. a Representative pictures of phosphorylated tau (AT8) stainings of the anterior part of superior retinas for each subject. Case numbers are indicated on the left and Braak Tau and Braak Amyloid stage are indicated on the right. b Positive gradient of phosphorylated tau staining towards the periphery in a representative AD case (#4)