Jaimie Hoh Kam

Viewing Ageing Eyes: Diverse Sites of Amyloid Beta Accumulation in the Ageing Mouse Retina and the Up-Regulation of Macrophages

Background Amyloid beta (Aβ) accumulates in the ageing central nervous system and is associated with a number of age-related diseases, including age-related macular degeneration (AMD) in the eye. AMD is characterised by accumulation of extracellular deposits called drusen in which Aβ is a key constituent. Aβ activates the complement cascade and its deposition is associated with activated macrophages. So far, little is known about the quantitative measurements of Aβ accumulation and definitions of its relative sites of ocular deposition in the normal ageing mouse. Methodology/Principal Findings We have traced Aβ accumulation quantitatively in the ageing mouse retina using immunohistochemistry and Western blot analysis. We reveal that it is not only deposited at Bruchs membrane and along blood vessels, but unexpectedly, it also coats photoreceptor outer segments. While Aβ is present at all sites of deposition from 3 months of age, it increases markedly from 6 months onward. Progressive accumulation of deposits on outer segments was confirmed with scanning electron microscopy, revealing age-related changes in their morphology. Such progress of accumulation of Aβ on photoreceptor outer segments with age was also confirmed in human retinae using immunohistochemistry. We also chart the macrophage response to increases in Aβ showing up-regulation in their numbers using both confocal laser imaging of the eye in vivo followed by in vitro immunostaining. With age macrophages become bloated with cellular debris including Aβ, however, their increasing numbers fail to stop Aβ accumulation. Conclusions Increasing Aβ deposition in blood vessels and Bruchs membrane will impact upon retinal perfusion and clearance of cellular waste products from the outer retina, a region of very high metabolic activity. This accumulation of Aβ may contribute to the 30% reduction of photoreceptors found throughout life and the shortening of those that remain. The coating of Aβ on outer segments may also have an impact upon visual function with age.

Vitamin D rejuvenates aging eyes by reducing inflammation, clearing amyloid beta and improving visual function

Vitamin D(3) plays a key role in immune regulation and may protect against the aging process. A focal point for age-related changes is the outer retina of the eye where there is high metabolic demand resulting in a gradual increase in extracellular deposition, inflammation, and cell loss giving rise to visual decline. Here, we demonstrate that vitamin D(3) administration for only 6 weeks in aged mice significantly impacts on this aging process. Treated mice showed significant reductions in retinal inflammation and levels of amyloid beta (Aβ) accumulation, which is a hallmark of aging. They also had significant reductions in retinal macrophage numbers and marked shifts in their morphology. These changes were reflected in a significant improvement in visual function, revealing that vitamin D(3) is a route to avoiding the pace of age-related visual decline. Excess amyloid beta deposition and inflammation are risk factors leading to age-related macular degeneration (AMD), the largest cause of blindness in those older than 50 years in developed countries. Recently, vitamin D(3) has been linked epidemiologically to protection against age-related macular degeneration. Hence, vitamin D(3) enrichment is likely to represent a beneficial route for those at risk.

Complement Factor H Is Critical in the Maintenance of Retinal Perfusion

Vascular pathologies are known to be associated with age-related macular degeneration. Recently, age-related macular degeneration was associated with a single-nucleotide substitution of the complement factor H (CFH) gene, part of the alternative pathway of the complement system, a critical element in the innate immune response. Such polymorphisms are found in more than 50% of cases of age-related macular degeneration. Here we show that the absence of CFH causes an autoimmune response that targets the vascular endothelium of both the inner and outer retinal vascular networks. In CFH-knockout (cfh(-/-)) mice, C3 and C3b, key components of the complement system, are progressively deposited on retinal vessels, which subsequently become restricted and wither, resulting in a reduction of retinal blood supply. This result leads to increased oxygen stress. While such effects are not systemic, these structural changes are mirrored in functional changes with a substantial decline in retinal blood flow dynamics. When the system is challenged functionally by laser-induced choroidal neovascularization, fluorescein leakage was significantly smaller in cfh(-/-) mice compared with controls, likely due to reduced retinal perfusion. These data reveal that in both the presence and absence of exogenous challenge to the innate immune system, CFH is required to maintain normal levels of retinal perfusion. It is likely that C3 and C3b accumulation in the aged CFH-deficient retina is associated with complement-mediated retinal endothelium destruction.

Biallelic Mutations in PLA2G5, Encoding Group V Phospholipase A2, Cause Benign Fleck Retina

Flecked-retina syndromes, including fundus flavimaculatus, fundus albipunctatus, and benign fleck retina, comprise a group of disorders with widespread or limited distribution of yellow-white retinal lesions of various sizes and configurations. Three siblings who have benign fleck retina and were born to consanguineous parents are the basis of this report. A combination of homozygosity mapping and exome sequencing helped to identify a homozygous missense mutation, c.133G>T (p.Gly45Cys), in PLA2G5, a gene encoding a secreted phospholipase (group V phospholipase A(2)). A screen of a further four unrelated individuals with benign fleck retina detected biallelic variants in the same gene in three patients. In contrast, no loss of function or common (minor-allele frequency>0.05%) nonsynonymous PLA2G5 variants have been previously reported (EVS, dbSNP, 1000 Genomes Project) or were detected in an internal database of 224 exomes (from subjects with adult onset neurodegenerative disease and without a diagnosis of ophthalmic disease). All seven affected individuals had fundoscopic features compatible with those previously described in benign fleck retina and no visual or electrophysiological deficits. No medical history of major illness was reported. Levels of low-density lipoprotein were mildly elevated in two patients. Optical coherence tomography and fundus autofluorescence findings suggest that group V phospholipase A(2) plays a role in the phagocytosis of photoreceptor outer-segment discs by the retinal pigment epithelium. Surprisingly, immunohistochemical staining of human retinal tissue revealed localization of the protein predominantly in the inner and outer plexiform layers.

Systemic Administration of Abeta mAb Reduces Retinal Deposition of Abeta and Activated Complement C3 in Age-Related Macular Degeneration Mouse Model

Age-related macular degeneration (AMD) is a leading cause of legal blindness in the Western world. There are effective treatments for the vascular complications of neo-vascular AMD, but no effective therapies are available for the dry/atrophic form of the disease. A previously described transgenic CFH-gene deficient mouse model, (cfh−/−), shows hallmarks of early AMD. The ocular phenotype has been further analysed to demonstrate amyloid beta (Aβ) rich basement membrane deposits associated with activated complement C3. Cfh−/− mice were treated systemically in both prophylactic and therapeutic regimes with an anti-Aβ monoclonal antibody (mAb), 6F6, to determine the effect on the cfh−/− retinal phenotype. Prophylactic treatment with 6F6 demonstrated a dose dependent reduction in the accumulation of both Aβ and activated C3 deposition. A similar reduction in the retinal endpoints could be seen after therapeutic treatment. Serum Aβ levels after systemic administration of 6F6 show accumulation of Aβ in the periphery suggestive of a peripheral sink mechanism. In summary, anti-Aβ mAb treatment can partially prevent or reverse ocular phenotypes of the cfh−/− mouse. The data support this therapeutic approach in humans potentially modulating two key elements in the pathogenesis of AMD – Aβ and activated, complement C3.

Near-infrared light increases ATP, extends lifespan and improves mobility in aged Drosophila melanogaster

Ageing is an irreversible cellular decline partly driven by failing mitochondrial integrity. Mitochondria accumulate DNA mutations and reduce ATP production necessary for cellular metabolism. This is associated with inflammation. Near-infrared exposure increases retinal ATP in old mice via cytochrome c oxidase absorption and reduces inflammation. Here, we expose fruitflies daily to 670 nm radiation, revealing elevated ATP and reduced inflammation with age. Critically, there was a significant increase in average lifespan: 100–175% more flies survived into old age following 670 nm exposure and these had significantly improved mobility. This may be a simple route to extending lifespan and improving function in old age.

Mitochondrial decline precedes phenotype development in the complement factor H mouse model of retinal degeneration but can be corrected by near infrared light.

Mitochondria produce adenosine triphosphate (ATP), critical for cellular metabolism. ATP declines with age, which is associated with inflammation. Here, we measure retinal and brain ATP in normal C57BL/6 and complement factor H knockout mice (Cfh(-/-)), which are proposed as a model of age-related macular degeneration. We show a significant premature 30% decline in retinal ATP in Cfh(-/-) mice and a subsequent shift in expression of a heat shock protein that is predominantly mitochondrial (Hsp60). Changes in Hsp60 are associated with stress and neuroprotection. We find no differences in brain ATP between C57BL/6 and Cfh(-/-) mice. Near infrared (NIR) increases ATP and reduces inflammation. ATP decline in Cfh(-/-) mice was corrected with NIR which also shifted Hsp60 labeling patterns. ATP decline in Cfh(-/-) mice occurs before inflammation becomes established and photoreceptor loss occurs and may relate to disease etiology. However, ATP levels were corrected with NIR. In summary, we provide evidence for a mitochondrial basis for this disease in mice and correct this with simple light exposure known to improve mitochondrial function.

To unite or divide: mitochondrial dynamics in the murine outer retina that preceded age related photoreceptor loss

Mitochondrial function declines with age and is associated with age-related disorders and cell death. In the retina this is critical as photoreceptor energy demands are the greatest in the body and aged cell loss large (~30%). But mitochondria can fuse or divide to accommodate changing demands. We explore ageing mitochondrial dynamics in young (1 month) and old (12 months) mouse retina, investigating changes in mitochondrial fission (Fis1) and fusion (Opa1) proteins, cytochrome C oxidase (COX III), which reflects mitochondrial metabolic status, and heat shock protein 60 (Hsp60) that is a mitochondrial chaperon for protein folding. Western blots showed each protein declined with age. However, within this, immunostaining revealed increases of around 50% in Fis1 and Opa1 in photoreceptor inner segments (IS). Electron microscope analysis revealed mitochondrial fragmentation with age and marked changes in morphology in IS, consistent with elevated dynamics. COX III declined by approximately 30% in IS, but Hsp60 reductions were around 80% in the outer plexiform layer. Our results are consistent with declining mitochondrial metabolism. But also with increased photoreceptor mitochondrial dynamics that differ from other retinal regions, perhaps reflecting attempts to maintain function. These changes are the platform for age related photoreceptor loss initiated after 12 months.

Topical cyclodextrin reduces amyloid beta and inflammation improving retinal function in ageing mice

Retinal ageing results in chronic inflammation, extracellular deposition, including that of amyloid beta (Aβ) and declining visual function. In humans this can progress into age-related macular degeneration (AMD), which is without cure. Therapeutic approaches have focused on systemic immunotherapies without clinical resolution. Here, we show using aged mice that 2-Hydroxypropyl-β-cyclodextrin, a sugar molecule given as eye drops over 3 months results in significant reductions in Aβ by 65% and inflammation by 75% in the aged mouse retina. It also elevates retinal pigment epithelium specific protein 65 (RPE65), a key molecule in the visual cycle, in aged retina. These changes are accompanied by a significant improvement in retinal function measured physiologically. 2-Hydroxypropyl-β-cyclodextrin is as effective in reducing Aβ and inflammation in the complement factor H knockout (Cfh(-/-)) mouse that shows advanced ageing and has been proposed as an AMD model. β-cyclodextrin is economic, safe and may provide an efficient route to reducing the impact of retinal ageing.

Aged complement factor H knockout mice kept in a clean barriered environment have reduced retinal pathology.

Age-related macular degeneration (AMD) is the largest cause of visual loss in those over 60 years in the West and is a condition increasing in prevalence. Many diseases result from genetic/environmental interactions and 50% of AMD cases have an association with polymorphisms of the complement system including complement factor H. Here we explore interactions between genetic predisposition and environmental conditions in triggering retinal pathology in two groups of aged complement factor H knock out (Cfh(-/-)) mice. Mice were maintained over 9 months in either a conventional open environment or a barriered pathogen free environment. Open environment Cfh(-/-) mice had significant increases in subretinal macrophage numbers, inflammatory and stress responses and reduced photoreceptor numbers over mice kept in a pathogen free environment. Hence, environmental factors can drive retinal disease in these mice when linked to complement deficits impairing immune function. Both groups of mice had similar levels of retinal amyloid beta accumulation. Consequently there is no direct link between this and inflammation in Cfh(-/-) mice.