Rebuilding the Retina: Prospects for Müller Glial-mediated Self-repair

Retinal degeneration is a leading cause of untreatable blindness in the industrialised world. It is typically irreversible and there are few curative treatments available. The use of stem cells to generate new retinal neurons for transplantation purposes has received significant interest in recent years and is beginning to move towards clinical trials. However, such approaches are likely to be most effective for relatively focal areas of repair. An intriguing complementary approach is endogenous self-repair. Retinal cells from the ciliary marginal zone (CMZ), retinal pigment epithelium (RPE) and Müller glial cells (MG) have all been shown to play a role in retinal repair, typically in lower vertebrates. Among them, MG have received renewed interest, due to their distribution throughout (centre to periphery) the neural retina and their potential to re-acquire a progenitor-like state following retinal injury with the ability to proliferate and generate new neurons. Triggering these innate self-repair mechanisms represents an exciting therapeutic option in treating retinal degeneration. However, these cells behave differently in mammalian and non-mammalian species, with a considerably restricted potential in mammals. In this short review, we look at some of the recent progress made in our understanding of the signalling pathways that underlie MG-mediated regeneration in lower vertebrates, and some of the challenges that have been revealed in our attempts to reactivate this process in the mammalian retina

Müller glia activation in response to inherited retinal degeneration is highly varied and disease-specific

Despite different aetiologies, most inherited retinal disorders culminate in photoreceptor loss, which induces concomitant changes in the neural retina, one of the most striking being reactive gliosis by Müller cells. It is typically assumed that photoreceptor loss leads to an upregulation of glial fibrilliary acidic protein (Gfap) and other intermediate filament proteins, together with other gliosis-related changes, including loss of integrity of the outer limiting membrane (OLM) and deposition of proteoglycans. However, this is based on a mix of both injury-induced and genetic causes of photoreceptor loss. There are very few longitudinal studies of gliosis in the retina and none comparing these changes across models over time. Here, we present a comprehensive spatiotemporal assessment of features of gliosis in the degenerating murine retina that involves Müller glia. Specifically, we assessed Gfap, vimentin and chondroitin sulphate proteoglycan (CSPG) levels and outer limiting membrane (OLM) integrity over time in four murine models of inherited photoreceptor degeneration that encompass a range of disease severities (Crb1rd8/rd8, Prph2+/Δ307, Rho-/-, Pde6brd1/rd1). These features underwent very different changes, depending upon the disease-causing mutation, and that these changes are not correlated with disease severity. Intermediate filament expression did indeed increase with disease progression in Crb1rd8/rd8 and Prph2+/Δ307, but decreased in the Prph2+/Δ307 and Pde6brd1/rd1 models. CSPG deposition usually, but not always, followed the trends in intermediate filament expression. The OLM adherens junctions underwent significant remodelling in all models, but with differences in the composition of the resulting junctions; in Rho-/- mice, the adherens junctions maintained the typical rod-Müller glia interactions, while in the Pde6brd1/rd1 model they formed predominantly between Müller cells in late stage of degeneration. Together, these results show that gliosis and its associated processes are variable and disease-dependent

A comprehensive atlas of Aggrecan, Versican, Neurocan and Phosphacan expression across time in wildtype retina and in retinal degeneration

As photoreceptor cells die during retinal degeneration, the surrounding microenvironment undergoes significant changes that are increasingly recognized to play a prominent role in determining the efficacy of therapeutic interventions. Chondroitin Sulphate Proteoglycans (CSPGs) are a major component of the extracellular matrix that have been shown to inhibit neuronal regrowth and regeneration in the brain and spinal cord, but comparatively little is known about their expression in retinal degeneration. Here we provide a comprehensive atlas of the expression patterns of four individual CSPGs in three models of inherited retinal degeneration and wildtype mice. In wildtype mice, Aggrecan presented a biphasic expression, while Neurocan and Phosphacan expression declined dramatically with time and Versican expression remained broadly constant. In degeneration, Aggrecan expression increased markedly in Aipl1-/- and Pde6brd1/rd1, while Versican showed regional increases in the periphery of Rho-/- mice. Conversely, Neurocan and Phosphacan broadly decrease with time in all models. Our data reveal significant heterogeneity in the expression of individual CSPGs. Moreover, there are striking differences in the expression patterns of specific CSPGs in the diseased retina, compared with those reported following injury elsewhere in the CNS. Better understanding of the distinct distributions of individual CSPGs will contribute to creating more permissive microenvironments for neuro-regeneration and repair

Seasonal, annual and decadal change in tadpole populations in tropical Australian streams

Abstract Declines due to fungal disease (chytridiomycosis) have affected many stream-dwelling frog species, especially in the tropics, leading to reduced abundance and diversity of their tadpoles. Studies in the Australian Wet Tropics have demonstrated that some frog species have declined or disappeared, while others have persisted. To assess the occurrence of stream-breeding frogs, we monitored tadpole populations of five frog species in Wet Tropics streams in the early 1990s (uplands, before chytridomycosis emergence), and in 2011-2013 (uplands and lowlands, after chytridiomycosis emergence), and investigated environmental factors that might influence tadpole abundance. Riffle-dwelling tadpoles of two frog species disappeared from the upland stream site during the 1990s, reflecting reported losses of adult populations. Tadpoles of one upland pool species initially declined but had recovered by 2011-2013. Samples from the lowlands in 2011 to 2013 indicated no similar loss. Chytridiomycosis was the likely cause of changes in tadpole abundances between the two survey periods, given its known occurrence and documented effects on adult frogs in these systems; however, we did not measure its prevalence in this study. Tadpole populations fluctuated seasonally, with abundances highest in spring and summer, reflecting the timing of frog reproduction. The most important biophysical influence on the assemblages that we measured was current velocity. Tadpole peak abundances suggest that they make a substantial contribution at the consumer level of food webs, and that their loss has altered food webs substantially in upland streams.</jats:p

Harnessing the Potential of Human Pluripotent Stem Cells and Gene Editing for the Treatment of Retinal Degeneration

PURPOSE OF REVIEW: A major cause of visual disorders is dysfunction and/or loss of the light-sensitive cells of the retina, the photoreceptors. To develop better treatments for patients, we need to understand how inherited retinal disease mutations result in the dysfunction of photoreceptors. New advances in the field of stem cell and gene editing research offer novel ways to model retinal dystrophies in vitro and present opportunities to translate basic biological insights into therapies. This brief review will discuss some of the issues that should be taken into account when carrying out disease modelling and gene editing of retinal cells. We will discuss (i) the use of human induced pluripotent stem cells (iPSCs) for disease modelling and cell therapy; (ii) the importance of using isogenic iPSC lines as controls; (iii) CRISPR/Cas9 gene editing of iPSCs; and (iv) in vivo gene editing using AAV vectors. RECENT FINDINGS: Ground-breaking advances in differentiation of iPSCs into retinal organoids and methods to derive mature light sensitive photoreceptors from iPSCs. Furthermore, single AAV systems for in vivo gene editing have been developed which makes retinal in vivo gene editing therapy a real prospect. SUMMARY: Genome editing is becoming a valuable tool for disease modelling and in vivo gene editing in the retina

Measurement of the bone endocortical region using clinical CT.

The extent of the endocortical region and cortical bone mineral density (cBMD) throughout the proximal femur are of interest as both have been linked to fracture risk and osteoporosis treatment response. Non-invasive in-vivo clinical CT-based techniques capable of measuring the cortical bone attributes of thickness, density and mass over a bone surface have already been proposed. Several studies have robustly shown these methods to be capable of producing cortical thickness measurements to a sub-millimetre accuracy. Unfortunately, these methods are unable to provide high quality cBMD estimates, and are not designed to measure any attributes over the endocortical region of cortical bone. In this paper, we develop a cortical bone mapping based technique capable of providing an improved cBMD estimate and a measure of the endocortical width, while maintaining similar quality cortical thickness and trabecular bone mineral density (tBMD) estimates. The performance of the technique was assessed using a paired dataset of ex-vivo QCT and HR-pQCT scans across 72 proximal femurs. The HR-pQCT scans were analysed using a new method developed for this study: high resolution tissue classification (HRTC). In HRTC the cortical, endocortical and sub-surface trabecular bone features are extracted from the partially resolvable microarchitectural details in the HR-pQCT scan. We demonstrate that measurement of the endocortical extent from QCT is possible with an accuracy of -0.15±0.71mm, and that local cBMD can be measured down to densities of 300 mg/cm3

Tracking neuronal motility in live murine retinal explants

The developing retina undergoes dynamic organizational changes involving significant intra-retinal motility of the encompassing cells. Here, we present a protocol for tracking retinal cell motility in live explanted mouse retinae. Although originally applied to rod and cone photoreceptors, this strategy is applicable to any fluorescently labeled cell in mouse retinae and other similar experimental retinal models. Careful tissue handling is critical for the successful acquisition of high-quality live imaging data. Further instructions for semi-automated in silico data handling are provided. For complete details on the use and execution of this protocol, please refer to Aghaizu et al. (2021)

Constraining the provenance of the Stonehenge 'Altar Stone': Evidence from automated mineralogy and U–Pb zircon age dating

The Altar Stone at Stonehenge is a greenish sandstone thought to be of Late Silurian-Devonian (‘Old Red Sandstone’) age. It is classed as one of the bluestone lithologies which are considered to be exotic to the Salisbury Plain environ, most of which are derived from the Mynydd Preseli, in west Wales. However, no Old Red Sandstone rocks crop out in the Preseli; instead a source in the Lower Old Red Sandstone Cosheston Subgroup at Mill Bay to the south of the Preseli, has been proposed. More recently, on the basis of detailed petrography, a source for the Altar Stone much further to the east, towards the Wales-England border, has been suggested. Quantitative analyses presented here compare mineralogical data from proposed Stonehenge Altar Stone debris with samples from Milford Haven at Mill Bay, as well as with a second sandstone type found at Stonehenge which is Lower Palaeozoic in age. The Altar Stone samples have contrasting modal mineralogies to the other two sandstone types, especially in relation to the percentages of its calcite, kaolinite and barite cements. Further differences between the Altar Stone sandstone and the Cosheston Subgroup sandstone are seen when their contained zircons are compared, showing differing morphologies and U-Pb age dates having contrasting populations. These data confirm that Mill Bay is not the source of the Altar Stone with the abundance of kaolinite in the Altar Stone sample suggesting a source further east, towards the Wales-England border. The disassociation of the Altar Stone and Milford Haven undermines the hypothesis that the bluestones, including the Altar Stone, were transported from west Wales by sea up the Bristol Channel and adds further credence to a totally land-based route, possibly along a natural routeway leading from west Wales to the Severn estuary and beyond. This route may well have been significant in prehistory, raising the possibility that the Altar Stone was added en route to the assemblage of Preseli bluestones taken to Stonehenge around or shortly before 3000 BC. Recent strontium isotope analysis of human and animal bones from Stonehenge, dating to the beginning of its first construction stage around 3000 BC, are consistent with the suggestion of connectivity between this western region of Britain and Salisbury Plain.This study appears to be the first application of quantitative automated mineralogy in the provenancing of archaeological lithic material and highlights the potential value of automated mineralogy in archaeological provenancing investigations, especially when combined with complementary techniques, in the present case zircon age dating