The Alzheimer's-related amyloid beta peptide is internalised by R28 neuroretinal cells and disrupts the microtubule associated protein 2 (MAP-2)

Age-related Macular Degeneration (AMD) is a common, irreversible blinding condition that leads to the loss of central vision. AMD has a complex aetiology with both genetic as well as environmental risks factors, and share many similarities with Alzheimer's disease. Recent findings have contributed significantly to unravelling its genetic architecture that is yet to be matched by molecular insights. Studies are made more challenging by observations that aged and AMD retinas accumulate the highly pathogenic Alzheimer's-related Amyloid beta (A?) group of peptides, for which there appears to be no clear genetic basis. Analyses of human donor and animal eyes have identified retinal A? aggregates in retinal ganglion cells (RGC), the inner nuclear layer, photoreceptors as well as the retinal pigment epithelium. A? is also a major drusen constituent; found correlated with elevated drusen-load and age, with a propensity to aggregate in retinas of advanced AMD. Despite this evidence, how such a potent driver of neurodegeneration might impair the neuroretina remains incompletely understood, and studies into this important aspect of retinopathy remains limited. In order to address this we exploited R28 rat retinal cells which due to its heterogeneous nature, offers diverse neuroretinal cell-types in which to study the molecular pathology of A?. R28 cells are also unaffected by problems associated with the commonly used RGC-5 immortalised cell-line, thus providing a well-established model in which to study dynamic A? effects at single-cell resolution. Our findings show that R28 cells express key neuronal markers calbindin, protein kinase C and the microtubule associated protein-2 (MAP-2) by confocal immunofluorescence which has not been shown before, but also calretinin which has not been reported previously. For the first time, we reveal that retinal neurons rapidly internalised A?1-42, the most cytotoxic and aggregate-prone amongst the A? family. Furthermore, exposure to physiological amounts of A?1-42 for 24 h correlated with impairment to neuronal MAP-2, a cytoskeletal protein which regulates microtubule dynamics in axons and dendrites. Disruption to MAP-2 was transient, and had recovered by 48 h, although internalised A? persisted as discrete puncta for as long as 72 h. To assess whether A? could realistically localise to living retinas to mediate such effects, we subretinally injected nanomolar levels of oligomeric A?1-42 into wildtype mice. Confocal microscopy revealed the presence of focal A? deposits in RGC, the inner nuclear and the outer plexiform layers 8 days later, recapitulating naturally-occurring patterns of A? aggregation in aged retinas. Our novel findings describe how retinal neurons internalise A? to transiently impair MAP-2 in a hitherto unreported manner. MAP-2 dysfunction is reported in AMD retinas, and is thought to be involved in remodelling and plasticity of post-mitotic neurons. Our insights suggest a molecular pathway by which this could occur in the senescent eye leading to complex diseases such as AMD

‘I enjoyed it because … you could do whatever you wanted and be creative’: three principles for participatory research and pedagogy

The complexity of many children’s lives can result in their ideas being neither understood nor included in mainstream opportunities for learning, particularly children who are living with disadvantage. With a focus on developing ethical and inclusive principles for participatory research and pedagogy, this paper reports on a pilot project where we worked with young, hard-to-reach individuals across four sites in England to enable them to design and carry out research about their experiences and views of disadvantage. Here, we present snapshots of the young participants’ choices of research topics and methods, which reflected their own lives and interests, and led to powerful visualizations of the complexity of child and youth disadvantage. Reflecting back on the project, we discuss effective ways to initiate and sustain participatory research that can enable young researchers to be involved as active and empowered agents at every stage of the research process. We also consider the implications for developing participatory pedagogy, with researchers working alongside educators to create school cultures that foster belonging and genuinely support all students’ expertise and ways of knowing by looking beyond the school buildings and into their lives in the wider community

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The complexities underlying age-related macular degeneration: could amyloid beta play an important role?

Age-related macular degeneration (AMD) causes irreversible loss of central vision for which there is no effective treatment. Incipient pathology is thought to occur in the retina for many years before AMD manifests from midlife onwards to affect a large proportion of the elderly. Although genetic as well as non-genetic/environmental risks are recognized, its complex aetiology makes it difficult to identify susceptibility, or indeed what type of AMD develops or how quickly it progresses in different individuals. Here we summarize the literature describing how the Alzheimer's-linked amyloid beta (Aβ) group of misfolding proteins accumulate in the retina. The discovery of this key driver of Alzheimer's disease in the senescent retina was unexpected and surprising, enabling an altogether different perspective of AMD. We argue that Aβ fundamentally differs from other substances which accumulate in the ageing retina, and discuss our latest findings from a mouse model in which physiological amounts of Aβ were subretinally-injected to recapitulate salient features of early AMD within a short period. Our discoveries as well as those of others suggest the pattern of Aβ accumulation and pathology in donor aged/AMD tissues are closely reproduced in mice, including late-stage AMD phenotypes, which makes them highly attractive to study dynamic aspects of Aβ-mediated retinopathy. Furthermore, we discuss our findings revealing how Aβ behaves at single-cell resolution, and consider the long-term implications for neuroretinal function. We propose Aβ as a key element in switching to a diseased retinal phenotype, which is now being used as a biomarker for late-stage AMD

A mouse model to study Aβ-driven pathology in the ageing retina

Purpose : The Alzheimer’s-linked Amyloid beta (Aβ) peptide is reported to be deposited in aged retinas. Aβ has been implicated in key stages of Age-related Macular Degeneration (AMD), yet its role remains poorly understood. Here we employ a mouse model to study the in-vivo effects of Aβ to delineate its mechanisms of action and to understand how Aβ triggers/drives retinal pathology with age. Methods : Aβ was characterised by negative stain TEM and immunogold labelling. C57BL/6 mice were subretinally injected with 3µL of oligomeric Aβ1-42 (625nM, n=5) or vehicle control (n=3). At 8 days post-injection eyes were enucleated, OCT-embedded and cryosectioned at 16µM intervals for histological analysis. H&E staining and confocal immunofluorescence analysed retinal morphology in response to Aβ exposure and reported Aβ localisation. Exclusion criteria included a 200µM radius from the injection site to omit areas of mechanical trauma. Morphometric analysis was performed blind using OlyVIA and ImageJ. Data is expressed as means ± SEM with a statistical significance of *P ≤ 0.05. Results : TEM and Dot Blot assay enabled us to identify a window in which Aβ is reported to be most toxic. Fundus images showed large areas of pathology in Aβ exposed mice (9582 ± 4831) compared to controls (48.33 ± 8.97) which were indistinguishable from non-injected littermates, p=0.12. Serial line scans of Aβ injected mice revealed a 2-fold increase in RPE hypopigmentation associated with photoreceptor outer segment (POS) loss, RPE disorganisation and RPE hypertrophy compared to controls. Similarly, confocal data showed POS and inner segment disorganisation compared to well-preserved retinal architecture in control retinas. Aβ was detected in multiple retinal locations including POS, the RPE/choroid interface and the outer and inner plexiform layers. Conclusions : Our findings demonstrate that subretinal Aβ injections faithfully recapitulate key features of early AMD including dysfunctional RPE and damaged photoreceptors. Critically, we found no obvious indication of apoptosis or disruption of the blood-retinal barrier indicating gradual cellular impairment over time. Previously published literature showing static images of Abeta in human post-mortem eyes tantalisingly correlates the presence of Aβ with high drusen-loads and AMD. Our model therefore represents a powerful tool to investigate the dynamic nature of Aβ-mediated pathology in living retinas

Oligomeric Aβ1-42 induces an AMD-like phenotype and accumulates in lysosomes to impair RPE function

Alzheimer’s disease-associated amyloid beta (Aβ) proteins accumulate in the outer retina with increasing age and in eyes of age-related macular degeneration (AMD) patients. To study Aβ-induced retinopathy, wild-type mice were injected with nanomolar human oligomeric Aβ1-42, which recapitulate the Aβ burden reported in human donor eyes. In vitro studies investigated the cellular effects of Aβ in endothelial and retinal pigment epithelial (RPE) cells. Results show subretinal Aβ-induced focal AMD-like pathology within 2 weeks. Aβ exposure caused endothelial cell migration, and morphological and barrier alterations to the RPE. Aβ co-localized to late-endocytic compartments of RPE cells, which persisted despite attempts to clear it through upregulation of lysosomal cathepsin B, revealing a novel mechanism of lysosomal impairment in retinal degeneration. The rapid upregulation of cathepsin B was out of step with the prolonged accumulation of Aβ within lysosomes, and contrasted with enzymatic responses to internalized photoreceptor outer segments (POS). Furthermore, RPE cells exposed to Aβ were identified as deficient in cargo-carrying lyso-somes at time points that are critical to POS degradation. These findings imply that Aβ accumulation within late-endocytic compartments, as well as lysosomal deficiency, impairs RPE function over time, contributing to visual defects seen in aging and AMD eyes