Knockdown of thioredoxin interacting protein in Müller cells attenuates photoreceptor apoptosis in streptozotocin-induced diabetic mouse model.

We explored the effect of inhibition of thioredoxin interacting protein (Txnip) on neuroprotection in Müller cells under high glucose. Wild-type (WT) and Txnip knockout (Txnip-/-) mice were used to establish a streptozotocin (STZ)-induced diabetes model and a Müller cells high glucose model. We detected BDNF expression and PI3K/AKT/CREB pathway activation levels in the retina and Müller cells of each group in vivo and in vitro experiments. The Txnip-/- STZ group showed higher expression of BDNF and phosphorylation of PI3K/AKT/CREB in retina, and less retinal photoreceptor apoptosis was observed in Txnip-/- diabetic group than in WT. After using an inhibitor of PI3K signaling pathway, BDNF expression was reduced; In vitro co-cultured with Müller cells in different groups, 661 W cells showed different situations, Txnip-/- Müller cells maximum downregulated Cleaved-caspase 3 expression in 661 W, accompanied by an increase in Bcl-2/Bax ratio. These findings indicate that inhibiting endogenous Txnip in mouse Müller cells can promote their expression and secretion of BDNF, thereby reducing HG induced photoreceptor apoptosis and having important neuroprotective effects on DR. The regulation of BDNF expression by Txnip may be achieved by activating the PI3K/AKT/CREB pathway. This study suggests that regulating Txnip may be a potential target for DR treatment.

Gene Augmentation for Autosomal Dominant CRX-Associated Retinopathies.

The cone-rod homeobox (CRX) protein is a key transcription factor essential for photoreceptor function and survival. Mutations in human CRX gene are linked to a wide spectrum of blinding diseases ranging from mild macular dystrophy to severe Leber congenital amaurosis (LCA), cone-rod dystrophy (CRD), and retinitis pigmentosa (RP). These diseases are still incurable and mostly inherited in an autosomal dominant form. Dysfunctional mutant CRX protein interferes with the function of wild-type CRX protein, demonstrating the dominant negative effect. At present, gene augmentation is the most promising treatment strategy for hereditary diseases. This study aims to review the pathogenic mechanisms of various CRX mutations and propose two therapeutic strategies to rescue sick photoreceptors in CRX-associated retinopathies, namely, Tet-On-hCRX system and adeno-associated virus (AAV)-mediated gene augmentation. The outcome of proposed studies will guide future translational research and suggest guidelines for therapy evaluation in terms of treatment safety and efficacy.

Retinitis Pigmentosa: Progress in Molecular Pathology and Biotherapeutical Strategies.

Retinitis pigmentosa (RP) is genetically heterogeneous retinopathy caused by photoreceptor cell death and retinal pigment epithelial atrophy that eventually results in blindness in bilateral eyes. Various photoreceptor cell death types and pathological phenotypic changes that have been disclosed in RP demand in-depth research of its pathogenic mechanism that may account for inter-patient heterogeneous responses to mainstream drug treatment. As the primary method for studying the genetic characteristics of RP, molecular biology has been widely used in disease diagnosis and clinical trials. Current technology iterations, such as gene therapy, stem cell therapy, and optogenetics, are advancing towards precise diagnosis and clinical applications. Specifically, technologies, such as effective delivery vectors, CRISPR/Cas9 technology, and iPSC-based cell transplantation, hasten the pace of personalized precision medicine in RP. The combination of conventional therapy and state-of-the-art medication is promising in revolutionizing RP treatment strategies. This article provides an overview of the latest research on the pathogenesis, diagnosis, and treatment of retinitis pigmentosa, aiming for a convenient reference of what has been achieved so far.

PKM2 Is a Potential Diagnostic and Therapeutic Target for Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a major cause of blindness that is difficult to diagnose and treat. PKM2, a subtype of pyruvate kinase, is strongly associated with oxidative stress and is expressed in photoreceptors. We investigated whether PKM2 reduces photoreceptor cell apoptosis and evaluated possible antiapoptotic mechanisms in RP. We established RP models by exposing 661W cells to blue light and modulated PKM2 activity using a PKM2 inhibitor. We measured the apoptosis rates using calcein-acetoxymethyl ester/propidium iodide double staining and Cell Counting Kit-8, the oxidative stress levels using a reactive oxygen species assay, and the changes in protein expression by western blotting. Photodamage increased PKM2 expression, cellular oxidative stress, and apoptosis of 661W cells. PKM2 inhibition significantly reduced the levels of apoptosis and oxidative stress induced by photodamage. Our data suggest that PKM2 is a potential disease marker and therapeutic target for RP.

Selecting Channelrhodopsin Constructs for Optimal Visual Restoration in Differing Light Conditions.

Channelrhodopsin (ChR)-based optogenetics is one promising approach to restore vision in photoreceptor degenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Currently, a large number of ChRs from different alga species as well as engineered variants are available. They vary with their light response properties like peak sensitive wavelength (λmax), current amplitude, and kinetics. Therefore, it is important to choose an appropriate ChR for practical applications, such as vision restoration. Here we describe a standard laboratory protocol for characterizing properties of ChRs in in vitro in human embryonic kidney (HEK) cells. Based on such characterization, we also discuss the criteria for selecting optimal ChRs for optogenetic vision restoration.

An allosteric interleukin-1 receptor modulator mitigates inflammation and photoreceptor toxicity in a model of retinal degeneration.

BACKGROUND: Inflammation and particularly interleukin-1ß (IL-1ß), a pro-inflammatory cytokine highly secreted by activated immune cells during early AMD pathological events, contribute significantly to retinal neurodegeneration. Here, we identify specific cell types that generate IL-1ß and harbor the IL-1 receptor (IL-1R) and pharmacologically validate IL-1ß's contribution to neuro-retinal degeneration using the IL-1R allosteric modulator composed of the amino acid sequence rytvela (as well as the orthosteric antagonist, Kineret) in a model of blue light-induced retinal degeneration. METHODS: Mice were exposed to blue light for 6 h and sacrificed 3 days later. Mice were intraperitoneally injected with rytvela, Kineret, or vehicle twice daily for 3 days. The inflammatory markers F4/80, NLRP3, caspase-1, and IL-1ß were assessed in the retinas. Single-cell RNA sequencing was used to determine the cell-specific expression patterns of retinal Il1b and Il1r1. Macrophage-induced photoreceptor death was assessed ex vivo using retinal explants co-cultured with LPS-activated bone marrow-derived macrophages. Photoreceptor cell death was evaluated by the TUNEL assay. Retinal function was assessed by flash electroretinography. RESULTS: Blue light markedly increased the mononuclear phagocyte recruitment and levels of inflammatory markers associated with photoreceptor death. Co-localization of NLRP3, caspase-1, and IL-1ß with F4/80+ mononuclear phagocytes was clearly detected in the subretinal space, suggesting that these inflammatory cells are the main source of IL-1ß. Single-cell RNA sequencing confirmed the immune-specific expression of Il1b and notably perivascular macrophages in light-challenged mice, while Il1r1 expression was found primarily in astrocytes, bipolar, and vascular cells. Retinal explants co-cultured with LPS/ATP-activated bone marrow-derived macrophages displayed a high number of TUNEL-positive photoreceptors, which was abrogated by rytvela treatment. IL-1R antagonism significantly mitigated the inflammatory response triggered in vivo by blue light exposure, and rytvela was superior to Kineret in preserving photoreceptor density and retinal function. CONCLUSION: These findings substantiate the importance of IL-1ß in neuro-retinal degeneration and revealed specific sources of Il1b from perivascular MPs, with its receptor Ilr1 being separately expressed on surrounding neuro-vascular and astroglial cells. They also validate the efficacy of rytvela-induced IL-1R modulation in suppressing detrimental inflammatory responses and preserving photoreceptor density and function in these conditions, reinforcing the rationale for clinical translation.

Retina Organoid Transplants Develop Photoreceptors and Improve Visual Function in RCS Rats With RPE Dysfunction.

Purpose: To study if human embryonic stem cell-derived photoreceptors could survive and function without the support of retinal pigment epithelium (RPE) after transplantation into Royal College of Surgeons rats, a rat model of retinal degeneration caused by RPE dysfunction. Methods: CSC14 human embryonic stem cells were differentiated into primordial eye structures called retinal organoids. Retinal organoids were analyzed by quantitative PCR and immunofluorescence and compared with human fetal retina. Retinal organoid sheets (30-70 day of differentiation) were transplanted into immunodeficient RCS rats, aged 44 to 56 days. The development of transplant organoids in vivo in relation to the host was examined by optical coherence tomography. Visual function was assessed by optokinetic testing, electroretinogram, and superior colliculus electrophysiologic recording. Cryostat sections were analyzed for various retinal, synaptic, and donor markers. Results: Retinal organoids showed similar gene expression to human fetal retina transplanted rats demonstrated significant improvement in visual function compared with RCS nonsurgery and sham surgery controls by ERGs at 2 months after surgery (but not later), optokinetic testing (up to 6 months after surgery) and electrophysiologic superior colliculus recordings (6-8 months after surgery). The transplanted organoids survived more than 7 months; developed photoreceptors with inner and outer segments, and other retinal cells; and were well-integrated within the host. Conclusions: This study, to our knowledge, is the first to show that transplanted photoreceptors survive and function even with host's dysfunctional RPE. Our findings suggest that transplantation of organoid sheets from stem cells may be a promising approach/therapeutic for blinding diseases.

Shihu Yeguang Pill protects against bright light-induced photoreceptor degeneration in part through suppressing photoreceptor apoptosis.

Photoreceptor cells are first-order retinal neurons that directly contribute to the formation of vision. Photoreceptor degeneration is the primary cause of vision impairment during the course of retinopathies such as retinitis pigmentosa and age-related macular degeneration, for which photoreceptor-targeted therapies are currently unavailable. Shihu Yeguang Pill (SYP), a classic formula in traditional Chinese medicine, has a long histology of clinical application for the treatment of a wide range of retinopathies in China. However, whether SYP is pharmacological effective at protecting photoreceptor cells is unclear. The current study thus directly addressed the pharmacological implications of SYP in photoreceptor degeneration in a mouse model characterized by bright light-induced retinal degeneration. Non-invasive full-retinal assessment was carried out to evaluate the effect of SYP on the retinal structure and function through optical coherence tomography and electroretinography, respectively. In addition, photoreceptor apoptosis, second-order neuron impairment and reactive changes in retinal microglial and müller cells, hallmark pathologies associated with photoreceptor degeneration, were assessed using immunohistochemistry and real-time PCR analyses. The results showed that SYP treatment attenuated bright light-induced impairment of the retinal structure and function. Moreover, SYP treatment suppressed photoreceptor apoptosis, alleviated the impairment of bipolar and horizontal cells and mitigated the reactive changes of müller and microglial cells in the bright light-exposed retinas. Real-time PCR analyses showed that dysregulated expression of pro-apoptotic c-fos and c-jun and anti-apoptotic bcl-2 as well as proinflammatory TNF-α in the bright light-exposed retinas was partially normalized as a result of SYP treatment. In summary, the work here demonstrates for the first time that SYP treatment protects the retinas from developing bright light-induced photoreceptor degeneration and associated alterations in second-order neurons and glial cells. The findings here thus provide experimental evidence to better support the mechanism-guided clinical application of SYP in the treatment of related retinal degenerative diseases.

Automated Quantification of Photoreceptor alteration in macular disease using Optical Coherence Tomography and Deep Learning.

Diabetic macular edema (DME) and retina vein occlusion (RVO) are macular diseases in which central photoreceptors are affected due to pathological accumulation of fluid. Optical coherence tomography allows to visually assess and evaluate photoreceptor integrity, whose alteration has been observed as an important biomarker of both diseases. However, the manual quantification of this layered structure is challenging, tedious and time-consuming. In this paper we introduce a deep learning approach for automatically segmenting and characterising photoreceptor alteration. The photoreceptor layer is segmented using an ensemble of four different convolutional neural networks. En-face representations of the layer thickness are produced to characterize the photoreceptors. The pixel-wise standard deviation of the score maps produced by the individual models is also taken to indicate areas of photoreceptor abnormality or ambiguous results. Experimental results showed that our ensemble is able to produce results in pair with a human expert, outperforming each of its constitutive models. No statistically significant differences were observed between mean thickness estimates obtained from automated and manually generated annotations. Therefore, our model is able to reliable quantify photoreceptors, which can be used to improve prognosis and managment of macular diseases.

Small molecule activation of metabolic enzyme pyruvate kinase muscle isozyme 2, PKM2, circumvents photoreceptor apoptosis.

Photoreceptor cell death is the ultimate cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. Similar to cancer cells, photoreceptors maintain pyruvate kinase muscle isoform 2 (PKM2) expression, which is a critical regulator in aerobic glycolysis. Unlike PKM1, which has constitutively high catalytic activity, PKM2 is under complex regulation. Recently, we demonstrated that genetically reprogramming photoreceptor metabolism via PKM2-to-PKM1 substitution is a promising neuroprotective strategy. Here, we explored the neuroprotective effects of pharmacologically activating PKM2 via ML-265, a small molecule activator of PKM2, during acute outer retinal stress. We found that ML-265 increased PKM2 activity in 661 W cells and in vivo in rat eyes without affecting the expression of genes involved in glucose metabolism. ML-265 treatment did, however, alter metabolic intermediates of glucose metabolism and those necessary for biosynthesis in cultured cells. Long-term exposure to ML-265 did not result in decreased photoreceptor function or survival under baseline conditions. Notably, though, ML-265-treatment did reduce entrance into the apoptotic cascade in in vitro and in vivo models of outer retinal stress. These data suggest that reprogramming metabolism via activation of PKM2 is a novel, and promising, therapeutic strategy for photoreceptor neuroprotection.

New Nrf2-Inducer Compound ITH12674 Slows the Progression of Retinitis Pigmentosa in the Mouse Model rd10.

BACKGROUND/AIMS: It is well established that oxidative stress and inflammation are common pathogenic features of retinal degenerative diseases. ITH12674 is a novel compound that induces the transcription factor Nrf2; in so doing, the molecule exhibits anti-inflammatory, and antioxidant properties, and affords neuroprotection in rat cortical neurons subjected to oxidative stress. We here tested the hypothesis that ITH12674 could slow the retinal degeneration that causes blindness in rd10 mice, a model of retinitis pigmentosa. METHODS: Animals were intraperitoneally treated with 1 or 10 mg/Kg ITH12674 or placebo from P16 to P30. At P30, retinal functionality and visual acuity were analyzed by electroretinography and optomotor test. By immunohistochemistry we quantified the photoreceptor rows and analyzed their morphology and connectivity. Oxidative stress and inflammatory state was studied by Western blot, and microglia reactivity was monitored by flow cytometry. The blood-brain barrier permeation of ITH12674 was evaluated using a PAMPA-BBB assay. RESULTS: In rd10 mice treated with 10 mg/Kg of the compound, the following changes were observed (with respect to placebo): (i) a decrease of vision loss with higher scotopic a- and b-waves; (ii) increased visual acuity; (iii) preservation of cone photoreceptors morphology, as well as their synaptic connectivity; (iv) reduced expression of TNF-α and NF-κB; (v) increased expression of p38 MAPK and Atg12-Atg5 complex; and (vi) decreased CD11c, MHC class II and CD169 positive cell populations. CONCLUSION: These data support the view that a Nrf2 inducer compound may arise as a new therapeutic strategy to combat retinal neurodegeneration. At present, we are chemically optimising compound ITH12674 with the focus on improving its neuroprotective potential in retinal neurodegenerative diseases.

Caspase-1-dependent inflammasomes mediate photoreceptor cell death in photo-oxidative damage-induced retinal degeneration.

Activation of the inflammasome is involved in the progression of retinal degenerative diseases, in particular, in the pathogenesis of Age-Related Macular Degeneration (AMD), with NLRP3 activation the focus of many investigations. In this study, we used genetic and pharmacological approaches to explore the role of the inflammasome in a mouse model of retinal degeneration. We identify that Casp1/11-/- mice have better-preserved retinal function, reduced inflammation and increased photoreceptor survivability. While Nlrp3-/- mice display some level of preservation of retinal function compared to controls, pharmacological inhibition of NLRP3 did not protect against photoreceptor cell death. Further, Aim2-/-, Nlrc4-/-, Asc-/-, and Casp11-/- mice show no substantial retinal protection. We propose that CASP-1-associated photoreceptor cell death occurs largely independently of NLRP3 and other established inflammasome sensor proteins, or that inhibition of a single sensor is not sufficient to repress the inflammatory cascade. Therapeutic targeting of CASP-1 may offer a more promising avenue to delay the progression of retinal degenerations.

Photoreceptor morphology and correlation with subretinal fluid chronicity associated with choroidal nevus.

BACKGROUND: Subretinal fluid (SRF) can be associated with choroidal nevus and can cause progressive change in the morphology of overlying photoreceptors. METHODS: A retrospective observational study was performed using optical coherence tomography to assess nevus and SRF features, as well as photoreceptor morphology over time. RESULTS: There were 232 choroidal nevi that presented with or developed SRF. Photoreceptor morphology at presentation was classified as normal (n=60, 26%), shaggy (elongated) (n=73, 31%), retracted (stalactite appearance) (n=76, 33%), or absent (n=23, 10%). There was a progression in photoreceptor morphology with increasing SRF chronicity (p=0.003). For nevus presenting with normal photoreceptors and later developed SRF (n=60), photoreceptors became shaggy in 29 (48%), retracted in 24 (40%), and absent in 7 (12%) after 15, 19 and 22 months, respectively. For nevus presenting with SRF and shaggy photoreceptors (n=73), progression to retracted photoreceptors occurred in 31 (42%) after a mean of 22 months; for nevus with SRF and retracted photoreceptors (n=76), progression to absent photoreceptors occurred in 19 (25%) after a mean of 34 months; and for nevus with absent photoreceptors (n=23), photoreceptor morphology showed no change after mean follow-up of 33 months. Risk of nevus growth to melanoma was not associated with photoreceptor morphology at presentation (p=0.19). CONCLUSION: In eyes with choroidal nevus and SRF, there is a longitudinal evolution in photoreceptor morphology from normal to shaggy to retracted then absent with increasing SRF chronicity. SRF chronicity, as indicated by photoreceptor morphology on presentation, did not correlate with nevus growth to melanoma.

Therapeutic effects of mesenchymal stem cell-derived exosomes on retinal detachment.

Retinal detachment (RD) induces ischemia and oxygen deficiency in the retina and results in multiple pathological events; photoreceptor cell degeneration and death is the eventual cause of vision decline. In this study, we investigated the therapeutic effects of mesenchymal stem cell-derived exosomes (MSC-Exos) in a rat retinal detachment (RD) model. The model was developed using a subretinal injection of 1% hyaluronic acid in male Sprague-Dawley rats. MSC-Exos were sub-retinally injected at the time of retinal separation to study their therapeutic function. The retinal expression levels of inflammatory cytokines TNF-α, IL-1ß, and MCP-1 were detected by RT-PCR, the autophagy-related protein 5 (Atg5) and microtubule-associated protein 1 light chain 3 beta (LC3) were detected by Western blot, and apoptosis was examined using TUNEL assays at 3 days following RD. Retinal structure was observed at 7 days post-RD. Proteomic analysis was also performed to detect proteins carried by MSC-Exos using iTRAQ-based technology combined with one-dimensional nano LC-nano-ESI- MS/MS. We found that expression of TNF-α and IL-1ß were significantly reduced, the LC3-II to LC3-I ratio was enhanced and cleavage of Atg5 was decreased after MSC-Exo treatment. Treatment with MSC-Exos also suppressed photoreceptor cell apoptosis and maintained normal retinal structure when compared to control groups. Proteomic analysis revealed that MSC-Exos contained proteins with anti-inflammatory, neuroprotective and anti-apoptotic effects. These results suggest that MSC-Exos have therapeutic effects on RD-induced retinal injury and can be used to reduce effects of retinal detachment on photoreceptor cell degeneration in patients.

Autophagy, lysosome dysfunction and mTOR inhibition in MNU-induced photoreceptor cell damage.

Progressive photoreceptor death is the main cause of retinal degeneration diseases. Determining the underlying mechanism of this process is essential for therapy improvement. Autophagy has long been considered to be involved in neuronal degeneration diseases, and the regulation of autophagy is thought to have potential implications for neurodegenerative disease therapies. However, whether autophagy is protective or destructive varies among diseases and is controversial. In the present study, we established an N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell damage model in vitro that faithfully replicated photoreceptor cell death in retinal degeneration diseases. Cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays. Reactive oxygen species (ROS) levels were assessed through 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence. Autophagy was confirmed by observing autophagosomes using transmission electron microscopy (TEM). A lysosome tracker was used to identify acidic lysosomes in cells. We also measured the expression of some proteins related to autophagy, apoptosis and lysosomal degradation by western blot and immunofluorescence assays. We found that MNU could decrease photoreceptor cell viability in a time- and dose-dependent manner, and this change was accompanied by concomitant increases in ROS and the expression of the apoptosis-inducing protein cleaved caspase-3. Moreover, autophagy was activated by MNU treatment during this process. Inhibition of autophagy with 3-methyladenine accelerated cell damage. Lysosome dysfunction was confirmed by autophagosome enlargement and increased cathepsin expression, which was accompanied by mTOR dephosphorylation. In conclusion, autophagy was activated through inhibition of the PI3K/mTOR pathway in the context of MNU-induced photoreceptor cell death. Prolonged mTOR dephosphorylation and autophagy activation resulted in autophagic vacuole accumulation, as indicated by inefficient degradation in lysosomes, and further led to apoptosis.

Treatment with Stem Cells from Human Exfoliated Deciduous Teeth and Their Derived Conditioned Medium Improves Retinal Visual Function and Delays the Degeneration of Photoreceptors.

Retinitis pigmentosa (RP) is a hereditary disease characterized by degeneration and the loss of photoreceptors. Stem cell-based therapy has emerged as a promising strategy for treating RP. Stem cells from exfoliated deciduous teeth (SHEDs), a type of mesenchymal stem cell from human exfoliated deciduous teeth, have the potential to differentiate into photoreceptor-like cells under specific induction in vitro. It has been confirmed that through paracrine secreta, SHEDs exert neurotrophic, angiogenic, immunoregulatory, and antiapoptotic functions in injured tissues. This study was designed to determine whether retinal-differentiated SHEDs and the conditioned medium derived from SHEDs (SHEDs-CM) have therapeutic effects in a mouse model of RP. The results showed that both SHEDs and SHEDs-CM improved electroretinogram responses, ameliorated photoreceptor degeneration, and maintained the structure of the outer segments of photoreceptors. The therapeutic effects were related to antiapoptotic activity of SHEDs and SHEDs-CM. Thus, SHEDs may be a promising stem cell source for treating retinal degeneration.

Adeno-Associated Viral Vectors as a Tool for Large Gene Delivery to the Retina.

Gene therapy using adeno-associated viral (AAV) vectors currently represents the most promising approach for the treatment of many inherited retinal diseases (IRDs), given AAV's ability to efficiently deliver therapeutic genes to both photoreceptors and retinal pigment epithelium, and their excellent safety and efficacy profiles in humans. However, one of the main obstacles to widespread AAV application is their limited packaging capacity, which precludes their use from the treatment of IRDs which are caused by mutations in genes whose coding sequence exceeds 5 kb. Therefore, in recent years, considerable effort has been made to identify strategies to increase the transfer capacity of AAV vectors. This review will discuss these new developed strategies, highlighting the advancements as well as the limitations that the field has still to overcome to finally expand the applicability of AAV vectors to IRDs due to mutations in large genes.

FREQUENT SUBCLINICAL MACULAR CHANGES IN COMBINED BRAF/MEK INHIBITION WITH HIGH-DOSE HYDROXYCHLOROQUINE AS TREATMENT FOR ADVANCED METASTATIC BRAF MUTANT MELANOMA: Preliminary Results From a Phase I/II Clinical Treatment Trial.

PURPOSE: To assess the potential ocular toxicity of a combined BRAF inhibition (BRAFi) + MEK inhibition (MEKi) + hydroxychloroquine (HCQ) regime used to treat metastatic BRAF mutant melanoma. METHODS: Patients with stage IV metastatic melanoma and BRAF V600E mutations (n = 11, 31-68 years of age) were included. Treatment was with oral dabrafenib, 150 mg bid, trametinib, 2 mg/day, and HCQ, 400 mg to 600 mg bid. An ophthalmic examination, spectral domain optical coherence tomography, near-infrared and short-wavelength fundus autofluorescence, and static perimetry were performed at baseline, 1 month, and q/6 months after treatment. RESULTS: There were no clinically significant ocular events; there was no ocular inflammation. The only medication-related change was a separation of the photoreceptor outer segment tip from the apical retinal pigment epithelium that could be traced from the fovea to the perifoveal retina noted in 9/11 (82%) of the patients. There were no changes in retinal pigment epithelium melanization or lipofuscin content by near-infrared fundus autofluorescence and short-wavelength fundus autofluorescence, respectively. There were no inner retinal or outer nuclear layer changes. Visual acuities and sensitivities were unchanged. CONCLUSION: BRAFi (trametinib) + MEKi (dabrafenib) + HCQ causes very frequent, subclinical separation of the photoreceptor outer segment from the apical retinal pigment epithelium without inner retinal changes or signs of inflammation. The changes suggest interference with the maintenance of the outer retinal barrier and/or phagocytic/pump functions of the retinal pigment epithelium by effective MEK inhibition.

In Vitro and In Vivo Proteomic Comparison of Human Neural Progenitor Cell-Induced Photoreceptor Survival.

Retinal degenerative diseases lead to blindness with few treatments. Various cell-based therapies are aimed to slow the progression of vision loss by preserving light-sensing photoreceptor cells. A subretinal injection of human neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) rat model of retinal degeneration has aided in photoreceptor survival, though the mechanisms are mainly unknown. Identifying the retinal proteomic changes that occur following hNPC treatment leads to better understanding of neuroprotection. To mimic the retinal environment following hNPC injection, a co-culture system of retinas and hNPCs is developed. Less cell death occurs in RCS retinal tissue co-cultured with hNPCs than in retinas cultured alone, suggesting that hNPCs provide retinal protection in vitro. Comparison of ex vivo and in vivo retinas identifies nuclear factor (erythroid-derived 2)-like 2 (NRF2) mediated oxidative response signaling as an hNPC-induced pathway. This is the first study to compare proteomic changes following treatment with hNPCs in both an ex vivo and in vivo environment, further allowing the use of ex vivo modeling for mechanisms of retinal preservation. Elucidation of the protein changes in the retina following hNPC treatment may lead to the discovery of mechanisms of photoreceptor survival and its therapeutic for clinical applications.

High-Temperature Requirement A 1 Causes Photoreceptor Cell Death in Zebrafish Disease Models.

Age-related macular degeneration (AMD) is an important cause of blindness. It is characterized by a retinal pigment epithelium (RPE) disorder that leads to death of photoreceptor cells (PRCs). AMD has a strong genetic association with high-temperature requirement A 1 (HTRA1). The relationship between HTRA1 and the AMD phenotype is unknown. In this study, we show that the expression of HTRA1 in PRCs, as well as in RPE, is increased by the disease-associated HTRA1 mutation and aging. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and quantitative PCR of apoptosis-associated caspases confirmed that PRC-specific overexpression of HTRA1 induced PRC death. Transgenic zebrafish overexpressing human HTRA1 in rod PRCs showed morphologic changes of the RPE, including PRC death and lipofuscin accumulation, features similar to those of early AMD. htra1 expression was also increased in a retinitis pigmentosa zebrafish model compared with wild type. In both fish lines, PRC death was rescued by the suppression of htra1 by the inhibitor 6-boroV. AKT-forkhead box O3 signaling downstream of HTRA1 was activated via a tumor growth factor ß signal, resulting in PRC death. These findings suggest that HTRA1 derived from PRCs is associated with early AMD via PRC death. HTRA1 is a potentially effective target for neuroprotective therapy of early AMD and other degenerative diseases of PRCs.