RNAi-mediated suppression of vimentin or glial fibrillary acidic protein prevents the establishment of Müller glial cell hypertrophy in progressive retinal degeneration.

Gliosis is a complex process comprising upregulation of intermediate filament (IF) proteins, particularly glial fibrillary acidic protein (GFAP) and vimentin, changes in glial cell morphology (hypertrophy) and increased deposition of inhibitory extracellular matrix molecules. Gliosis is common to numerous pathologies and can have deleterious effects on tissue function and regeneration. The role of IFs in gliosis is controversial, but a key hypothesized function is the stabilization of glial cell hypertrophy. Here, we developed RNAi approaches to examine the role of GFAP and vimentin in vivo in a murine model of inherited retinal degeneration, the Rhodopsin knockout (Rho-/- ) mouse. Specifically, we sought to examine the role of these IFs in the establishment of Müller glial hypertrophy during progressive degeneration, as opposed to (more commonly assessed) acute injury. Prevention of Gfap upregulation had a significant effect on the morphology of reactive Müller glia cells in vivo and, more strikingly, the reduction of Vimentin expression almost completely prevented these cells from undergoing degeneration-associated hypertrophy. Moreover, and in contrast to studies in knockout mice, simultaneous suppression of both GFAP and vimentin expression led to severe changes in the cytoarchitecture of the retina, in both diseased and wild-type eyes. These data demonstrate a crucial role for Vimentin, as well as GFAP, in the establishment of glial hypertrophy and support the further exploration of RNAi-mediated knockdown of vimentin as a potential therapeutic approach for modulating scar formation in the degenerating retina.

Müller Glia Reactivity and Development of Gliosis in Response to Pathological Conditions.

Within the mammalian retina, both Müller glia and astrocytes display reactivity in response to many forms of retinal injury and disease in a process termed gliosis. Reactive gliosis is a complex process that is considered to represent a cellular response to protect the retina from further damage and to promote its repair following pathological insult. It includes morphological, biochemical and physiological changes, which may vary depending on the type and degree of the initial injury. Not only does gliosis have numerous triggers, but also there is a great degree of heterogeneity in the glial response, creating multiple levels of complexity. For these reasons, understanding the process of glial scar formation and how this process differs in different pathological conditions and finding strategies to circumvent these barriers represent major challenges to the advancement of many ocular therapies.

Transplanted Donor- or Stem Cell-Derived Cone Photoreceptors Can Both Integrate and Undergo Material Transfer in an Environment-Dependent Manner.

Human vision relies heavily upon cone photoreceptors, and their loss results in permanent visual impairment. Transplantation of healthy photoreceptors can restore visual function in models of inherited blindness, a process previously understood to arise by donor cell integration within the host retina. However, we and others recently demonstrated that donor rod photoreceptors engage in material transfer with host photoreceptors, leading to the host cells acquiring proteins otherwise expressed only by donor cells. We sought to determine whether stem cell- and donor-derived cones undergo integration and/or material transfer. We find that material transfer accounts for a significant proportion of rescued cells following cone transplantation into non-degenerative hosts. Strikingly, however, substantial numbers of cones integrated into the Nrl-/- and Prph2rd2/rd2, but not Nrl-/-;RPE65R91W/R91W, murine models of retinal degeneration. This confirms the occurrence of photoreceptor integration in certain models of retinal degeneration and demonstrates the importance of the host environment in determining transplantation outcome.

Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration.

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases.

Gliosis Can Impede Integration Following Photoreceptor Transplantation into the Diseased Retina.

Retinal degenerations leading to the loss of photoreceptor (PR) cells are a major cause of vision impairment and untreatable blindness. There are few clinical treatments and none can reverse the loss of vision. With the rapid advances in stem cell biology and techniques in cell transplantation, PR replacement by transplantation represents a broad treatment strategy applicable to many types of degeneration. The number of donor cells that integrate into the recipient retina determines transplantation success, yet the degenerating retinae presents a number of barriers that can impede effective integration. Here, we briefly review recent advances in the field of PR transplantation. We then describe how different aspects of gliosis may impact on cell integration efficiency.

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.

Photoreceptor replacement therapy: challenges presented by the diseased recipient retinal environment.

Vision loss caused by the death of photoreceptors is the leading cause of irreversible blindness in the developed world. Rapid advances in stem cell biology and techniques in cell transplantation have made photoreceptor replacement by transplantation a very plausible therapeutic strategy. These advances include the demonstration of restoration of vision following photoreceptor transplantation and the generation of transplantable populations of donor cells from stem cells. In this review, we present a brief overview of the recent progress in photoreceptor transplantation. We then consider in more detail some of the challenges presented by the degenerating retinal environment that must play host to these transplanted cells, how these may influence transplanted photoreceptor cell integration and survival, and some of the progress in developing strategies to circumnavigate these issues.