Cyr61 activates retinal cells and prolongs photoreceptor survival in rd1 mouse model of retinitis pigmentosa

Subretinal injections with glial cell line‐derived neurotrophic factor (GDNF) rescue morphology as well as function of rod cells in mouse and rat animal models of retinitis pigmentosa. At the same time, it is postulated that this effect is indirect, mediated by activation of retinal Müller glial (RMG) cells. Here, we show that Cyr61/CCN1, one of the secreted proteins up‐regulated in primary RMG after glial cell line‐derived neurotrophic factor stimulation, provides neuroprotective and pro‐survival capacities: Recombinant Cyr61 significantly reduced photoreceptor (PR) cells death in organotypic cultures of Pde6b^rd1 retinas. To identify stimulated pathways in the retina, we treated Pde6b^rd1 retinal explants with Cyr61 and observed an overall increase in activated Erk1/2 and Stat3 signalling molecules characterized by activation‐site‐specific phosphorylation. To identify Cyr61 retinal target cells, we isolated primary porcine PR, RMG and retinal pigment epithelium (RPE) cells and exposed them separately to Cyr61. Here, RMG as well as RPE cells responded with induced phosphorylation of Erk1/2, Stat3 and Akt. In PR, no increase in phosphorylation in any of the studied proteins was detected, suggesting an indirect neuroprotective effect of Cyr61. Cyr61 may thus act as an endogenous pro‐survival factor for PR, contributing to the complex repertoire of neuroprotective activities generated by RMG and RPE cells.

     

PKG activity causes photoreceptor cell death in two retinitis pigmentosa models

Photoreceptor degeneration in retinitis pigmentosa is one of the leading causes of hereditary blindness in the developed world. Although causative genetic mutations have been elucidated in many cases, the underlying neuronal degeneration mechanisms are still unknown. Here, we show that activation of cGMP-dependent protein kinase (PKG) hallmarks photoreceptor degeneration in rd1 and rd2 human homologous mouse models. When induced in wild-type retinae, PKG activity was both necessary and sufficient to trigger cGMP-mediated photoreceptor cell death. Target-specific, pharmacological inhibition of PKG activity in both rd1 and rd2 retinae strongly reduced photoreceptor cell death in organotypic retinal explants. Likewise, inhibition of PKG in vivo, using three different application paradigms, resulted in robust photoreceptor protection in the rd1 retina. These findings suggest a pivotal role for PKG activity in cGMP-mediated photoreceptor degeneration mechanisms and highlight the importance of PKG as a novel target for the pharmacological intervention in RP.     

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.     

N-Acetylcysteine promotes long-term survival of cones in a model of retinitis pigmentosa

Retinitis pigmentosa (RP) is a major source of blindness caused by a large variety of mutations that lead to the death of rod photoreceptors. After rods die, cones gradually die from progressive oxidative damage. Several types of antioxidant formulations have been shown to reduce cone cell death over a relatively short-time frame, but in order for this strategy to be translated into a new treatment for patients with RP, prolonged effects will be needed. In this study, we determined that orally administered N-acetylcysteine (NAC) reduced cone cell death and preserved cone function by reducing oxidative damage in two models of RP, rd1(+/+) and rd10(+/+) mice. In rd10(+/+) mice, supplementation of drinking water with NAC promoted partial maintenance of cone structure and function for at least 6 months. Topical application of NAC to the cornea also reduced superoxide radicals in the retina and promoted survival and functioning of cones. Since oral and/or topical administration of NAC is feasible for long-term treatment in humans, and NAC has a good safety profile, it is reasonable to consider clinical trials to evaluate the effects of prolonged treatment with NAC in patients with RP.     

Ciliary neurotrophic factor blocks rod photoreceptor differentiation from postmitotic precursor cells in vitro

The development of photoreceptors in the mammalian retina is thought to be controlled by extrinsic signals. We have shown previously that ciliary neurotrophic factor (CNTF) potently inhibits photoreceptor differentiation in cultures of rat retina. The present study analyzes which developmental processes are affected by CNTF. Rod differentiation as determined by opsin and recoverin immunocytochemistry was effectively blocked by CNTF and leukemia inhibitory factor, but not by other neurotrophic agents tested. CNTF did not influence proliferation, cell death, or survival, and had no effect on the downregulation of nestin immunoreactivity in progenitor cells. Opsin-positive rods could be reverted to an opsin-negative state initially, but became unresponsive to CNTF later. No compensatory increase in the number of other cell types was observed. Application of neutralizing antibodies against CNTF revealed that rod development was partially blocked by an endogenous CNTF-like molecule in control cultures. Our results suggest that CNTF can act as a specific negative regulator of rod differentiation. Its action on photoreceptor precursor cells could serve to synchronize the maturation of photoreceptors, which are born over an extended period of time. Together with other stimulatory signals, CNTF may thus control the temporally and numerically correct integration of photoreceptors into the retinal network.     

PKM2 ablation enhanced retinal function and survival in a preclinical model of retinitis pigmentosa

Mammalian Genome - Retinitis pigmentosa (RP) is a neurodegenerative disorder that causes irreversible vision loss in over 1.5 million individuals world-wide. The genetic heterogeneity of RP...     

Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice

Autophagy is an essential recycling pathway implicated in neurodegeneration either as a pro-survival or a pro-death mechanism. Its role after axonal injury is still uncertain. Axotomy of the optic nerve is a classical model of neurodegeneration. It induces retinal ganglion cell death, a process also occurring in glaucoma and other optic neuropathies. We analyzed autophagy induction and cell survival following optic nerve transection (ONT) in mice. Our results demonstrate activation of autophagy shortly after axotomy with autophagosome formation, upregulation of the autophagy regulator Atg5 and apoptotic death of 50% of the retinal ganglion cells (RGCs) after 5 days. Genetic downregulation of autophagy using knockout mice for Atg4B (another regulator of autophagy) or with specific deletion of Atg5 in retinal ganglion cells, using the Atg5(flox/flox) mice reduces cell survival after ONT, whereas pharmacological induction of autophagy in vivo increases the number of surviving cells. In conclusion, our data support that autophagy has a cytoprotective role in RGCs after traumatic injury and may provide a new therapeutic strategy to ameliorate retinal diseases.     

Zinc-desferrioxamine attenuates retinal degeneration in the rd10 mouse model of retinitis pigmentosa

Iron-associated oxidative injury plays a role in retinal degeneration such as age-related macular degeneration and retinitis pigmentosa. The metallo-complex zinc-desferrioxamine (Zn/DFO) may ameliorate such injury by chelation of labile iron in combination with release of zinc. We explored whether Zn/DFO can affect the course of retinal degeneration in the rd10 mouse model of retinitis pigmentosa. Zn/DFO-treated animals showed significantly higher electroretinographic responses at 3 and 4.5 weeks of age compared with saline-injected controls. Corresponding retinal (photoreceptor) structural rescue was observed by quantitative histological and immunohistochemical techniques. When administered alone, the components of the complex, Zn and DFO, showed a lesser, partial effect. TBARS, a marker of lipid peroxidation, and levels of oxidative DNA damage as quantified by 8-OHdG immunostaining were significantly lower in Zn/DFO-treated retinas compared with saline-injected controls. Reduced levels of retinal ferritin as well as reduced iron content within ferritin molecules were measured in Zn/DFO-treated retinas. The data, taken together, suggest that the protective effects of the Zn/DFO complex are mediated through modulation of iron bioavailability, leading to attenuation of oxidative injury. Reducing iron-associated oxidative stress using complexes such as Zn/DFO may serve as a “common pathway” therapeutic approach to attenuate injury in retinal degeneration.     

Lysophosphatidylcholine acyltransferase 1 controls mitochondrial reactive oxygen species generation and survival of retinal photoreceptor cells

Due to their high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice bearing a frameshift mutation in the gene encoding lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration in mice; however, the mechanism by which this mutation affects degeneration is unclear. In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 not only decreased saturated PC production but also affected membrane lipid composition, presumably by altering saturated fatty acyl-CoA availability. Furthermore, we demonstrated that Lpcat1 deletion led to increased mitochondrial reactive oxygen species levels in photoreceptor cells, but not in other retinal cells, and did not affect the OS structure or trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC plays critical roles in photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration–related retinal diseases.     

A non‐canonical rhodopsin‐mediated insulin receptor signaling pathway in retinal photoreceptor neurons

We previously reported a ligand‐independent and rhodopsin‐dependent insulin receptor (IR) neuroprotective signaling pathway in both rod and cone photoreceptor cells, which is activated through protein–protein interaction. Our previous studies were performed with either retina or isolated rod or cone outer segment preparations and the expression of IR signaling proteins were examined. The isolation of outer segments with large portions of the attached inner segments is a technical challenge. Optiprep? density gradient medium has been used to isolate the cells and subcellular organelles, Optiprep? is a non‐ionic iodixanol‐based medium with a density of 1.320 g/mL. We employed this method to examine the expression of IR and its signaling proteins, and activation of one of the downstream effectors of the IR in isolated photoreceptor cells. Identification of the signaling complexes will be helpful for therapeutic targeting in disease conditions.     

Patterns of retinal light absorption related to retinitis pigmentosa mutants from in silico model structures of rhodopsin

Changes induced by mutations in rhodopsin that are associated with the degenerative visual disease retinitis pigmentosa result in an altered pattern of light absorption according to quantum mechanical simulations and reference experimental works. Eleven single-point mutations associated with retinitis pigmentosa at and in the proximity to the retinal binding pocket of rhodopsin have been modeled in silico and their spectra calculated with the NDOL (Neglect of Differential Overlap accounting L azimuthal quantum number) a priori method. The altered pattern of absorption found would lead to cumulative consequences in energy dissipation with aging. Different energy balances in the case of mutants at the very molecular level, compared to native nonmutated rhodopsin, can cause permanent cellular stress and would play a role in the progression of the retine degenerative process. It could explain the worsening of the pathological condition mostly in adults and suggests the probable beneficial effects of using quenching drugs and protection devices against excess of light in the early stages of life for avoiding or reducing potential damage.     

shRNA knockdown of guanylate cyclase 2e or cyclic nucleotide gated channel alpha 1 increases photoreceptor survival in a cGMP phosphodiesterase mouse model of retinitis pigmentosa

In vertebrate rods, dark and light conditions produce changes in guanosine 3′,5′‐cyclic monophosphate (cGMP) and calcium (Ca²⁺) levels, which are regulated by the opposing function of several proteins. During the recovery of a bright flash, guanylate cyclase (GUCY) helps raise cGMP to levels that open cGMP‐gated calcium sodium channels (CNG) to increase Na⁺ and Ca²⁺ influx in the outer segment. In contrast, light activates cGMP phosphodiesterase 6 (PDE6) causing rapid hydrolysis of cGMP, CNG closure, and reduced Na⁺ and Ca²⁺ levels. In Pde6b mouse models of retinitis pigmentosa (RP), photoreceptor death is preceded by abnormally high cGMP and Ca²⁺ levels, likely because of continued synthesis of cGMP by guanylate cyclases and unregulated influx of Ca²⁺ to toxic levels through CNG channels. To reverse the effects of Pde6b loss of function, we employed an shRNA knockdown approach to reduce the expression of Gucy2e or Cnga1 in Pde6b^H620Q photoreceptors prior to degeneration. Gucy2e‐ or Cnga1‐shRNA lentiviral‐mediated knockdown GUCY2E and CNGA1 expression increase visual function and photoreceptor survival in Pde6b^H620Q mice. We demonstrated that effective knockdown of GUCY2E and CNGA1 expression to counteract loss of PDE6 function may develop into a valuable approach for treating some patients with RP.     

Ceramide is the key mediator of oxidative stress-induced apoptosis in retinal photoreceptor cells

Nitric oxide and reactive oxygen species play a critical role in photoreceptor apoptosis. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. Here, we demonstrate that the sphingolipid ceramide is the key mediator of oxidative stress-induced apoptosis in 661W retinal photoreceptor cells. Treatment of 661W cells with the nitric oxide donor, sodium nitroprusside, activates acid sphingomyelinase. As a result, sphingomyelin is hydrolysed, which leads to an increase in the concentration of ceramide. We also show that ceramide is responsible for the activation of the mitochondrial apoptotic pathway in 661W photoreceptor cells and subsequent activation of the caspase cascade. Furthermore, we show for the first time that ceramide is responsible for the increased Ca2+ levels in the mitochondria and cytosol that precedes activation of the calpain-mediated apoptotic pathway. Additionally, we provide evidence that ceramide also activates the endolysosomal protease cathepsin D pathway. In summary, our findings show that ceramide controls the cell death decisions in photoreceptor cells and highlight the relevance of acid sphingomyelinase as a potential therapeutic target for the treatment of retinal pathologies.     

Enhancing survival of photoreceptor cells in vivo using the synthetic progestin Norgestrel

Retinal degenerations such as Retinitis Pigmentosa remain difficult to treat given the diverse array of genes responsible for their aetiology. Rather than concentrate on specific genes, our focus is on identifying therapeutic avenues for the treatment of retinal disease that target general survival mechanisms or pathways. Norgestrel is a synthetic progestin commonly used in hormonal contraception. Here, we report a novel anti-apoptotic role for Norgestrel in diseased mouse retinas in vivo. Dosing with Norgestrel protects photoreceptor cells from undergoing apoptosis in two distinct models of retinal degeneration; the light damage model and the Pde6b(rd10) model. Photoreceptor rescue was assessed by analysis of cell number, structural integrity and function. Improvements in cell survival of up to 70% were achieved in both disease models, indicating that apoptosis had been halted or at least delayed. A speculative mechanism of action for Norgestrel involves activation of survival pathways in the retina. Indeed, Norgestrel increases the expression of basic fibroblast growth factor which is known to both promote cell survival and inhibit apoptosis. In summary, our results demonstrate significant protection of photoreceptor cells which may be attributed to Norgestrel mediated activation of endogenous survival pathways within the retina.