25 research outputs found

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

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    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

    The role of high-fat diet and oxidative stress in driving the structural and functional deficits associated with Age-related Macular Degeneration

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    Age-related Macular Degeneration (AMD) is the leading cause of irreversible blindness in the developed world. Current treatments are only suitable for a proportion of patients and are usually ineffective in the long-term. This is partially due to an incomplete understanding of the underlying aetiologies that drive pathology in the Retinal Pigment Epithelium (RPE) and adjacent tissues of the outer retina. Many risk factors for AMD, including consumption of a high fat diet (HFD), increase oxidative stress in the retina. I studied the link between a HFD and structural changes to the tissues in the outer retina. Next, I investigated how these pathogenic changes could cause disease phenotypes in the RPE at a single cell resolution. To achieve these objectives, I used serial block-face scanning electron microscopy (SBSEM) to study healthy tissues in the mouse outer retina. An entire patch of RPE and overlying photoreceptors were reconstructed in 3D, and comparisons were made between mono-nucleate and bi-nucleate RPE cells, of which the latter is associated with early AMD. This novel approach provided new data on the structure and arrangement of RPE and photoreceptors, as well as their relationship to each other. 3D approaches have not been extensively used before, thus we have gained new types of structural information on these tissues. In mice fed a HFD, I used conventional TEM alongside confocal immunofluorescence imaging to study the pathogenic changes in tissues of the outer retina. Detailed studies of HFD mouse eyes have not been carried out to this extent before. My findings revealed HFD-induced changes to the structure of outer retinal tissues, showing the effects of an unhealthy diet and oxidative stress in living eyes. Next, my work delved into these pathogenic mechanisms at single-cell resolution. A HFD is associated with oxidative stress and impaired acidification of intracellular compartments in the lysosomalautophagy pathway. Using confocal-immunofluorescence microscopy and conventional TEM, I studied how these insults impaired the trafficking of photoreceptor outer segments (POS) in cultured RPE cells. My discoveries reveal the trafficking and breakdown of POS cargos in early and late endosomes/ phagosomes, and their eventual transport to lysosomes and autophagy bodies in healthy RPE cells. My data also provides novel mechanistic insights into how HFD associated oxidative stress and impaired acidification alters POS trafficking, potentially contributing to early stages of RPE pathology. Notably, it appears that damage may be confined to only a proportion of RPE lysosomes, suggesting that at least some compartments in the endo-lysosomal pathway remains functional, even in diseased cells. Collectively, my work has revealed novel structural and functional insights into pathogenic mechanism in the RPE and adjacent tissues of the outer retina, which could significantly influence the way in which disease processes in the aging retina are understood. Such insights are critical if effective treatments against AMD are to be devised in the futur

    Serial block face scanning electron microscopy reveals novel organizational details of the retinal pigment epithelium

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    Advances in imaging have led to the development of several new types of microscopes such as serial block face scanning electron microscopy (SBF-SEM), lightsheet microscopy, as well as X-ray micro-computed tomography (micro-CT), which enables the study of samples in fundamentally different ways. Significantly, these are now commercially available, which facilitates their widespread use in research. With SBF-SEM, fixed and resin-embedded specimens can be serially sectioned and imaged to construct a 3D dataset of the ultrastructure of cells and tissues at high resolution. We used this technique on perfusion-fixed C57BL/6 mouse eyes to image the outer retina. Our findings revealed novel organizational details of the retinal pigment epithelium (RPE) (Keeling et al., 2020b); a specialized cell monolayer that maintains the overlying photoreceptors and also forms the outer blood-retinal-barrier. RPE cells were found to look after far more photoreceptors than was widely assumed. 3D-data enabled measurements of the RPE cytoplasmic and nuclear volumes, the length and angle of microvilli on the apical RPE surface, as well as sub-RPE spaces under the basolateral membrane. The study also compared between mono-nucleate vs. bi-nucleate RPE cells, whilst the use of computing microinstructions (macros) provided information on interactions between adjacent cells in the RPE monolayer. Analysis of SBF-SEM stacks showed several hundred mitochondria which were rendered in 3D, providing information on their volume and spatial distribution in healthy RPE. Mitochondria were found in varying shapes and sizes, and predominantly localized to the mid and basal-zones of cells. The capabilities of SBF-SEM alongside other imaging techniques are being increasingly harnessed by investigators to gain novel insights into the organization of cells and tissues in the eye. These findings also help improve the current understanding of pathology linked with common blinding conditions such as age-related macular degeneration (AMD), as well as rare forms of retinopathy which leads to irreversible sight-los

    Use of a handheld electrophysiology device (RETeval, LKC) to identify visual pathway decussation defects

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    Electrodiagnostic testing (EDTs) require large equipment and trained clinical scientists so are restricted to specialist centres, and often have long waiting lists. Pattern and flash visual evoked potentials (fVEP) have been utilised in the assessment of visual pathway integrity, including chiasmal anomalies in albinism. In this qualitative clinical study, we assess the potential of the handheld RETeval to screen for excess nerve decussation and other post-chiasmal defects

    Impaired Cargo Clearance in the Retinal Pigment Epithelium (RPE) Underlies Irreversible Blinding Diseases

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    Chronic degeneration of the Retinal Pigment Epithelium (RPE) is a precursor to pathological changes in the outer retina. The RPE monolayer, which lies beneath the neuroretina, daily internalises and digests large volumes of spent photoreceptor outer segments. Impaired cargo handling and processing in the endocytic/phagosome and autophagy pathways lead to the accumulation of lipofuscin and pyridinium bis-retinoid A2E aggregates and chemically modified compounds such as malondialdehyde and 4-hydroxynonenal within RPE. These contribute to increased proteolytic and oxidative stress, resulting in irreversible damage to post-mitotic RPE cells and development of blinding conditions such as age-related macular degeneration, Stargardt disease and choroideremia. Here, we review how impaired cargo handling in the RPE results in their dysfunction, discuss new findings from our laboratory and consider how newly discovered roles for lysosomes and the autophagy pathway could provide insights into retinopathies. Studies of these dynamic, molecular events have also been spurred on by recent advances in optics and imaging technology. Mechanisms underpinning lysosomal impairment in other degenerative conditions including storage disorders, α-synuclein pathologies and Alzheimer’s disease are also discussed. Collectively, these findings help transcend conventional understanding of these intracellular compartments as simple waste disposal bags to bring about a paradigm shift in the way lysosomes are perceived

    Use of a handheld electrophysiology device (RETeval, LKC) to identify visual pathway decussation defects

    No full text
    Purpose : electrodiagnostic testing (EDTs) require large equipment and trained clinical scientists so are restricted to specialist centres, and often have long waiting lists. Pattern and flash visual evoked potentials (fVEP) have been utilised in the assessment of visual pathway integrity, including chiasmal anomalies in albinism. In this qualitative clinical study, we assess the potential of the handheld RETeval to screen for excess nerve decussation and other post-chiasmal defects.Methods : 18 patients with suspected albinism or retro-chiasmal lesions (aged 0-15yrs, mean 5) were selected from those undergoing routine EDTs at a regional referral centre. Standard paediatric EDTs were performed, followed by fVEP using the RETeval. Control data was collected from 12 volunteers (aged 26-54yrs, mean 32). Right and left occipital electrodes were placed midway between Oz (10-20 system) and either ear. The ground electrode was placed at Fz. FVEPs of 3cd.s/m2 were recorded from 10 brief (&lt;5ms) 1Hz flashes. The left occipital VEP waveform was subtracted from that of the right occiput, creating a single channel response for each eye separately. RETeval data was compared to results obtained from the standard paediatric protocol using a photic stimulator (Grass PS 22). Pearson’s correlation was used to calculate the extent of asymmetry, where a value of -1 showed complete asymmetry whilst a value of +1 showed absolute symmetry in occipital VEP distribution. Data between controls and patients were compared using a Mann-Whitney U test.Results : FVEP testing using both the standard protocol and the RETeval identified asymmetry in 11 patients with suspected albinism. The Pearsons correlate of the albinism cohort (-0.504 ± 0.208) was compared to the control data (0.184 ± 0.339) and a Mann-Whitney U test showed a significant difference between the two groups (p&lt;0.001). This demonstrates the effectiveness of the RETeval in detecting crossed asymmetry secondary to excessive nerve decussation in albinism. The RETeval also detected 7 cases of left and right retro-chiasmal lesions that showed an uncrossed asymmetry.Conclusions : this study adds to the growing body of work investigating the use of the handheld RETeval device for prioritising patients waiting for in-depth EDTs. This data shows the RETeval has the potential to be used as a screening tool for the detection of chiasmal and retro-chiasmal anomalies.<br/

    Eyetracking-enhanced VEP for nystagmus

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    Visual evoked potential (VEP) testing is an essential first stage in the diagnostic workup of patients with infantile nystagmus (IN). VEPs are an important factor in the diagnosis of albinism, which accompanies IN in ~28% of cases, as well as chiasmal, optic nerve and neurometabolic disease that can all present with IN. VEPs are also used to assess prognostic visual ability in cases of retinal dystrophy and optic nerve disease. Despite being used regularly in those with IN, VEP testing requires patients to keep the eyes still; something that people with IN naturally cannot do. Fixation instability during VEP testing is believed to reduce VEP signal amplitude, and as a result, the reliability of VEP may be reduced in one of the patient groups that needs it the most. This study investigates whether VEP signal quality (amplitude) can be improved in people with IN, by triggering acquisition only during the foveating (slow) periods of the nystagmus waveform
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