The concept 'the retina as an additional tool for AD diagnosis' has been increasingly explored, since some AD patients present visual alterations before the appearance of the first cognitive symptoms. Moreover, the retina and the brain share the same embryonic origin.
We recently reported the existence of structural and functional changes in the brain and retina of 3xTg-AD mice (15, 16). These alterations occur as early as 4 months of age. Because of the detection of these early changes in this animal model of AD, in the present work we investigated the presence of potential early molecular and cellular alterations in the retina and brain that may underlie the structural and functional changes detected in this model. Moreover, we intended to check whether the potential changes in the retina and brain were similar, as well as to check whether some changes could eventually occur in the retina before they appear in the brain.
Increased Aβ levels were detected in the hippocampus and cortex of 3xTg-AD mice at 4 and 8 months, despite no changes detected in APP and BACE at both time points, with the exception of increased levels of APP in the hippocampus at 8 months. These results suggest that the increased levels of Aβ detected in the brain may result from an increase of BACE activity or an impairment of Aβ removal. Several evidences point out mechanisms of Aβ clearance as a potential therapeutic strategy to modulate AD (29–31). In the retina, no changes were detected in Aβ levels. Actually, the presence of Aβ in the retina of animal models of AD is still a matter of controversy (reviewed in (3). In fact, we tried different approaches and techniques to detect Aβ in the retina and we were only successful by using a specific protocol of homogenization abovementioned. It was recently reported Aβ staining in retinal slices of 3xTg-AD mice, at 5 post-natal weeks (32). We also found a similar result, however, when we incubated both retinal slices and whole mounts only with the secondary antibody, we observed the same staining pattern. Thus, the Aβ staining previously reported may actually result from precipitation of the secondary antibody or unspecific binding. In fact, 3xTg-AD mice are known to be one of the animal models of AD with lower Aβ expression in the retina (33).
We also checked the hyperphosphorylated tau protein in the hippocampus, cortex and retina of 3xTg-AD mice. For that, we used an antibody that recognizes tau protein phosphorylated at serine 396, a phosphorylation site associated with early AD (34). We detected increased p-tau levels in the hippocampus and retina of 3xTg-AD mice, at both time points, while in the cortex this increase was only observed at 4 months. Similarly, others reported increased p-tau in the retinas of 3xTg-AD mice already at pre-symptomatic stages (32). However, it is important to highlight that these authors used a different antibody that recognizes the protein tau phosphorylated at serine 202 and threonine 205, which is more associated with the later stages of AD.
Regarding barriers integrity, our results indicate that claudin-5, occludin and ZO-1 levels are not affected in the retina and brain of 3xTg-AD mice at early stages (4 and 8 months of age). Additionally, we did not detect albumin extravasation from the blood to brain or retinal parenchyma. In summary, our results show that at early time points the BRB and BBB are not compromised in 3xTg-AD mice. From the best of our knowledge, this is a pioneering study assessing simultaneously the integrity of BBB and BRB in an AD animal model. Previous studies were mainly focused in BBB, and still no consistent results were reported (35–38). In the retina, attenuated and disorganized ZO-1 and occludin staining was reported in 5xFAD mice, an AD animal model with a more aggressive phenotype, at 8 months (39).
We also assessed cell loss, and synaptic and neurotransmitters changes. Cholinergic neurons are the most affected in AD, and their degeneration contributes for the memory loss (24). We did not observe changes in the protein levels of ChAT in any region analyzed of 3xTg-AD mice at 4 and 8 months, suggesting that cholinergic neurons and the synthesis of acetylcholine are not affected in this animal model at these early stages. In 3xTg-AD mice, others demonstrated a decrease in the number of ChAT-positive cells in the basal forebrain already at 4 months (40). These apparent contradictory results could be due to the assessment of distinct regions: basal forebrain versus hippocampus and cortex. Additionally, others also reported a decrease in the number of ChAT+ cells in the retina of APP/ PS1 mice, associated with an increase of cell death by apoptosis, but this was observed at 13–16 months (41). In fact, we cannot discard the possibility that the 3xTg-AD mice can also present changes in the number of ChAT+ cells at an older age.
We assessed cell death in the hippocampus, cortex and retina to clarify whether cell death could occurs simultaneously in the retina and brain of 3xTg-AD mice at the early stages. We did not detect any evidence of cell death by apoptosis in all regions analyzed. Formerly, it was described an increase in the number of cells stained for cleaved caspase-3 both in the retina and hippocampus of 3xTg-AD mice at 5 weeks of age that disappear at 30–40 weeks (7–10 months) (32). However, caspase-3 has been also implicated in the regulation of synaptic plasticity (42, 43) and in mitochondria function (44). Grimaldi and colleagues claimed that increased staining for caspase-3 indicates neuronal apoptosis (32), but they did not show any evidence of cell death in the retina and brain of 3xTg-AD mice.
Loss of RGCs in AD patients has also been described (reviewed in (3). We did not find a significant decrease in the number of RGCs, at 4 and 8 months. Additionally, we did not find changes in the function of RGCs in 3xTg-AD mice (16), therefore suggesting that RGCs are not affected in this animal model at the early stages of pathology.
Regarding synaptic changes, our results suggest that the retina and brain are not similarly affected, at 4 months, in this animal model. At 8 months, we did not detect any changes in the levels of the synaptic proteins evaluated in the three regions analyzed. Although others detected decreased synaptophysin and PSD95 levels in the hippocampus and cortex of 3xTg-AD mice at 4 and 7 months (45), most studies did not observe changes in synaptic proteins at 6, 8 and 9 months in this AD animal model (46–48). At 4 months we detected an increase in the content of several synaptic proteins, which has been corroborated by other studies, at early stages (57). This increase might be due to a compensatory mechanism. Also, in postmortem brains from AD patients it was described an increase in several synaptic proteins at early stages of the pathology (Braak 3 and 4) that did not persist at later stages (Braak 5 and 6) (49). These studies corroborate our findings, suggesting that a biphasic synaptic protein response during AD progression is likely to occur. These evidences lead us to hypothesize that compensatory mechanisms (increase of synaptic proteins) might occur to keep homeostasis and cell survival. However, with the disease progression the synaptic deficit reaches a certain threshold where the brain cannot compensate anymore.
Several reports have demonstrated that AD patients present a disturbance in glutamate and GABA levels, leading to a neuronal circuitry disruption, and consequently affecting memory (50–55). Changes in neurotransmission occur prior to cognitive decline (25). We detected a decrease of GABA in the cortex of 3xTg-AD mice at 4 months, but no more significant changes were observed in the GABA and glutamate levels in all regions analyzed. Others reported a decrease in vesicular glutamate transporter type I (vGluT1) density, without neuronal damage, in the hippocampus of 3xTg-AD mice, at 4 months (56). At the early stages the total glutamate levels do not change, but with the alterations in VGluT1 the glutamate transport could be compromised and therefore the glutamate levels at the synaptic cleft.
Evidences point out the glial cells as key elements in the AD pathophysiology (reviewed in (57). Regarding GFAP, our results suggest the occurrence of differential glial fluctuations over time in the areas analyzed. It was recently reported an increase of astrocytic reactivity in the retina of 3xTg-AD mice at 5–10 and 30–40 weeks (32), which is not in agreement with our findings. However, others also reported inconsistent glial changes in the brain and retina in APP/PS1 mice (58). Moreover, we did not find significant changes in vimentin, suggesting no significant changes in Müller cells, at the early stages. Others also reported no changes in Müller cells in 3xTg-AD (59) and in APP/PS1 mice (60), at early stages. Conversely, evidence of an increase of GFAP staining in the Müller cells end-feet in the retina of 3xTg-AD mice at 9 months of age (32) suggests an increase of Müller cells reactivity. Taking into account our results and previous findings, it appears that Müller cells reactivity may occur in AD, but at later stages.
During an insult microglial cells increase their proliferative rate, change their morphology, and acquire a reactive state in order to reestablish the homeostasis (61). No changes were detected in the number of microglial cells neither in the hippocampus nor in the retina of 3xTg-AD mice at early stages. Former studies also reported no changes in the number of microglial cells in the retina (32) and brain of 3xTg-AD mice, at early stages (62). We also assessed microglial morphology, i.e., the number and length of processes. In the hippocampus of 3xTg-AD mice we observed a hypertrophy of microglial cells (only at 4 months), while in the retina microglial cells display an atrophic morphology. Moreover, at 8 months, retinal microglial cells featured a biphasic morphological structure since presented atrophy in the last orders and a hypertrophy in the initial ones. These results suggest a complex microglial remodeling in AD and also indicate there is a differential microglia remodeling in the retina and hippocampus during AD pathology. To our knowledge, this study is the first one that simultaneously analyzed the microglia structure in the brain and retina in the context of AD. It was reported an increase of microglial cells complexity in the retina of 3xTg-AD mice at early stages (5–10 weeks) (32). This apparent contradictory results could be explained because they used a different approach for 3D microglia reconstruction (imageJ plug-in) and the time point assessed was different (5–10 weeks versus 4 months in our work). We recently reported changes in retinal microglia of 3xTg-AD mice at older ages. Microglia acquired a different morphology and orientation along the retina, and their localization changed from a parallel to a perpendicular position relative to the retinal surface (63). In the brain of AD patients, it was found a reduction in the length of processes and arborized area of microglial cells, indicative of microglia reactivity (64). This group of findings led us to hypothesize that in the early stages microglial cells adopt a hyper-ramified morphology trying to maintain the appropriate brain surveillance. However, with disease progression, microglia acquire a less complex morphology accompanied with a senescent state.