**4. The degenerating retina: sight loss in old age**

situated on the roof of the midbrain and is involved in controlling eye movement; the supra‐ chiasmatic nucleus (SCN) which is a small, wing-shaped structure within the hypothalamus located directly above the optic chiasm that is involved in circadian rhythm; and nuclei within the midbrain that are implicated in controlling pupil diameter (**Figure 2**). Of note, several of these aforementioned visual centres have been associated with AD pathology. For instance, the SC has been shown to progressively accumulate both Alzheimer-associated senile plaques as well as neurofibrillary tangles (NFT). AD-linked changes have also been reported in the SCN including a reduction in the size, cell number and accumulation of NFTs, which may collectively contribute to the wide range of visual complications reported in these patients [15,

**3. Visual abnormalities in Alzheimer's disease and in patients with**

abnormalities in patients with AD and dementia [29].

A variety of visual abnormalities have been reported in patients with AD ranging from visuoconstructional and visuo-perceptual dysfunctions, object agnosia, prosopagnosia to visual hallucinations as well as simultanagnosia [5, 18, 19]. Patients with posterior cortical atrophy (PCA), which is associated with degeneration of the posterior cortex, also report particular difficulties with visual tasks often presenting with visual agnosia, visual neglect and visual hallucinations [20, 21]. However, compared to memory loss, visual deficits have received little attention and are thus poorly understood. Patients in the spectrum of, and leading to, clinical AD show marked reductions in the number of RGC [22], narrowing of venous blood column diameter and reduced venous blood flow rates [23], as well as optic nerve abnormalities such as loss of axonal densities [24], RGCs [25] and increased receptor expression for advances glycation end products [26]. Abnormalities are also observed in regions of AD brains that synapse with the optic nerve and/or are associated with the visual processing pathway (**Figure 2**). Some examples include the loss of myelin, diminished nucleolar volume in the LGN, as well as accumulation of lipofuscin (autofluorescent pigment granules) [27]. AD brains also show the presence of NFT in the SC [28] (which receives ~10% of the RGC axons), as well as the presence of Aβ/neuritic plaques in regions of the brain that are implicated in visual attention and the control of eye movement. Aβ deposition has also been reported in the lens of AD patients [29]. Additionally, histopathological evidence from post-mortem brains reveals significant pathological changes in the visual processing regions of the brain including the loss of pyramidal cells and reduced myelin in the outer laminae of the visual cortex [30–33]. A comprehensive list of such neurological changes in areas of the brain associated with vision has been described by Armstrong (2009) indicating the extensive nature of psyco-visual

Not surprisingly, a large number of AD transgenic animal models also develop visual deficits and have therefore been used to investigate retinal pathologies. These include, but are not limited to, the Tg2576 [34], APPswe/PS1M146L and APPswe/PSΔE9 [35], 5xFAD [36, 37], and P301S [38] mice. A wide range of retinal changes have been documented in these animals,

17].

**dementia**

90 Update on Dementia

Despite the growing number of Alzheimer's patients reporting visual complications, this has received comparatively little attention. This may be due to several reasons including incom‐ plete diagnosis, associated complications, old age and cognitive impairment of patients as well as lack of medical devices or tools to obtain a clear clinical diagnosis. Consequently, the breadth and diversity of visual abnormalities in AD and dementia patients is yet to be fully recognised. For example, analysis of published literature in NCBI PubMed using keywords such as 'vision and dementia' (without the inclusion of any further search parameters) yielded only 447 citations in a 10-year period (between 2006 and June 2016), which is surprisingly few given the frequency of visual abnormalities reported in these patients. In contrast, search terms such as 'memory loss and cognitive decline' yield 3062 citations over a similar period. Understanding how the ageing retina becomes susceptible to degeneration and how it may affect the visual pathway and/or perception by the brain could provide insights into the molecular and cellular basis underlying visual abnormalities in AD. Here then is an opportunity to gain further insights into how the world may be perceived by those suffering from AD and dementia. Patients with retinopathies not only have damaged retinal tissues but also show impairments in how visual information is relayed to and processed by the brain. For instance, visual hallucinations classified under the term Charles Bonnet syndrome have been reported in patients with late-onset visual disorders such as AMD [41]. Indeed, almost half of AMD patients experience visual hallucinations, whilst a third report hallucinations that are distress‐ ing, intrusive and interfere with daily activities [41, 42]. AMD affects approximately 50 million individuals globally [8, 43]. Unlike rare diseases caused by single-gene mutations, AMD is a complex multifactorial disease which in many ways shows striking parallels with AD [4, 44, 45].

AMD exhibits an age-dependent prevalence with one in three individuals exhibiting some sign of early disease by their seventh decade [43]. Currently, there are over half million AMD patients in the United Kingdom (source: Macular Society), with comparable incidence rates in Europe and other Western populations as reported by the European Eye Study (EUREYE) [46]. This puts a significant strain on national healthcare budgets with the direct annual cost of AMD exceeding US\$254 billion globally [14]. This figure is predicted to rise three-fold over the next 20 years as a result of increased life expectancy and reduced mortality rates [43]. This common, irreversible blinding condition derives its name from the macula; the anatomical region affected in disease. This specialised region, which we have introduced earlier, resides at the centre of the retina, temporal to the optic disk and is responsible for visual acuity and image resolution (mediating focused central vision). Patients with AMD therefore suffer loss of centrally mediated sight [45]. As the majority of patients are typically in the latter stages of life, this has a disproportionate social impact, similar to some social issues encountered by patients with AD and dementia [43].

The early stage of AMD is typically asymptomatic, and like AD, can remain so for many years before clinical diagnosis. Hence, most patients with early AMD exhibit few or no obvious visual symptoms [47], although a recent study found indications of early macular pathology even in those aged between 35 and 44 [48]. A major pathological hallmark of early AMD is the focal deposition of lipid-rich extracellular aggregates between the RPE and Bruch's membrane (**Figure 1**) [49]. With increasing age, such aggregates termed 'drusen' become common within the periphery of normal healthy retinas as hard structures with well-defined borders [50]. In contrast, patients with larger, soft drusen showing ill-defined borders (~125 μM) in the macula region are considered to be at a higher risk of developing AMD [51]. Late AMD presents as two distinct phenotypes; classified as geographic atrophy (dry) and neovascular (wet) AMD. If early stages of the disease are excluded, the numbers of dry and wet AMD patients are broadly similar [52]. Dry AMD is typified by gradual impairment of macular RPE cells and death of overlying photoreceptors. By contrast, wet AMD is characterised by growth of new leaky blood vessels from the underlying choroid (**Figure 1**). This results in accumulation of fluid/sub-retinal swelling and scaring of the macula due to disruption of the outer BRB [45]. The growth of new vessels in wet AMD may be managed in most cases through monthly intravitreal injections of vascular endothelial growth factor (VEGF) inhibitors. In contrast, dry AMD which affects the majority of AMD patients currently has no effective treatment [8]. Significant advances have been made in recent years to identify the genetic landscape of AMD and related retinopathies [45]. However, this new knowledge has yet to provide insights into key disease mechanisms, or translate into effective treatments against advancing blindness. It is therefore vital to gain a better understanding of disease processes in the ageing retina before effective AMD treatments can be developed. The recent discovery of the Alzheimer'sassociated Aβ peptide, a well-known neurodegenerative agent associated in key stages of AMD, has opened up the possibilities of studying sight loss from a novel perspective. Such studies in a highly accessible tissue such as the retina could lead to a better understanding of Aβ mechanisms as well as new insights into AD and dementia.

#### **5. Age-related macular degeneration and Aβ**

The healthy retina is constitutively exposed to Aβ. In fact, recent findings demonstrate that Aβ synthesis occurs at local sites within the retinal environment including the RPE and RGCs [53, 54]. The RPE is considered to be the principal source of Aβ in the posterior eye; a tissue which also expresses APP [55]. The RPE also expresses the necessary factors for regulating Aβ synthesis including β- and γ-secretase, as well as the Aβ-degrading enzyme neprilysin [56, 57]. Furthermore, studies of mouse and bovine ocular fluids show the presence of Aβ in picomolar to nanomolar quantities within both aqueous and vitreous humours [58].

centre of the retina, temporal to the optic disk and is responsible for visual acuity and image resolution (mediating focused central vision). Patients with AMD therefore suffer loss of centrally mediated sight [45]. As the majority of patients are typically in the latter stages of life, this has a disproportionate social impact, similar to some social issues encountered by

The early stage of AMD is typically asymptomatic, and like AD, can remain so for many years before clinical diagnosis. Hence, most patients with early AMD exhibit few or no obvious visual symptoms [47], although a recent study found indications of early macular pathology even in those aged between 35 and 44 [48]. A major pathological hallmark of early AMD is the focal deposition of lipid-rich extracellular aggregates between the RPE and Bruch's membrane (**Figure 1**) [49]. With increasing age, such aggregates termed 'drusen' become common within the periphery of normal healthy retinas as hard structures with well-defined borders [50]. In contrast, patients with larger, soft drusen showing ill-defined borders (~125 μM) in the macula region are considered to be at a higher risk of developing AMD [51]. Late AMD presents as two distinct phenotypes; classified as geographic atrophy (dry) and neovascular (wet) AMD. If early stages of the disease are excluded, the numbers of dry and wet AMD patients are broadly similar [52]. Dry AMD is typified by gradual impairment of macular RPE cells and death of overlying photoreceptors. By contrast, wet AMD is characterised by growth of new leaky blood vessels from the underlying choroid (**Figure 1**). This results in accumulation of fluid/sub-retinal swelling and scaring of the macula due to disruption of the outer BRB [45]. The growth of new vessels in wet AMD may be managed in most cases through monthly intravitreal injections of vascular endothelial growth factor (VEGF) inhibitors. In contrast, dry AMD which affects the majority of AMD patients currently has no effective treatment [8]. Significant advances have been made in recent years to identify the genetic landscape of AMD and related retinopathies [45]. However, this new knowledge has yet to provide insights into key disease mechanisms, or translate into effective treatments against advancing blindness. It is therefore vital to gain a better understanding of disease processes in the ageing retina before effective AMD treatments can be developed. The recent discovery of the Alzheimer'sassociated Aβ peptide, a well-known neurodegenerative agent associated in key stages of AMD, has opened up the possibilities of studying sight loss from a novel perspective. Such studies in a highly accessible tissue such as the retina could lead to a better understanding of

patients with AD and dementia [43].

92 Update on Dementia

Aβ mechanisms as well as new insights into AD and dementia.

The healthy retina is constitutively exposed to Aβ. In fact, recent findings demonstrate that Aβ synthesis occurs at local sites within the retinal environment including the RPE and RGCs [53, 54]. The RPE is considered to be the principal source of Aβ in the posterior eye; a tissue which also expresses APP [55]. The RPE also expresses the necessary factors for regulating Aβ synthesis including β- and γ-secretase, as well as the Aβ-degrading enzyme neprilysin [56,

**5. Age-related macular degeneration and Aβ**

The retina is a particularly useful tissue to study Aβ pathology as it is continuously exposed to high photo-oxidative stresses throughout life, an ideal environment for Aβ accumulation [44, 59, 60]. Hence, it is not surprising that the Aβ burden in the retina increases with advancing age. To date, age-dependent accumulation of Aβ has been shown in multiple retinal locations including photoreceptors, RPE, Bruch's membrane and within the inner and outer retinal vasculature [39, 55, 61, 62]. This pattern of Aβ accumulation has been reported both in rodent models and in donor human eyes. For instance, Aβ deposits on photoreceptor were shown to be abundant on mature POS or outer tips which are phagocytosed by RPE cells as part of the daily visual cycle. Studies in wild-type mice show such Aβ-enriched outer tips of photorecep‐ tors to be enlarged, possibly due to impaired internalisation of POS by senescent RPE [39]. The use of antibodies that recognise Aβ as well as APP also show immunoreactivity within the cytoplasm of RPE cells that are adjacent to drusen [55]. Numerous studies also reveal the presence of Aβ within drusen, which links a key clinical hallmark of AMD with Aβ [44, 55, 61–63]. Aβ within drusen have been shown organised into assemblies of approximately 2–10 μm in diameter. These spheres referred to as 'amyloid vesicles' were shown to have a concen‐ tric ring-like interior, permeated with Aβ immunoreactivity [55].

Interestingly, studies of post-mortem tissues show that the ageing human retina plays host to a variety of Aβ assemblies. The use of various antibodies including 4G8, 6E10, WO1, WO2, OC, A11 and 82E1 has revealed the presence of non-fibrillar oligomers, protofibrils and mature amyloid fibrils [55, 61, 62]. Furthermore, different Aβ structures were evident in different locations within amyloid vesicles. For example, in studies using 4G8, 6E10, WO1 and WO2 (which specifically recognise mature Aβ assemblies including protofibrils and mature fibrils), immunoreactivity was typically observed within the outer shell of amyloid vesicles [55, 61, 63]. Conversely, studies investigating Aβ oligomers (antibodies A11 and M204) showed preferential accumulation at the centre of drusen in close proximity to the inner collagenous layer of Bruch's membrane. Here, Aβ oligomers constituted the most abundant Aβ assembly within drusen [39, 62]. Moreover, the presence of Aβ within drusen appeared to correlate with drusen load as well as increasing age [63]. One study using a small number of patient samples found that Aβ deposition were only present within drusen of AMD patients; supporting the likelihood that Aβ accumulation is associated with more advanced forms of AMD [64].

As the RPE monolayer, which is strategically juxtaposed between the neuroretina and the outer retinal vasculature (**Figure 1**), appears to be the main focus of Aβ deposition, it is not surprising that Aβ has profound effects on its function. Of critical importance is the role of the RPE in maintaining the immune-privileged state of the retina via the outer BRB. Oligomeric Aβ1–42 has been shown to impair both early zonular occludens (ZO-1) and mid-to-late occludin tight junctions in the RPE as well as induce actin cytoskeletal disorganisation. This suggests that Aβ may compromise BRB integrity [65]. This is comparable to Aβ's mode of action in the AD brain which results in blood-brain barrier (BBB) disruption, increased BBB permeability and endothelial cell dysfunction [66]. In fact recent studies have also shown a downregulation of both ZO-1 and occludin upon application of Aβ to human cerebral microvascular endothelial cells. Such insights reveal striking parallels between neurodegenerative processes between the ageing retina and brain, and how Aβ may play a key role in both pathologies [7, 66]. Similarly, oligomeric Aβ exposure causes an upregulation of VEGF in both the brain and retina which has been linked with AD and AMD. VEGF is essential in maintaining hippocampal plasticity as well as cognitive function. However, VEGF upregulation is correlated with Aβ1–42 accumu‐ lation in AD brains resulting in neuronal cell death and BBB dysfunction [67]. In the eye, VEGF is primarily secreted by the RPE; the increased levels of which are correlated with the neovas‐ cular form of AMD [8]. Anti-VEGF inhibitors consequently form the current the mainstay of wet AMD treatments. Exposure of RPE cells to Aβ was shown to profoundly increase VEGF secretion, which may contribute to such an undesirable pro-angiogenic retinal environment [57].

Aβ also appears to play a central role in chronic inflammation of the ageing retina. Such pathology is similar to inflammatory conditions found in AD brains [68]. For example, transcriptome studies show enhanced complement gene expression in AD brains, particularly those of complement C1q and C3 proteins [69, 70]. AMD involves a similar chronic inflam‐ matory response that is as yet incompletely understood. Here, compliment associated proteins deposit within drusen alongside Aβ including compliment factor C3, compliment factor H and the membrane attack complex, including its constituents C5, C6, C7 and C9 [55, 71]. Conse‐ quently, Aβ is thought to promote a pro-inflammatory retinal microenvironment where it colocalises with complement factor H (CFH) and iC3b to induce compliment activation. Studies have also shown the elevation of pro-inflammatory IL-1β, IL-6, IL-8, TNF-α and caspase-1 upon intravitreal Aβ injection in C57BL/6 J mice, as well as an increase in IL-8 and MMP-9 secretion levels by RPE upon exposure to Aβ1–42 [72, 73]. Microglial activation and engulfment of Aβ have also been observed co-localised with retinal Aβ [39]. Similar pathology is also reported in the brains of patients with AD [68].

Unsurprisingly, key features of AMD observed in human donor eyes can be recapitulated by experimentally elevating retinal Aβ levels in wild-type mice. Our studies show that subretinal injection of human recombinant Aβ1–42 at physiological doses (nM range) in C57BL/6 mice induces RPE pigment abnormalities, RPE plasticity as well as photoreceptor outer segment loss, hallmarks of AMD (**Figure 3**). Critically, using the 82E1 antibody specific to human Aβ, we found experimentally introduced Aβ to co-localise to multiple retinal locations corresponding to points of Aβ immunoreactivity reported in eyes of both AD [74, 75] and AMD patients/mouse models [55, 61–63]. Hence, Aβ was shown to localise to RGC, the outer nuclear layer, photoreceptors as well as the RPE-Bruch's membrane interface [39]. Attempts by others to elevate Aβ1–42 levels in the rodent vitreous resulted in apoptotic cells in photoreceptor and nuclear layers as well as a significant reduction in RGC [76, 77]. However, our method of elevating the retinal Aβ load via subretinal injection appears to mimic the senescent eye more accurately (**Figure 3**), as the resulting phenotype certainly bears closer resemblance to human AMD [55, 63, 64]. Additional evidence for ocular Aβ pathology comes from studies implicating Aβ in other eye diseases such as supranuclear cataracts and glaucoma of which the latter is common amongst AD patients [4].
