**6. Similarities between AD and AMD**

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

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

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

[57].

94 Update on Dementia

reported in the brains of patients with AD [68].

common amongst AD patients [4].

Degenerative processes in the ageing retina and brain share many common features. A major pathological hallmark common to both AD and AMD is the formation of insoluble extracellular aggregates that share several histochemical and compositional properties. Proteomic analyses of the molecular components of senile plaques and drusen, for instance, has revealed common proteins including tau, clusterin, vitronectin, apolipoprotein E (ApoE), serum amyloid P (SAP), Aβ, metal ions, as well as pro-inflammatory factors and components of the compliment cascade [4, 44, 78]. Histochemically, such deposits also stain with thioflavin T and Congo red which confirms the presence of misfolded or amyloid proteins. However, there appears to be differences between Aβ structures found in senile plaques and drusen. For instance, whilst

**Figure 3.** Subretinally injected Aβ in our mouse model recapitulates key aspects of retinal degeneration observed in age-related macular degeneration (AMD). Wild-type C57BL/6 mice injected with nM concentrations of recombinant human Aβ1–42 recapitulated key features of AMD. At 8 days post-injection, retinas contained RPE pigment abnormali‐ ties, RPE hypertrophy as well as photoreceptor outer segment loss, in contrast to healthy retinas of vehicle injected mice. Confocal-immunofluorescence using the human Aβ-specific antibody 82E1 revealed focal Aβ deposits (green) in the inner nuclear layer and in the RPE-Bruch's membrane interface (arrows) corresponding to areas of Aβ synthesis/ accumulation reports in aged human retinas. RGC, retinal ganglion cells; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PIS, photoreceptor inner layer; POS, photoreceptor outer segments; RPE, retinal pigment epithelium; BM, Bruch's membrane; CH, choroid. DAPI (blue). Scale bar corresponds to 100 μm.

both types of extracellular protein deposits stain for Congo red, only senile plaques are positive for the apple green birefringence dye specific for anti-parallel β-pleated sheets [49, 63].

Commonalities between AD and AMD are also observed in the manner in which highly localised and significant damage occurs to lysosomes and mitochondria. In AD brains, these include substantial increases in the size/number of endosomes, autophagosomes and lyso‐ somes; accumulation of lysosomal dense bodies in dystrophic neurites, as well as changes in expression of lysosomal hydrolases such as cathepsins [2, 79]. Our studies as well as those of others have shown selective permeabilisation of lysosomal membranes and release of lytic content into the cytosol as a precursor to neuronal death, indicating a mechanism of early cellular compromise correlated with a specific vulnerability in certain neurons [2, 79, 80]. Analysis of fixed tissues from AMD patients show extrudes of senescent RPE cytoplasm with reactive lysosomes into the underlying Bruch's membrane, and the accumulation of incom‐ pletely digested POS from overlying photoreceptors as lipofuscin within lysosomes [14]. Senescent postmitotic RPE cells with lipofuscin-filled lysosomes are a characteristic feature of the ageing retina, and it accounts for as much as 20% of the cytoplasmic volume by the age of 80. Experiments using cell lines show the toxic nature of lipofuscin and its derivative Nretinylidene–N-retinylethanolamine (A2E) that disrupts the phagocytic mechanisms of RPE cells, impairs lysosomal proteases, inhibits the lysosomal ATPase proton pump and causes leakage of lysosomal contents into the cytosol [81]. Dysfunctional lysosomes with lipofuscin/A2E also generates reactive oxygen species (ROS), modify lipid peroxidation and forms high molecular weight components that are stable within lysosomes. Moreover, A2E causes detergent-like membrane disruption and inhibits lytic function. Healthy macula RPE cells contain high levels of lysosomal enzymes acid phosphatase and cathepsin D, relative to lysosomes from RPE cells in the nasal/mid-zone and peripheral retina [82]. Lysosomal enzyme activity decreases by up to 50% when exposed to lipofuscin [83], indicating the regional vulnerability of the macula in early AMD. Lysosomal damage may be further exacerbated by the highly photoxidative RPE environment, providing ideal conditions for ROS generation [84].

Mitochondria also show early damage in AD. Hence, post-mortem AD brains show signifi‐ cantly fewer mitochondria, abnormally enlarged as well as exceptionally small mitochondria, damaged cristae, changes to organelle physiology, fission/fusion rates and transport defects [85]. Mitochondrial abnormalities have also been linked to AMD primarily using studies of cell lines showing a decrease in the number/area of RPE mitochondria, changes in redox components and proteins involved in mitochondrial trafficking, increase in mitochondrial DNA repair and decreased RPE mitochondrial respiration. A2E specifically damages mito‐ chondria inducing RPE apoptosis [60]. Our previous studies of a variant form of cystatin C associated with AMD revealed a striking endoplasmic reticulum (ER)/Golgi to mitochondria mis-localisation, which may have long-term consequences for RPE mitochondria [86, 87]. Recently, a strong association between the mitochondrial ARMS2 variant protein and AMD was reported which appeared to drive AMD towards a neovascular phenotype [8].

Genetic risk factors between AMD and AD also indicate evidence of a shared aetiology. For instance, studies have revealed a substantial link between allelic variants encoding compo‐ both types of extracellular protein deposits stain for Congo red, only senile plaques are positive for the apple green birefringence dye specific for anti-parallel β-pleated sheets [49, 63].

Commonalities between AD and AMD are also observed in the manner in which highly localised and significant damage occurs to lysosomes and mitochondria. In AD brains, these include substantial increases in the size/number of endosomes, autophagosomes and lyso‐ somes; accumulation of lysosomal dense bodies in dystrophic neurites, as well as changes in expression of lysosomal hydrolases such as cathepsins [2, 79]. Our studies as well as those of others have shown selective permeabilisation of lysosomal membranes and release of lytic content into the cytosol as a precursor to neuronal death, indicating a mechanism of early cellular compromise correlated with a specific vulnerability in certain neurons [2, 79, 80]. Analysis of fixed tissues from AMD patients show extrudes of senescent RPE cytoplasm with reactive lysosomes into the underlying Bruch's membrane, and the accumulation of incom‐ pletely digested POS from overlying photoreceptors as lipofuscin within lysosomes [14]. Senescent postmitotic RPE cells with lipofuscin-filled lysosomes are a characteristic feature of the ageing retina, and it accounts for as much as 20% of the cytoplasmic volume by the age of 80. Experiments using cell lines show the toxic nature of lipofuscin and its derivative Nretinylidene–N-retinylethanolamine (A2E) that disrupts the phagocytic mechanisms of RPE cells, impairs lysosomal proteases, inhibits the lysosomal ATPase proton pump and causes leakage of lysosomal contents into the cytosol [81]. Dysfunctional lysosomes with lipofuscin/A2E also generates reactive oxygen species (ROS), modify lipid peroxidation and forms high molecular weight components that are stable within lysosomes. Moreover, A2E causes detergent-like membrane disruption and inhibits lytic function. Healthy macula RPE cells contain high levels of lysosomal enzymes acid phosphatase and cathepsin D, relative to lysosomes from RPE cells in the nasal/mid-zone and peripheral retina [82]. Lysosomal enzyme activity decreases by up to 50% when exposed to lipofuscin [83], indicating the regional vulnerability of the macula in early AMD. Lysosomal damage may be further exacerbated by the highly photoxidative RPE environment, providing ideal conditions for ROS generation

Mitochondria also show early damage in AD. Hence, post-mortem AD brains show signifi‐ cantly fewer mitochondria, abnormally enlarged as well as exceptionally small mitochondria, damaged cristae, changes to organelle physiology, fission/fusion rates and transport defects [85]. Mitochondrial abnormalities have also been linked to AMD primarily using studies of cell lines showing a decrease in the number/area of RPE mitochondria, changes in redox components and proteins involved in mitochondrial trafficking, increase in mitochondrial DNA repair and decreased RPE mitochondrial respiration. A2E specifically damages mito‐ chondria inducing RPE apoptosis [60]. Our previous studies of a variant form of cystatin C associated with AMD revealed a striking endoplasmic reticulum (ER)/Golgi to mitochondria mis-localisation, which may have long-term consequences for RPE mitochondria [86, 87]. Recently, a strong association between the mitochondrial ARMS2 variant protein and AMD

was reported which appeared to drive AMD towards a neovascular phenotype [8].

Genetic risk factors between AMD and AD also indicate evidence of a shared aetiology. For instance, studies have revealed a substantial link between allelic variants encoding compo‐

[84].

96 Update on Dementia

nents of the alternative compliment cascade and the risk of developing AMD including factor H, factor B and C3 [88–91]. Evidence for a similar genetic predisposition in AD has been reported where polymorphisms within the CFH allele have been linked with an increased risk of AD [92]. The large number of compliment cascade components that have been reported within drusen and senile plaques, as well as the fact that chronic inflammation is a key driver in both AD and AMD indicates that similar inflammatory responses may be involved in the aetiologies of both AD and AMD. A strong genetic link has also been associated with ApoE, a polymorphic gene encoding proteins ApoE2, ApoE3 and ApoE4 involved in lipid metabolism. Amongst these, ApoE4 is somewhat confusingly associated with a lower risk of developing AMD, whilst conferring an increased susceptibility to AD. Although the reason for this is not clear, the positively charged nature of ApoE4 is speculated to interact somewhat differently with Bruch's membrane in the outer retina compared to its behaviour in the brain [93, 94]. The opposite holds true with regard to ApoE2, which is protective in AD but is associated with a higher risk of developing AMD. The reason for this also remains elusive [4]. Collectively, it appears that ApoE dysregulation may affect Aβ metabolism/clearance in the retina and brain in somewhat different ways, but which nonetheless triggers or drives pathology in these respective tissues [93, 95, 96]. Several environmental factors are also shared between AD and AMD that are thought confer increased susceptibility. These include cigarette smoking and diet, as well as conditions such as high blood pressure, heart disease, stroke, diabetes, high cholesterol levels and obesity [97–99]. In fact, a recent study conducted by the World Health Organisation (WHO) revealed that smoking, which is the most prominent environmental risk factor for AMD, almost doubled the risk of developing dementia [http://www.who.int/ tobacco/publications/en/]. The growing awareness of these shared pathologies in the senescent brain and retina as well as the increasing sophistication of detection methods are beginning to uncover closer links between AD and AMD. For instance, a recent study revealed the increased risk of AMD amongst AD patients [100].

Collectively, this body of evidence strongly suggests a significant overlap between the aetiologies of AMD and AD. This is not surprising, given the extensive visual complications being reported in patients with AD and dementia. Initial detection typically relies on selfdiagnosis and/or observations by friends and family and is therefore often inconsistent, adding to the potential delay in recognising these neuropathological conditions in a timely manner. Hence, ocular studies have been proposed alongside studies to identify common biomarkers so that those at greater risks may be identified relatively early before progressing to more advanced stages.
