Nonsense-mediated mRNA decay efficiency varies in choroideremia providing a target to boost small molecule therapeutics.

Choroideremia (CHM) is an x-linked recessive chorioretinal dystrophy, with 30% caused by nonsense mutations in the CHM gene resulting in an in-frame premature termination codon (PTC). Nonsense-mediated mRNA decay (NMD) is the cell's natural surveillance mechanism that detects and destroys PTC-containing transcripts, with UPF1 being the central NMD modulator. NMD efficiency can be variable amongst individuals with some transcripts escaping destruction, leading to the production of a truncated non-functional or partially functional protein. Nonsense suppression drugs, such as ataluren, target these transcripts and read-through the PTC, leading to the production of a full length functional protein. Patients with higher transcript levels are considered to respond better to these drugs, as more substrate is available for read-through. Using Quantitative reverse transcription PCR (RT-qPCR), we show that CHM mRNA expression in blood from nonsense mutation CHM patients is 2.8-fold lower than controls, and varies widely amongst patients, with 40% variation between those carrying the same UGA mutation [c.715 C>T; p.(R239*)]. These results indicate that although NMD machinery is at work, efficiency is highly variable and not wholly dependent on mutation position. No significant difference in CHM mRNA levels was seen between two patients' fibroblasts and their induced pluripotent stem cell-derived retinal pigment epithelium. There was no correlation between CHM mRNA expression and genotype, phenotype or UPF1 transcript levels. NMD inhibition with caffeine was shown to restore CHM mRNA transcripts to near wild-type levels. Baseline mRNA levels may provide a prognostic indicator for response to nonsense suppression therapy, and caffeine may be a useful adjunct to enhance treatment efficacy where indicated.

Vascular changes in diabetic retinopathy-a longitudinal study in the Nile rat.

Diabetic retinopathy is the most common microvascular complication of diabetes and is a major cause of blindness, but an understanding of the pathogenesis of the disease has been hampered by a lack of accurate animal models. Here, we explore the dynamics of retinal cellular changes in the Nile rat (Arvicanthis niloticus), a carbohydrate-sensitive model for type 2 diabetes. The early retinal changes in diabetic Nile rats included increased acellular capillaries and loss of pericytes that correlated linearly with the duration of diabetes. These vascular changes occurred in the presence of microglial infiltration but in the absence of retinal ganglion cell loss. After a prolonged duration of diabetes, the Nile rat also exhibits a spectrum of retinal lesions commonly seen in the human condition including vascular leakage, capillary non-perfusion, and neovascularization. Our longitudinal study documents a range and progression of retinal lesions in the diabetic Nile rat remarkably similar to those observed in human diabetic retinopathy, and suggests that this model will be valuable in identifying new therapeutic strategies.

Arl3 and RP2 regulate the trafficking of ciliary tip kinesins.

Ciliary trafficking defects are the underlying cause of many ciliopathies, including Retinitis Pigmentosa (RP). Anterograde intraflagellar transport (IFT) is mediated by kinesin motor proteins; however, the function of the homodimeric Kif17 motor in cilia is poorly understood, whereas Kif7 is known to play an important role in stabilizing cilia tips. Here we identified the ciliary tip kinesins Kif7 and Kif17 as novel interaction partners of the small GTPase Arl3 and its regulatory GTPase activating protein (GAP) Retinitis Pigmentosa 2 (RP2). We show that Arl3 and RP2 mediate the localization of GFP-Kif17 to the cilia tip and competitive binding of RP2 and Arl3 with Kif17 complexes. RP2 and Arl3 also interact with another ciliary tip kinesin, Kif7, which is a conserved regulator of Hedgehog (Hh) signaling. siRNA-mediated loss of RP2 or Arl3 reduced the level of Kif7 at the cilia tip. This was further validated by reduced levels of Kif7 at cilia tips detected in fibroblasts and induced pluripotent stem cell (iPSC) 3D optic cups derived from a patient carrying an RP2 nonsense mutation c.519C > T (p.R120X), which lack detectable RP2 protein. Translational read-through inducing drugs (TRIDs), such as PTC124, were able to restore Kif7 levels at the ciliary tip of RP2 null cells. Collectively, our findings suggest that RP2 and Arl3 regulate the trafficking of specific kinesins to cilia tips and provide additional evidence that TRIDs could be clinically beneficial for patients with this retinal degeneration.

Translational read-through of the RP2 Arg120stop mutation in patient iPSC-derived retinal pigment epithelium cells.

Mutations in the RP2 gene lead to a severe form of X-linked retinitis pigmentosa. RP2 patients frequently present with nonsense mutations and no treatments are currently available to restore RP2 function. In this study, we reprogrammed fibroblasts from an RP2 patient carrying the nonsense mutation c.519C>T (p.R120X) into induced pluripotent stem cells (iPSC), and differentiated these cells into retinal pigment epithelial cells (RPE) to study the mechanisms of disease and test potential therapies. RP2 protein was undetectable in the RP2 R120X patient cells, suggesting a disease mechanism caused by complete lack of RP2 protein. The RP2 patient fibroblasts and iPSC-derived RPE cells showed phenotypic defects in IFT20 localization, Golgi cohesion and Gß1 trafficking. These phenotypes were corrected by over-expressing GFP-tagged RP2. Using the translational read-through inducing drugs (TRIDs) G418 and PTC124 (Ataluren), we were able to restore up to 20% of endogenous, full-length RP2 protein in R120X cells. This level of restored RP2 was sufficient to reverse the cellular phenotypic defects observed in both the R120X patient fibroblasts and iPSC-RPE cells. This is the first proof-of-concept study to demonstrate successful read-through and restoration of RP2 function for the R120X nonsense mutation. The ability of the restored RP2 protein level to reverse the observed cellular phenotypes in cells lacking RP2 indicates that translational read-through could be clinically beneficial for patients.

Hsp90 inhibition protects against inherited retinal degeneration.

The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1(-/-) cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function.

Stem cells in retinal regeneration: past, present and future.

Stem cell therapy for retinal disease is under way, and several clinical trials are currently recruiting. These trials use human embryonic, foetal and umbilical cord tissue-derived stem cells and bone marrow-derived stem cells to treat visual disorders such as age-related macular degeneration, Stargardt's disease and retinitis pigmentosa. Over a decade of analysing the developmental cues involved in retinal generation and stem cell biology, coupled with extensive surgical research, have yielded differing cellular approaches to tackle these retinopathies. Here, we review these various stem cell-based approaches for treating retinal diseases and discuss future directions and challenges for the field.

Using induced pluripotent stem cells to understand retinal ciliopathy disease mechanisms and develop therapies.

The photoreceptor cells in the retina have a highly specialised sensory cilium, the outer segment (OS), which is important for detecting light. Mutations in cilia-related genes often result in retinal degeneration. The ability to reprogramme human cells into induced pluripotent stem cells and then differentiate them into a wide range of different cell types has revolutionised our ability to study human disease. To date, however, the challenge of producing fully differentiated photoreceptors in vitro has limited the application of this technology in studying retinal degeneration. In this review, we will discuss recent advances in stem cell technology and photoreceptor differentiation. In particular, the development of photoreceptors with rudimentary OS that can be used to understand disease mechanisms and as an important model to test potential new therapies for inherited retinal ciliopathies.

Array-based discovery of aptamer pairs.

Affinity reagent pairs that recognize distinct epitopes on a target protein can greatly improve the sensitivity and specificity of molecular detection. Importantly, such pairs can be conjugated to generate reagents that achieve two-site "bidentate" target recognition, with affinities greatly exceeding either monovalent component. DNA aptamers are especially well-suited for such constructs, because they can be linked via standard synthesis techniques without requiring chemical conjugation. Unfortunately, aptamer pairs are difficult to generate, primarily because conventional selection methods preferentially yield aptamers that recognize a dominant "hot spot" epitope. Our array-based discovery platform for multivalent aptamers (AD-MAP) overcomes this problem to achieve efficient discovery of aptamer pairs. We use microfluidic selection and high-throughput sequencing to obtain an enriched pool of aptamer sequences. Next, we synthesize a custom array based on these sequences, and perform parallel affinity measurements to identify the highest-affinity aptamer for the target protein. We use this aptamer to form complexes that block the primary binding site on the target, and then screen the same array with these complexes to identify aptamers that bind secondary epitopes. We used AD-MAP to discover DNA aptamer pairs that bind distinct sites on human angiopoietin-2 with high affinities, even in undiluted serum. To the best of our knowledge, this is the first work to discover new aptamer pairs using arrays. We subsequently conjugated these aptamers with a flexible linker to construct ultra-high-affinity bidentate reagents, with equilibrium dissociation constants as low as 97 pM: >200-fold better than either component aptamer. Functional studies confirm that both aptamers critically contribute to this ultrahigh affinity, highlighting the promise of such reagents for research and clinical use.

Replenishing IRAK-M expression in retinal pigment epithelium attenuates outer retinal degeneration.

Chronic inflammation is a constitutive component of many age-related diseases, including age-related macular degeneration (AMD). Here, we identified interleukin-1 receptor-associated kinase M (IRAK-M) as a key immunoregulator in retinal pigment epithelium (RPE) that declines during the aging process. Rare genetic variants of IRAK3, which encodes IRAK-M, were associated with an increased likelihood of developing AMD. In human samples and mouse models, IRAK-M abundance in the RPE declined with advancing age or exposure to oxidative stress and was further reduced in AMD. Irak3-knockout mice exhibited an increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M led to a disruption in RPE cell homeostasis, characterized by compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of adeno-associated virus (AAV)-expressing human IRAK3 rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in Irak3-knockout mice. Our data show that replenishment of IRAK-M in the RPE may redress dysregulated pro-inflammatory processes in AMD, suggesting a potential treatment for retinal degeneration.

Melanopsin contributions to irradiance coding in the thalamo-cortical visual system.

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a subset of retinal ganglion cells expressing the photopigment melanopsin (mRGCs). These mRGCs are known to drive such reflex light responses as circadian photoentrainment and pupillomotor movements. By contrast, until now there has been no direct assessment of their contribution to conventional visual pathways. Here, we address this deficit. Using new reporter lines, we show that mRGC projections are much more extensive than previously thought and extend across the dorsal lateral geniculate nucleus (dLGN), origin of thalamo-cortical projection neurons. We continue to show that this input supports extensive physiological light responses in the dLGN and visual cortex in mice lacking rods+cones (a model of advanced retinal degeneration). Moreover, using chromatic stimuli to isolate melanopsin-derived responses in mice with an intact visual system, we reveal strong melanopsin input to the ∼40% of neurons in the LGN that show sustained activation to a light step. We demonstrate that this melanopsin input supports irradiance-dependent increases in the firing rate of these neurons. The implication that melanopsin is required to accurately encode stimulus irradiance is confirmed using melanopsin knockout mice. Our data establish melanopsin-based photoreception as a significant source of sensory input to the thalamo-cortical visual system, providing unique irradiance information and allowing visual responses to be retained even in the absence of rods+cones. These findings identify mRGCs as a potential origin for aspects of visual perception and indicate that they may support vision in people suffering retinal degeneration.

Exposure to war and conflict: The individual and inherited epigenetic effects on health, with a focus on post-traumatic stress disorder.

War and conflict are global phenomena, identified as stress-inducing triggers for epigenetic modifications. In this state-of-the-science narrative review based on systematic principles, we summarise existing data to explore the outcomes of these exposures especially in veterans and show that they may result in an increased likelihood of developing gastrointestinal, auditory, metabolic and circadian issues, as well as post-traumatic stress disorder (PTSD). We also note that, despite a potential "healthy soldier effect", both veterans and civilians with PTSD exhibit the altered DNA methylation status in hypothalamic-pituitary-adrenal (HPA) axis regulatory genes such as NR3C1. Genes associated with sleep (PAX8; LHX1) are seen to be differentially methylated in veterans. A limited number of studies also revealed hereditary effects of war exposure across groups: decreased cortisol levels and a heightened (sex-linked) mortality risk in offspring. Future large-scale studies further identifying the heritable risks of war, as well as any potential differences between military and civilian populations, would be valuable to inform future healthcare directives.

Replenishing Age-Related Decline of IRAK-M Expression in Retinal Pigment Epithelium Attenuates Outer Retinal Degeneration.

Unchecked, chronic inflammation is a constitutive component of age-related diseases, including age-related macular degeneration (AMD). Here we identified interleukin-1 receptor-associated kinase (IRAK)-M as a key immunoregulator in retinal pigment epithelium (RPE) that declines with age. Rare genetic variants of IRAK-M increased the likelihood of AMD. IRAK-M expression in RPE declined with age or oxidative stress and was further reduced in AMD. IRAK-M-deficient mice exhibited increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M disrupted RPE cell homeostasis, including compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of AAV-expressing IRAK-M rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in IRAK-M-deficient mice. Our data support that replenishment of IRAK-M expression may redress dysregulated pro-inflammatory processes in AMD, thereby treating degeneration.

Spatiotemporal control of actomyosin contractility by MRCKß signaling drives phagocytosis.

Phagocytosis requires actin dynamics, but whether actomyosin contractility plays a role in this morphodynamic process is unclear. Here, we show that in the retinal pigment epithelium (RPE), particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3, triggering activation of MRCKß/myosin-II and its coeffector N-WASP, membrane deformation, and cup formation. Continued MRCKß/myosin-II activity then drives recruitment of a mechanosensing bridge, enabling cytoskeletal force transmission, cup closure, and particle internalization. In vivo, MRCKß is essential for RPE phagocytosis and retinal integrity. MerTK-independent activation of MRCKß signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. MRCKß is also required for efficient particle translocation from the cortex into the cell body in Fc receptor-mediated phagocytosis. Thus, conserved MRCKß signaling at the cortex controls spatiotemporal regulation of actomyosin contractility to guide distinct phases of phagocytosis in the RPE and represents the principle phagocytic effector pathway downstream of MerTK.

Investigation of PTC124-mediated translational readthrough in a retinal organoid model of AIPL1-associated Leber congenital amaurosis.

Leber congenital amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterized by severe sight impairment in infancy and rapidly progressing degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6) compared with control or CRISPR-corrected organoids. Increased levels of cGMP were detected. The translational readthrough-inducing drug (TRID) PTC124 was investigated as a potential therapeutic agent. LCA4 retinal organoids exhibited low levels of rescue of full-length AIPL1. However, this was insufficient to fully restore PDE6 in photoreceptors and reduce cGMP. LCA4 retinal organoids are a valuable platform for in vitro investigation of novel therapeutic agents.

Nidek MP1 is able to detect subtle decline in function in inherited and age-related atrophic macular disease with stable visual acuity.

PURPOSE: To investigate whether the Nidek MP1 microperimeter (NAVIS software Version 1.7; Nidek Technologies, Padua, Italy) can detect functional decline in progressive atrophic macular disease with stable visual acuity. METHODS: Nine eyes of nine patients with stable acuity but progressive inherited or age-related atrophic macular disease evident on fundus autofluorescence imaging were reviewed. Each patient underwent 3 consecutive microperimetry tests at baseline, 6 months, and 12 months. Acuity, fixation, and microperimetry tests were performed at each visit. Changes in acuity, fixation stability, and macular sensitivity were analyzed. To detect regional change in retinal sensitivity, the test grid was divided into clusters based on either topographical or functional features. The mean sensitivities within each zone were also compared across the three visits. RESULTS: In this cohort, there was no significant change in visual acuity, fixation stability, and macular sensitivity over 1 year. However, significant decline in mean sensitivity within the central macula and test loci adjacent to dense scotoma was found (P = 0.004 and 0.002, respectively). In contrast, mean sensitivity elsewhere remained stable. CONCLUSION: The MP1 can detect significant change in regional retinal sensitivity within 12 months in patients with progressive atrophic macular disease and stable acuity. Individualized analysis of regional sensitivity may be a useful method for quantifying microperimetry.

Molecular characterization and functional analysis of phagocytosis by human embryonic stem cell-derived RPE cells using a novel human retinal assay.

PURPOSE: To examine the ability of retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (HESC) to phagocytose photoreceptor outer segments, and to determine whether exposure to human retina induces any morphological changes in these cells. METHODS: HESC-RPE cells were derived from a super-confluent preparation of the Shef1 HESC line. Pigmented colonies were isolated and expanded into pigmented monolayers on Matrigel matrix-coated dishes or filters. Cells were exposed to fluorescently labeled outer segments isolated from the porcine eye and assessed for phagocytic activity at regular intervals. Expression of molecules associated with RPE phagocytosis was analyzed by RT-PCR, immunocytochemistry, and western blot. The role of Mer Tyrosine Kinase (MERTK) in the phagocytosis of outer segments was investigated using antibodies directed against MERTK to block function. In a novel approach, cells were also exposed to fresh human neural retina tissue then examined by electron microscopy for evidence of phagocytosis and changes in cell morphology. RESULTS: HESC-derived RPE cells are capable of phagocytosing isolated porcine outer segments and express molecules associated with RPE-specific phagocytosis, including MERTK. Pre-incubation with antibodies against MERTK blocked phagocytosis of photoreceptor outer segments, but not polystyrene beads. HESC-RPE cells also phagocytosed outer segments in a novel human retinal explant system. Furthermore co-culture adjacent to human retina tissue in this preparation resulted in the appearance of features in HESC-derived RPE cells normally observed only as the RPE matures. CONCLUSIONS: The ingestion of photoreceptor outer segments from an isolated population and an artificial ex vivo human retina system demonstrates HESC-derived RPE cells are functional. HESC-derived RPE possess the relevant molecules required for phagocytosis, including MERTK, which is essential for the phagocytosis of outer segments but not latex beads. Furthermore, some changes observed in cell morphology after co-culture with human retina may have implications for understanding the full development and differentiation of RPE cells.

Long-term visual and microperimetry outcomes following autologous retinal pigment epithelium choroid graft for neovascular age-related macular degeneration.

BACKGROUND: To describe the 2- to 4-year visual and microperimetry outcomes of autologous retinal pigment epithelium (RPE)-choroid graft in patients with neovascular age-related macular degeneration (AMD). METHODS: In this retrospective cohort study, 12 patients with subfoveal neovascular AMD who had undergone autologous RPE-choroid graft between August 2004 and June 2005 were reviewed. Change in visual acuity (VA), contrast sensitivity (CS), fixation stability and retinal sensitivity on microperimetry after 2-3 years and the rates of late postoperative complications were examined. RESULTS: Patients were followed for 26-48 months (mean, 39). Median preoperative VA (logMAR) was 0.87 but declined to 1.43 (1 year), 1.46 (2 years) and 1.38 (3 years), P = 0.001. Median CS (logCS) was 0.75 preoperatively but declined to 0.45 at 2 years. Six patients had serial microperimetry. Fixation stability declined in 1 but improved in 2 patients. All 6 had decline in retinal sensitivity over the graft during follow up. Retinal detachment did not occur after 12 months but 8 developed epiretinal membrane, 12 had cystic retinal change over the graft and 4 developed recurrent choroidal neovascularization. However, 10 grafts retained autofluorescence signal at 18-48 months of follow up. CONCLUSIONS: Autologous RPE-choroid graft can maintain VA, stable fixation and retinal sensitivity in some patients for over 3 years. The spatial correlation between graft autofluorescence, outer retinal structures on optical coherence tomography and retinal sensitivity are consistent with photoreceptor cell rescue. However, we caution the use of this technique as there is high complication rate and delayed loss of retinal function.