Alzheimer’s Disease Hallmarks in the Retina of Animal Models
In agreement with the above findings in patients, the pathological hallmarks of AD were also described in numerous animal models of AD (see a summary in Table 1). Both the soluble and insoluble forms of Aβ were found in the retina of sporadic models and transgenic mice harboring familial AD (FAD) mutations (Ning et al., 2008; Dutescu et al., 2009; Liu et al., 2009; Perez et al., 2009; Koronyo-Hamaoui et al., 2011; Koronyo et al., 2012; Tsai et al., 2014; Du et al., 2015; Gupta et al., 2016; Hart et al., 2016; Habiba et al., 2020). Intriguingly, early manifestations of retinal Aβ plaques have also been detected prior to their occurrence in the brain (Koronyo-Hamaoui et al., 2011). Moreover, upon assessment of therapeutic response, researchers found that positive effects of immunotherapy on cerebral Aβ-plaque reduction were also reflected in the respective retinas in transgenic animal models of AD (ADtg) (Liu et al., 2009; Koronyo-Hamaoui et al., 2011; Koronyo et al., 2012; Yang et al., 2013; He et al., 2014; Gao et al., 2015; Parthasarathy et al., 2015). These studies illustrate the common retino-cerebral mechanisms of neuroprotection in response to therapy, which encourages the use of retinal imaging to noninvasively assess therapeutic efficacy in real time.
TABLE 1  Alzheimer’s pathological hallmarks in retinas of animal models.
Hallmarks  Animal species  Models and Genotypes  Retinal pathologies  References
APP or Aβ related  Drosophila  AβPP, dBACE-AβPPL, pGMR-Aβ42  Increased APP, Aβ, Aβ42 toxicity  Finelli et al., 2004; Greeve et al., 2004; Carmine-Simmen et al., 2009; Cutler et al., 2015
Mouse  Tg2576, hTgAPPtg/tg, APPSWE/PS1ΔE9, APPSWE/PS1M146L/L286V, 3xTg, 5xFAD, Tg-SwDI  Increased APP, Aβ deposits, Aβ42, Aβ40, vascular Aβ, Aβ oligomers  Ning et al., 2008; Dutescu et al., 2009; Liu et al., 2009; Perez et al., 2009; Alexandrov et al., 2011; Koronyo-Hamaoui et al., 2011; Koronyo et al., 2012; Williams et al., 2013; Yang et al., 2013; Zhao et al., 2013; Edwards et al., 2014; He et al., 2014; Park et al., 2014; Gao et al., 2015; More and Vince, 2015; Parthasarathy et al., 2015; Pogue et al., 2015; Gupta et al., 2016; Oliveira-Souza et al., 2017; Criscuolo et al., 2018; Hadoux et al., 2019; Habiba et al., 2020; Sidiqi et al., 2020
O. degus  Spontaneous  Increased APP, Aβ, Aβ oligomers  Inestrosa et al., 2005; Ardiles et al., 2012; Du et al., 2015
Rat  TgF344-AD  Increased Aβ  Tsai et al., 2014
NFT or pTau related  Drosophila  hTau  Accumulation of pTau  Grammenoudi et al., 2006; Chouhan et al., 2016
Mouse  Tg2576, APPSWE/PS1 ΔE9, APPSWE/PS1M146L/L286V, 3xTg, P301S, rTg4510  Accumulation of pTau, NFT  Liu et al., 2009; Schön et al., 2012; Yang et al., 2013; Zhao et al., 2013; Chiasseu et al., 2017; Grimaldi et al., 2018; Harrison et al., 2019
O. degus  Spontaneous  Accumulation of pTau  Du et al., 2015; Chang et al., 2020
APP, amyloid precursor protein; Aβ, amyloid-beta; pTau, phosphorylated Tau; NFT, neurofibrillary tangle; O. degus, Octodon Degus. ADtg animals typically express the transmembrane amyloid precursor protein (APP), the source of Aβ protein, in retinal neurons. Indeed, this protein has been found in the retinas of ADtg drosophila, various ADtg mice (Tg2576, hTgAPPtg/tg, APPSWE/PS1 ΔE9, and APPSWE/PS1M146L/L286V), and the naturally occurring sporadic rodent strain Octodon degus (O. degus) (Ning et al., 2008; Dutescu et al., 2009; Liu et al., 2009; Ardiles et al., 2012; Du et al., 2015). Retinal cytoplasmic APP was found to increase in ADtg models (Ning et al., 2008; Dutescu et al., 2009), but decrease in the sporadic O. degus with aging (Du et al., 2015). Ning and colleagues found APP immunoreactivity increased with age in cells of the INL and GCL – but not the ONL – as well as the neuropil of the IPL and OPL and the outer segments (OS) and RPE (Ning et al., 2008).
ADtg rodent models, including Tg2576, APP/PS1, 3xTg, 5xFAD mice, TgF344-AD rat, and O. degus, show cerebral accumulation of soluble and insoluble Aβ with age, corresponding to AD-like progression (Dutescu et al., 2009; Liu et al., 2009; Perez et al., 2009; Alexandrov et al., 2011; Koronyo-Hamaoui et al., 2011; Williams et al., 2013; Edwards et al., 2014; Park et al., 2014; Tsai et al., 2014; Du et al., 2015; More and Vince, 2015; Parthasarathy et al., 2015; Pogue et al., 2015). Alloforms of Aβ pathognomonic to AD (Aβ40 and particularly Aβ42) were found to be elevated in the retinas of ADtg rodents (Dutescu et al., 2009; Liu et al., 2009; Alexandrov et al., 2011; Williams et al., 2013; Park et al., 2014; Tsai et al., 2014; Du et al., 2015; Parthasarathy et al., 2015) and ADtg drosophila (Greeve et al., 2004; Carmine-Simmen et al., 2009). In addition, plaque and insoluble Aβ deposits were identified in retinas of Tg2576, APPSWE/PS1ΔE9, APPSWE/PS1M146L/L286V, 3xTg, and 5xFAD mice, and in O. degus (Ning et al., 2008; Dutescu et al., 2009; Liu et al., 2009; Perez et al., 2009; Alexandrov et al., 2011; Koronyo-Hamaoui et al., 2011; Koronyo et al., 2012; Williams et al., 2013; Yang et al., 2013; Zhao et al., 2013; Edwards et al., 2014; Du et al., 2015; More and Vince, 2015; Hadoux et al., 2019).
Tg2576 mouse retinas were cross-sectioned and analyzed for plaque pathology, which was found in approximately 85% of these transgenic mice but was absent in WT controls (Liu et al., 2009; Williams et al., 2013). Plaques were mostly found in the GCL, INL, and ONL (Liu et al., 2009; Williams et al., 2013). Yet, one study was unable to detect retinal Aβ pathology in a Tg2576 mouse with cerebral Aβ (Dutescu et al., 2009).
5xFAD mice present an aggressive model of amyloidosis, with several familial AD mutations that result in the overexpression of Aβ42. Studies in this model have demonstrated the presence of Aβ42 in ocular tissues including the retina as well as increases in Aβ40 in the RPE (Park et al., 2014; Parthasarathy et al., 2015; Hadoux et al., 2019). In the sporadic O. degus model of AD, Aβ deposition appears to be progressive, accumulating first in the GCL, NFL, INL and photoreceptors (Chang et al., 2020). Whole retinal histological examination via Aβ-specific staining revealed the most plaque burden in the central retina (Inestrosa et al., 2005; Du et al., 2015).
Importantly, Koronyo-Hamaoui et al. (2011) developed the first approach to visualize retinal Aβ deposits in live APPSWE/PS1ΔE9 mice using curcumin as a specific Aβ-labeling fluorophore and optical Micron rodent retinal imager (Phoenix Technology Group, LLC). The identity of Aβ deposits detected in vivo by curcumin was validated by subsequent ex vivo labeling of the respective whole-mount retinas using anti-Aβ monoclonal antibody. Notably, retinal plaques were observed prior to detection in the corresponding brains (Koronyo-Hamaoui et al., 2011). In addition, following intravenous injection of curcumin, flatmount retinas from 2.5-month-old mice were isolated and stained with 4G8 monoclonal antibody, further confirming that the curcumin spots are the same Aβ-specific deposits (Koronyo-Hamaoui et al., 2011). A recent study by Sidiqi and colleagues corroborated the previous reports (Koronyo-Hamaoui et al., 2011; Koronyo et al., 2012, 2017) and demonstrated via retinal curcumin imaging in APPSWE/PS1ΔE9 mice the accumulation of retinal Aβ plaques with disease progression, which positively correlated with Aβ load in the brain (Sidiqi et al., 2020). Cross-sectional analysis of adult APP/PS1 ADtg mice retinas revealed Aβ deposits in the innermost layers as well as in the choroid and surrounding scleral tissue; WT controls showed only minimal to no plaque deposition (Koronyo-Hamaoui et al., 2011). Studies using the same ADtg model and Tg344F-AD rats (with the same double transgenes) also validated these findings (Ning et al., 2008; Liu et al., 2009; Perez et al., 2009; Tsai et al., 2014).
The initial discovery of tau in the adult mammalian retina dates back to 1988. In this early study, tau was predominantly found in horizontal cells residing in the OPL of adult rat retinas (Tucker and Matus, 1988). On the other hand, disease-associated pTau species were detected somatodendritically in RGCs of a commonly studied double transgenic mouse model of AD, known as the APPSWE/PS1ΔE9 mouse (Yang et al., 2013), and in the GCL to ONL of Tg2576 ADtg mice (Liu et al., 2009). In line with these observations, intracellular aggregates of pTau have also been detected in retinas of the APPSWE/PS1M146L/L286V mouse model of AD (Zhao et al., 2013). Further, recent analyses of young, pre-symptomatic 3xTg mouse retinas showed retinal Aβ plaques and tau tangles in the RGC layer (Grimaldi et al., 2018).
In the South American rodent O. degus, previously reported to develop several spontaneous AD-like pathologies without genetic manipulation, elevated levels of pTau were primarily detected in the GCL to NFL regions of the retina in both adult and aged animals (Du et al., 2015). In a subsequent study by the same group, early punctate AT8 immunoreactivity in the IPL was reported in young degus, compared with the denser expression in IPL to NFL of juvenile and adult animals (Chang et al., 2020). Interestingly, p(tau)-positive aggregates both appeared and propagated to other inner retinal layers earlier than Aβ deposits in these animals (Chang et al., 2020). Similarly, retinal accumulation of total tau and epitope-specific hyperphosphorylation were also reported to precede onset of behavioral deficits and brain tauopathy as early as 3 months of age in the 3xTg mouse model of AD (Chiasseu et al., 2017).
Several live imaging modalities have been developed and optimized for detection of proteinaceous aggregates and metabolic hyperspectral mapping of retinal tissue in rodent models of AD (Koronyo-Hamaoui et al., 2011; Koronyo et al., 2012, 2017; Schön et al., 2012; More and Vince, 2015; Sidiqi et al., 2020). Using longitudinal scanning laser ophthalmology, Schön et al. visualized and monitored pTau-containing RGCs in the P301S mouse model of tauopathy from 2 to 6.5 months of age and found a steady growth in pTau-positive cell counts (Schön et al., 2012).
In the rTg(tauP301L)4510 tauopathy mouse model of frontotemporal dementia (FTD), accumulation of both tau and pTau have been observed in RGCs as well as the IPL and INL (Harrison et al., 2019). These areas are associated with reduced neuronal density and optic nerve degeneration in these mice (Harrison et al., 2019). In an experimental rat model of optic nerve crush, injury-induced impaired autophagy was followed by an increase in hyperphosphorylated tau (pSer396), which co-localized with apoptotic markers in dying RGCs (Oku et al., 2019). Silencing the tau gene exerted neuroprotective effects in this model, indicating that tauopathy following retinal injury similar to that observed in the brain plays an essential role in neuronal atrophy (Oku et al., 2019).
AD-related tauopathies have also been investigated in a limited number of non-murine animal models of aging or AD. Selective expression of normal human tau in adult drosophila retina leads to progressive loss of ERG responses without a considerable effect on retinal structure or neuronal density, although TEM analysis later revealed signs of tau-induced retinal synaptotoxicity and abnormal photoreceptor morphology (Chouhan et al., 2016). In the same study, human Aβ expression in a separate group of flies caused an age-dependent loss of retinal neurons without altering ERG signals (Chouhan et al., 2016). In the UAS-Gal4 drosophila model of AD, species of pTau, phosphorylated at different epitopes to varying degrees and in a cell type-specific manner, have also been detected in both the retina and brain (Grammenoudi et al., 2006). In a small number of young and aged primates, total tau expression in the outer retina was observed in the OPL, ONL and inner segment of photoreceptors, whereas AT8-positive pTau was localized predominantly in the OPL and cones (Aboelnour et al., 2017). In older primates, pTau staining in retinal cones also appeared stronger compared with younger animals (Aboelnour et al., 2017). Altogether, it is apparent that tau expression and pTau accumulation may vary across several species commonly used to model human AD, and future studies should aim to elucidate these differences.
Together with these positive findings, it is important to note that the expression pattern of both APP and tau transgenes in the brain and retina of animal models of AD should not be misinterpreted as a precise reflection of the human disease. The layer and cellular burden of pathological Aβ and pTau aggregates is most likely governed by the promoter-driven expression of their corresponding transgenes. For instance, expression of the aggregate-prone mutant tau species in the P301S tauopathy mouse model is driven by the murine Thy1 promoter, which leads to the development of pathology in selected CNS cell types and consequently specific regions (Allen et al., 2002). In the retina, Thy1 is uniquely expressed by RGCs (Schmid et al., 1995) and therefore the intracellular aggregation of hyperphosphorylated tau in these cells and their nerve fibers is expected. This may underlie the common disparity in findings from stereological studies attempting to characterize regional and cellular susceptibility to AD pathology in CNS tissue from human versus animal models. Recent developments as well as ongoing efforts to fully characterize complete gene replacement animal models will be invaluable in addressing such limitations in several fields.