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Retinal ganglion cell (RGC) damage serves as a key indicator of various retinal degenerative diseases, including diabetic retinopathy (DR), glaucoma, retinal arterial and retinal vein occlusions, as well as inflammatory and traumatic optic neuropathies. Despite the growing body of data on the RGC proteomics associated with these conditions, there has been no dedicated study conducted to compare the molecular signaling pathways involved in the mechanism of neuronal cell death. Therefore, we launched the study using two different insults leading to RGC death: glutamate excitotoxicity and optic nerve crush (ONC). C57BL/6 mice were used for the study and underwent NMDA- and ONC-induced damage. Twenty-four hours after ONC and 1 h after NMDA injection, we collected RGCs using CD90.2 coupled magnetic beads, prepared protein extracts, and employed LC-MS for the global proteomic analysis of RGCs. Statistically significant changes in proteins were analyzed to identify changes to cellular signaling resulting from the treatment. We identified unique and common alterations in protein profiles in RGCs undergoing different types of cellular stresses. Our study not only identified both unique and shared proteomic changes but also laid the groundwork for the future development of a therapeutic platform for testing gene candidates for DR and glaucoma., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Previously, the RXR agonist UAB126 demonstrated therapeutic potential to treat obese mice by controlling blood glucose levels (BGL) and altering the expression of genes associated with lipid metabolism and inflammatory response. The purpose of the study was to assess the effects of UAB126 on the progression of diabetic retinopathy (DR) in rodent models of type 1 diabetes (T1D), streptozotocin-induced, and type 2 diabetes (T2D), in db/db mice. UAB126 treatment was delivered either by oral gavage for 6 weeks or by topical application of eye drops for 2 weeks. At the end of the treatment, the retinal function of diabetic mice was assessed by electroretinography (ERG), and their retinal tissue was harvested for protein and gene expression analyses. Bone-marrow cells were isolated and differentiated into bone marrow-derived macrophages (BMDMs). The glycolysis stress test and the 2-DG glucose uptake analysis were performed. Our results demonstrated that in the UAB126-treated diabetic BMDMs, the ECAR rate and the 2-DG uptake were improved as compared to untreated diabetic BMDMs. In UAB126-treated diabetic mice, hyperglycemia was reduced and associated with the preservation of ERG amplitudes and enhanced AMPK activity. Retinas from diabetic mice treated with topical UAB126 demonstrated an increase in Rxr and Ppar and the expression of genes associated with lipid metabolism. Altogether, our data indicate that RXR activation is beneficial to preclinical models of DR.

Introduction: As a metabolic disease, diabetes often leads to health complications such as heart failure, nephropathy, neurological disorders, and vision loss. Diabetic retinopathy (DR) affects as many as 100 million people worldwide. The mechanism of DR is complex and known to impact both neural and vascular components in the retina. While recent advances in the field have identified major cellular signaling contributing to DR pathogenesis, little has been reported on the protein post-translational modifications (PTM) - known to define protein localization, function, and activity - in the diabetic retina overall. Protein glycosylation is the enzymatic addition of carbohydrates to proteins, which can influence many protein attributes including folding, stability, function, and subcellular localization. O -linked glycosylation is the addition of sugars to an oxygen atom in amino acids with a free oxygen atom in their side chain (i.e., threonine, serine). To date, more than 100 congenital disorders of glycosylation have been described. However, no studies have identified the retinal O -linked glycoproteome in health or disease. With a critical need to expedite the discovery of PTMomics in diabetic retinas, we identified both global changes in protein levels and the retinal O -glycoproteome of control and diabetic mice., Methods: We used liquid chromatography/mass spectrometry-based proteomics and high throughput screening to identify proteins differentially expressed and proteins differentially O -glycosylated in the retinas of wildtype and diabetic mice., Results: Changes in both global expression levels of proteins and proteins differentially glycosylated in the retinas of wild-type and diabetic mice have been identified. We provide evidence that diabetes shifts both global expression levels and O -glycosylation of metabolic and synaptic proteins in the retina., Discussion: Here we report changes in the retinal proteome of diabetic mice. We highlight alterations in global proteins involved in metabolic processes, maintaining cellular structure, trafficking, and neuronal processes. We then showed changes in O -linked glycosylation of individual proteins in the diabetic retina., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Starr, Zhylkibayev, Mobley and Gorbatyuk.)

Posttranslational modifications (PTMs) are known to constitute a key step in protein biosynthesis and in the regulation of protein functions. Recent breakthroughs in protein purification strategies and current proteome technologies make it possible to identify the proteomics of healthy and diseased retinas. Despite these advantages, the research field identifying sets of posttranslationally modified proteins (PTMomes) related to diseased retinas is significantly lagging, despite knowledge of the major retina PTMome being critical to drug development. In this review, we highlight current updates regarding the PTMomes in three retinal degenerative diseases-namely, diabetic retinopathy (DR), glaucoma, and retinitis pigmentosa (RP). A literature search reveals the necessity to expedite investigations into essential PTMomes in the diseased retina and validate their physiological roles. This knowledge would accelerate the development of treatments for retinal degenerative disorders and the prevention of blindness in affected populations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Starr and Gorbatyuk.)

Worldwide, more than 200 million people are estimated to be exposed to unsafe levels of arsenic. Chronic exposure to unsafe levels of groundwater arsenic is responsible for multiple human disorders, including dermal, cardiovascular, neurological, pulmonary, renal, and metabolic conditions. Consumption of rice and seafood (where high levels of arsenic are accumulated) is also responsible for human exposure to arsenic. The toxicity of arsenic compounds varies greatly and may depend on their chemical form, solubility, and concentration. Surprisingly, synthetic organoarsenicals are extremely toxic molecules which created interest in their development as chemical warfare agents (CWAs) during World War I (WWI). Among these CWAs, adamsite, Clark I, Clark II, and lewisite are of critical importance, as stockpiles of these agents still exist worldwide. In addition, unused WWII weaponized arsenicals discarded in water bodies or buried in many parts of the world continue to pose a serious threat to the environment and human health. Metabolic inhibition, oxidative stress, genotoxicity, and epigenetic alterations including micro-RNA-dependent regulation are some of the underlying mechanisms of arsenic toxicity. Mechanistic understanding of the toxicity of organoarsenicals is also critical for the development of effective therapeutic interventions. This review provides comprehensive details and a critical assessment of recently published data on various chemical forms of arsenic, their exposure, and implications on human and environmental health., (© 2022. The Author(s).)

The UPR is sustainably activated in degenerating retinas, leading to translational inhibition via p-eIF2α. Recent findings have demonstrated that ablation of growth arrest and DNA damage-inducible protein 34 (GADD34), a protein phosphatase 1 regulatory subunit permitting translational machinery operation through p-eIF2α elevation, does not impact the rate of translation in fast-degenerating rd 16 mice. The current study aimed to validate whether P23H RHO mice degenerating at a slower pace manifest translational attenuation and whether GADD34 ablation impacts the rate of retinal degeneration via further suppression of retinal protein synthesis and apoptotic cell death. For this study, mice were examined with ERG and histological analyses. The molecular assessment was conducted in the naïve and LPS-challenged mice using Western blot and qRT-PCR analyses. Thus, this study demonstrates that the P23H RHO retinas manifest translational attenuation. However, GADD34 ablation resulted in a more prominent p-eIF2a increase without impacting the translation rate. GADD34 deficiency also led to a reduction in scotopic ERG amplitudes and an increased number of TUNEL-positive cells. Molecular analysis revealed that GADD34 deficiency reduces the expression of p-STAT3 and Il-6 while increasing the expression of Tnfa . Overall, the data indicate that GADD34 plays a multifunctional role. Under chronic UPR activation, GADD34 acts as a feedback player, dephosphorylating p-eIF2a, although this role does not seem to be critical. Additionally, GADD34 controls cytokine expression and STAT3 activation. Perhaps these molecular events are particularly important in controlling the pace of retinal degeneration.

Diabetic retinopathy (DR) is an ocular complication of diabetes mellitus (DM), a metabolic disorder characterized by elevation in blood glucose level. The pathogenesis of DR includes vascular, neuronal, and inflammatory components leading to activation of complex cellular molecular signaling. If untreated, the disease can culminate in vision loss that eventually leads to blindness. Animal models mimicking different aspects of DM complications have been developed to study the development and progression of DR. Despite the significant contribution of the developed DR models to discovering the mechanisms of DR and the recent achievements in the research field, the sequence of cellular events in diabetic retinas is still under investigation. Partially, this is due to the complexity of molecular mechanisms, although the lack of availability of models that adequately mimic all the neurovascular pathobiological features observed in patients has also contributed to the delay in determining a precise molecular trigger. In this review, we provide an update on the status of animal models of DR to help investigators choose an appropriate system to validate their hypothesis. We also discuss the key cellular and physiological events of DR in these models.

The use of chemical warfare agents is prohibited but they have been used in recent Middle Eastern conflicts. Their accidental exposure (e.g. arsenical lewisite) is also known and causes extensive painful cutaneous injury. However, their molecular pathogenesis is not understood. Here, we demonstrate that a nexus of stress granules (SGs), integrated stress, and RNA binding proteins (RBPs) Roquin and Reganse-1 play a key role. Lewisite and its prototype phenylarsine oxide (PAO) induce SG assembly in skin keratinocytes soon after exposure, which associate with various RBPs and translation-related proteins. SG disassembly was detected several hours after exposure. The dynamics of SG assembly-disassembly associates with the chemical insult and cell damage. Enhanced Roquin and Regnase-1 expression occurs when Roquin was recruited to SGs and Regnase-1 to the ribosome while in the disassembling SGs their expression is decreased with consequent induction of inflammatory mediators. SG-targeted protein translational control is regulated by the phosphorylation-dependent activation of eukaryotic initiation factors 2α (eIF2α). Treatment with integrated stress response inhibitor (ISRIB), which blocks eIF2α phosphorylation, impacted SG assembly dynamics. Topical application of ISRIB attenuated the inflammation and tissue disruption in PAO-challenged mice. Thus, the dynamic regulation of these pathways provides underpinning to cutaneous injury and identify translational therapeutic approach for these and similar debilitating chemicals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Srivastava, Mishra, Muzaffar, Gorbatyuk, Agarwal, Mukhtar and Athar.)

Existing animal models with rod-dominant retinas have shown that hyperglycemia injures neurons, but it is not yet clearly understood how blue cone photoreceptors and retinal ganglion cells (RGCs) deteriorate in patients because of compromised insulin tolerance. In contrast, northern tree shrews ( Tupaia Belangeri ), one of the closest living relatives of primates, have a cone-dominant retina with short wave sensitivity (SWS) and long wave sensitivity (LWS) cones. Therefore, we injected animals with a single streptozotocin dose (175 mg/kg i.p.) to investigate whether sustained hyperglycemia models the features of human diabetic retinopathy (DR). We used the photopic electroretinogram (ERG) to measure the amplitudes of A and B waves and the photopic negative responses (PhNR) to evaluate cone and RGC function. Retinal flat mounts were prepared for immunohistochemical analysis to count the numbers of neurons with antibodies against cone opsins and RGC specific BRN3a proteins. The levels of the proteins TRIB3, ISR-1, and p-AKT/p-mTOR were measured with western blot. The results demonstrated that tree shrews manifested sustained hyperglycemia leading to a slight but significant loss of SWS cones (12%) and RGCs (20%) 16 weeks after streptozotocin injection. The loss of BRN3a-positive RGCs was also reflected by a 30% decline in BRN3a protein expression. These were accompanied by reduced ERG amplitudes and PhNRs. Importantly, the diabetic retinas demonstrated increased expression of TRIB3 and level of p-AKT/p-mTOR axis but reduced level of IRS-1 protein. Therefore, a new non-primate model of DR with SWS cone and RGC dysfunction lays the foundation to better understand retinal pathophysiology at the molecular level and opens an avenue for improving the research on the treatment of human eye diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gorbatyuk, Pitale, Saltykova, Dorofeeva, Zhylkibayev, Athar, Fuchs, Samuels and Gorbatyuk.)

The current understanding of the molecular pathogenesis of diabetic retinopathy does not provide a mechanistic link between early molecular changes and the subsequent progression of the disease. In this study, we found that human diabetic retinas overexpressed TRIB3 and investigated the role of TRIB3 in diabetic retinal pathobiology in mice. We discovered that TRIB3 controlled major molecular events in early diabetic retinas via HIF1α-mediated regulation of retinal glucose flux, reprogramming cellular metabolism, and governing of inflammatory gene expression. These early molecular events further defined the development of neurovascular deficit observed in mice with diabetic retinopathy. TRIB3 ablation in the streptozotocin-induced mouse model led to significant retinal ganglion cell survival and functional restoration accompanied by a dramatic reduction in pericyte loss and acellular capillary formation. Under hypoxic conditions, TRIB3 contributed to advanced proliferative stages by significant upregulation of GFAP and VEGF expression, thus controlling gliosis and aberrant vascularization in oxygen-induced retinopathy mouse retinas. Overall, our data reveal that TRIB3 is a master regulator of diabetic retinal pathophysiology that may accelerate the onset and progression of diabetic retinopathy to proliferative stages in humans and present TRIB3 as a potentially novel therapeutic target for diabetic retinopathy., (© 2021 by the American Diabetes Association.)

Various retinal degenerative disorders manifest in alterations of the AKT/mTOR axis. Despite this, consensus on the therapeutic targeting of mTOR in degenerating retinas has not yet been achieved. Therefore, we investigated the role of AKT/mTOR signaling in rd16 retinas, in which we restored the AKT/mTOR axis by genetic ablation of pseudokinase TRB3, known to inhibit phosphorylation of AKT and mTOR. First, we found that TRB3 ablation resulted in preservation of photoreceptor function in degenerating retinas. Then, we learned that the mTOR downstream cellular pathways involved in the homeostasis of photoreceptors were also reprogrammed in rd16 TRB3 -/- retinas. Thus, the level of inactivated translational repressor p-4E-BP1 was significantly increased in these mice along with the restoration of translational rate. Moreover, in rd16 mice manifesting decline in p-mTOR at P15, we found elevated expression of Beclin-1 and ATG5 autophagy genes. Thus, these mice showed impaired autophagy flux measured as an increase in LC3 conversion and p62 accumulation. In addition, the RFP-EGFP-LC3 transgene expression in rd16 retinas resulted in statistically fewer numbers of red puncta in photoreceptors, suggesting impaired late autophagic vacuoles. In contrast, TRIB3 ablation in these mice resulted in improved autophagy flux. The restoration of translation rate and the boost in autophagosome formation occurred concomitantly with an increase in total Ub and rhodopsin protein levels and the elevation of E3 ligase Parkin1. We propose that TRB3 may retard retinal degeneration and be a promising therapeutic target to treat various retinal degenerative disorders.

Skin squamous cell carcinomas (SCCs) are a major cause of death in patients who have undergone or will undergo organ transplantation. Moreover, these neoplasms cause significant disease and economic burden and diminish patients' life quality. However, no effective treatment or intervention strategies are available. In this study, we investigated the pathologic role of 5'-cap translation, which is regulated by the formation of a ternary initiation factor complex involving eIF4E, eIF4G, and eIF4A1. We detected increased expression of phosphorylated eIF4E, eIF4G, and eIF4A1 in human and murine skin SCCs. The increase in these ternary initiation factor complex proteins was associated with enhanced eIF4E translation targets cyclin D1 and c-Myc. Conversely, small interfering RNA-mediated depletion of eIF4E in human SCC cells (A431 and SCC-13) reduced eIF4G and proteins that regulate the cell cycle and proliferation. Notably, inhibition of Raf/MAPK/extracellular signal-regulated kinase signaling decreased eIF4E and phosphorylated eIF4E accumulation and significantly diminished cell-cycle gene expression and tumor volume of A431-derived xenograft tumors. Furthermore, disrupting the eIF4E with an allosteric inhibitor of eIF4E and eIF4G binding, 4EGI-1, decreased the eIF4E/eIF4G expression and reduced the proliferation. Finally, combined inhibition of the Raf/MAPK/extracellular signal-regulated kinase axis and eIF4E impaired 5'-cap‒dependent translation and abrogated tumor cell proliferation. These data demonstrate that 5'-cap‒dependent translation is a potential therapeutic target for abrogating lethal skin SCCs in patients who have undergone or will undergo organ transplantation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Physiological equilibrium in the retina depends on coordinated work between rod and cone photoreceptors and can be compromised by the expression of mutant proteins leading to inherited retinal degeneration (IRD). IRD is a diverse group of retinal dystrophies with multifaceted molecular mechanisms that are not fully understood. In this review, we focus on the contribution of chronically activated unfolded protein response (UPR) to inherited retinal pathogenesis, placing special emphasis on studies employing genetically modified animal models. As constitutively active UPR in degenerating retinas may activate pro-apoptotic programs associated with oxidative stress, pro-inflammatory signaling, dysfunctional autophagy, free cytosolic Ca 2+ overload, and altered protein synthesis rate in the retina, we focus on the regulatory mechanisms of translational attenuation and approaches to overcoming translational attenuation in degenerating retinas. We also discuss current research on the role of the UPR mediator PERK and its downstream targets in degenerating retinas and highlight the therapeutic benefits of reprogramming PERK signaling in preclinical animal models of IRD. Finally, we describe pharmacological approaches targeting UPR in ocular diseases and consider their potential applications to IRD., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We compared the phenotypes of three mutant AAV2 viruses containing mutations in arginine amino acids (R585, R588 and R484) previously shown to be involved in AAV2 heparan sulfate binding. The transduction efficiencies of wild type and mutant viruses were determined in the eye, the brain and peripheral organs following subretinal, striatal and intravenous injection, respectively, in mice and rats. We found that each of the three mutants (the single mutant R585A; the double mutant R585, 588A; and the triple mutant R585, 588, 484A) had a unique phenotype compared to wt and each other. R585A was completely defective for transducing peripheral organs via intravenous injection, suggesting that R585A may be useful for targeting peripheral organs by substitution of peptide ligands in the capsid surface. In the brain, all three mutants displayed widespread transduction, with the double mutant R585, 588A displaying the greatest spread and the greatest number of transduced neurons. The double mutant was also extremely efficient for retrograde transport, while the triple mutant was almost completely defective for retrograde transport. This suggested that R484 may be directly involved in interaction with the transport machinery. Finally, the double mutant also displayed improved transduction of the eye compared to wild type and the other mutants., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: We reported previously that retinas of mice with inherited retinal degeneration make less protein than retinas of normal mice. Despite recent studies suggesting that diminished protein synthesis rates may contribute to neurologic disorders, a direct link between protein synthesis rates and the progression of neurodegeneration has not been established. Moreover, it remains unclear whether reduced protein synthesis could be involved in retinal pathogenesis. Dysregulation of AKT/mTOR signaling has been reported in the retina during retinal degeneration, but to what extent this signaling contributes to translational attenuation in these mice remains uncertain., Methods: C57BL/6J and rd16 mice were subcutaneously injected with anisomycin to chronically inhibit protein synthesis rates. An AAV2 construct encoding constitutively active 4ebp1 was subretinally delivered in wildtype animals to lower protein synthesis rates. 4ebp1/2 were knocked out in rd16 mice., Results: Anisomycin treatment lowered retinal translation rates, accelerated retinal degeneration in rd16 mice, and initiated cell death in the retinas of C57BL/6J mice. AAV-mediated transfer of constitutively active 4ebp1-4A into the subretinal space of wildtype animals inhibited protein synthesis, and led to reduced electroretinography amplitudes and fewer ONL nuclei. Finally, we report that restoring protein synthesis rates by knocking out 4ebp1/2 was associated with an approximately 2-fold increase in rhodopsin levels and a delay in retinal degeneration in rd16 mice., Conclusions: Our study indicates that protein synthesis inhibition is likely not a cell defense mechanism in the retina by which deteriorating photoreceptors survive, but may be harmful to degenerating retinas, and that restoring protein synthesis may have therapeutic potential in delaying the progression of retinal degeneration.

Activation of the unfolded protein response has been detected in various animal models of retinal degeneration. The PERK branch converges on eIF2α to regulate protein synthesis. We previously reported that diseased retinas produce less protein as they degenerate. We also proposed that the majority of this reduction in protein synthesis may not be due to control of eIF2α. Nevertheless, multiple research groups have reported that modulating eIF2α levels may be a viable strategy in the treatment of neurodegenerative diseases. Here, using two genetic approaches, a systemic Gadd34 knockout and a photoreceptor conditional Perk knockout, to alter p-eIF2α levels in rd16 mice, we demonstrate not only that degenerating retinas may not use this mechanism to signal for a decline in protein synthesis rates but also that modulation of p-eIF2α levels is insufficient to delay retinal degeneration.

Arsenic is a mitochondrial toxin, and its derivatives, such as arsenic trioxide (ATO), can trigger endoplasmic reticulum (ER) and the associated unfolded protein response (UPR). Here, we show that arsenic induction of the UPR triggers ATF4, which is involved in regulating this ER-mitochondrial crosstalk that is important for the molecular pathogenesis of arsenic toxicity. Employing ATF4 +/+ and ATF4 -/- MEFs, we show that ATO induces UPR and impairs mitochondrial integrity in ATF4 +/+ MEF cells which is largely ablated upon loss of ATF4. Following ATO treatment, ATF4 activates NADPH oxidase by promoting assembly of the enzyme components Rac-1/P47 phox /P67 phox , which generates ROS/superoxides. Furthermore, ATF4 is required for triggering Ca ++ /calpain/caspase-12-mediated apoptosis following ATO treatment. The IP3R inhibitor attenuates Ca ++ /calpain-dependent apoptosis, as well as reduces m-ROS and MMP disruption, suggesting that ER-mitochondria crosstalk involves IP3R-regulated Ca ++ signaling. Blockade of m-Ca ++ entry by inhibiting m-VDAC reduces ATO-mediated UPR in ATF4 +/+ cells. Additionally, ATO treatment leads to p53-regulated mitochondrial apoptosis, where p53 phosphorylation plays a key role. Together, these findings indicate that ATO-mediated apoptosis is regulated by both ER and mitochondria events that are facilitated by ATF4 and the UPR. Thus, we describe novel mechanisms by which ATO orchestrates cytotoxic responses involving interplay of ER and mitochondria., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Activating transcription factor 4 (ATF4) is a member of the PERK signaling pathway, which directly binds endoplasmic reticulum stress target genes and plays a crucial role in both adaptations to stress and activation of apoptosis. Previous publications demonstrated conflicting evidence on the role of ATF4 in the pathogenesis of neurodegenerative disorders. In this study, we used recombinant adeno-associate virus (rAAV)-mediated gene transfer to investigate if the sustained up-regulation of ATF4 launches a pro-survival or pro-death trend in the dopamine (DA) cells of the substantia nigra pars compacta in a rat model of Parkinson-like neurodegeneration induced by human alpha-synuclein (αS) overexpression. We showed that ATF4 does not protect nigral DA neurons against an αS-induced pathology. Moreover, the rAAV-mediated overexpression of ATF4 resulted in severe nigra-striatal degeneration via activation of caspases 3/7., (Published by Elsevier Ireland Ltd.)

RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease -mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop (-/-) background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin.

Age-related structural changes and gradual loss of key enzymes significantly affect the ability of the endoplasmic reticulum (ER) to facilitate proper protein folding and maintain homeostasis. In this work, we present several lines of evidence supporting the hypothesis that the age-related decline in expression of the ER chaperone glucose-regulated protein 78 (GRP78) could be related to the development of Parkinson's disease. We first determined that old (24 months) rats exhibit significantly lower levels of GRP78 protein in the nigrostriatal system as compared with young (2 months) animals. Then using recombinant adeno-associate virus-mediated gene transfer, we found that GRP78 downregulation by specific small interfering RNAs (siRNAs) aggravates alpha-synuclein (α-syn) neurotoxicity in nigral dopamine (DA) neurons. Moreover, the degree of chaperone decline corresponds with the severity of neurodegeneration. Additionally, comparative analysis of nigral tissues obtained from old and young rats revealed that aging affects the capacity of nigral DA cells to upregulate endogenous GRP78 protein in response to human α-syn neurotoxicity. Finally, we demonstrated that a sustained increase of GRP78 protein over the course of 9 months protected aging nigral DA neurons in the α-syn-induced rat model of Parkinson's-like neurodegeneration. Our data indicate that the ER chaperone GRP78 may have therapeutic potential for preventing and/or slowing age-related neurodegeneration., (Published by Elsevier Inc.)

Rhodopsin, a G-protein coupled receptor, most abundant protein in retinal rod photoreceptors, is glycosylated at asparagines-2 and 15 on its N-terminus. To understand the role of rhodopsin's glycosylation in vivo, we generated and characterized a transgenic mouse model that expresses a non-glycosylated form of rhodopsin. We show that lack of glycosylation triggers a dominant form of progressive retinal degeneration. Electron microscopic examination of retinas at postnatal day 17 revealed the presence of vacuolar structures that distorted rod photoreceptor outer segments and became more prominent with age. Expression of non-glycosylated rhodopsin alone showed that it is unstable and is regulated via ubiquitin-mediated proteasomal degradation at the base of outer segments. We observed similar vacuolization in outer segments of transgenic mice expressing human rhodopsin with a T17M mutation (hT17M), suggesting that the mechanism responsible for the degenerative process in mice expressing the non-glycosylated rhodopsin and the RHO(hT17M) mice is likely the cause of phenotype observed in retinitis pigmentosa patients carrying T17M mutation., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1β, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1β was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1β. We observed ~19% reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1β.

We previously reported activation of the unfolded protein response (UPR) in P23H rhodopsin (RHO) retinas with autosomal dominant retinitis pigmentosa (ADRP). Knowing that the UPR can trigger Ca(2+) release from the endoplasmic reticulum and regulate cellular signaling we examined the level of Ca(2+)-regulated proteins. We also looked for changes in the expression of Bcl2 family proteins, autophagy proteins and the mTOR/AKT pathways, as well as for the induction of mitochondria-associated apoptosis in the P23H RHO retina. Our data demonstrated that the elevation of calpain and caspase-12 activity was concomitantly observed with a decrease in the BCL2-XL/BAX ratio and an increase in mTor levels in the P23H-3 RHO retina suggesting a vulnerability of P23H RHO photoreceptors to apoptosis. The translocation of BAX to the mitochondria, as well as the release of cytochrome C and AIF into the cytosol supports this conclusion and indicates the involvement of mitochondria-induced apoptosis in the progression of ADRP. The level of autophagy proteins in general was found to be decreased in the P21-P30 P23H RHO retina. Injections of rapamycin, however, protected the P23H RHO rod photoreceptors from experiencing physiological decline. Despite this fact, the downregulation of mTOR did not alter the level of autophagy proteins. Our results imply that in addition to activation of the UPR during ADRP progression, photoreceptors also experience alterations in major proapoptotic pathways., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The glucose regulated protein 78 (GRP78), also known as BiP, is the endoplasmatic reticulum (ER) homologue of HSP70, which plays a dual role in the ER by controlling protein folding, in order to prevent aggregation, and by regulating the signaling of the unfolded protein response (UPR). Most neurodegenerative disorders including Parkinson's, Alzheimer's diseases and progressive retinal degeneration are characterized by activation of the UPR and modified expression of GRP78. The expression levels and activity of GRP78 are altered with age raising the question of whether the lack of GRP78 could be a predisposing factor for many neurodegenerative disorders associated with age including PD, Alzheimer and Age-related macular degeneration. Attempts to induce or upregulate GRP78 in animal models of neurodegeneration have recently been made with the help of pharmacological BiP protein Inducer X (BIX) and GRP78 cDNA delivery via adeno-associated virus (AAV) vectors. The results of these studies validate GRP78 as a new therapeutic target for treatments of forebrain ischemia, Parkinson disease and retinal degeneration. These data, together with the results from age-related studies, highlight the importance for developing drugs to induce elevation of endogenous GRP78 in order to increase cellular survival and extend functional longevity.

Purpose: Endoplasmic reticulum (ER) stress has been observed in animal models of retinitis pigmentosa expressing P23H rhodopsin. We compared levels of tightly induced ER stress genes, Binding of immunoglobulin protein (BiP) and CCAAT/enhancer-binding protein homologous protein (Chop), in seven additional models of retinal degeneration arising from genetic or environmental causes., Methods: Retinas from transgenic S334ter rhodopsin (lines 3, 4, and 5) and Royal College of Surgeons (RCS and RCS-p+) rats from postnatal (P) days 10 to 120 were analyzed. In a constant light (CL) model of retinal degeneration, BALB/c mice were exposed to 15,000 lux of CL for 0 to 8 hours. Retinal tissues from three to eight animals per experimental condition were collected for histologic and molecular analyses., Results: S334ter animals revealed significant increases in BiP, S334ter-3 (3.3× at P15), S334ter-4 (4× at P60), and S334ter-5 (2.2× at P90), and Chop, S334ter-3 (1.3× at P15), S334ter-4 (1.5× at P30), and S334ter-5 (no change), compared with controls. P23H-3 rats showed significant increase of BiP at P60 (2.3×) and Chop (1.6×). RCS and RCS-p+ rats showed significant increases in BiP at P60 (2.4×) and P20 (1.8×), respectively, but no statistically significant changes in Chop. BALB/c mice showed increases in BiP (1.5×) and Chop (1.3×) after 4 hours of CL. Increased levels of these ER stress markers correlated with photoreceptor cell loss., Conclusions: Our study reveals surprising increases in BiP and to a lesser degree Chop in retinal degenerations arising from diverse causes. We propose that manipulation of ER stress responses may be helpful in treating many environmental and heritable forms of retinal degeneration.

Accumulation of human wild-type (wt) α-synuclein (α-syn) induces neurodegeneration in humans and in experimental rodent models of Parkinson disease (PD). It also leads to endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). We overexpressed glucose regulated protein 78, also known as BiP (GRP78/BiP), to test the hypothesis that this ER chaperone modulates the UPR, blocks apoptosis, and promotes the survival of nigral dopamine (DA) neurons in a rat model of PD induced by elevated level of human α-syn. We determined that α-syn activates ER stress mediators associated with pancreatic ER kinase-like ER kinase (PERK) and activating transcription factor-6 (ATF6) signaling pathways as well as proaoptotic CCAAT/-enhancer-binding protein homologous protein (CHOP) in nigral DA neurons. At the same time, overexpression of GRP78/BiP diminished α-syn neurotoxicity by down regulating ER stress mediators and the level of apoptosis, promoted survival of nigral tyrosine hydroxylase (TH) positive cells and resulted in higher levels of striatal DA, while eliminating amphetamine induced behavioral asymmetry. We also detected a complex between GRP78/BiP and α-syn that may contribute to prevention of the neurotoxicity caused by α-syn. Our data suggest that the molecular chaperone GRP78/BiP plays a neuroprotective role in α-syn-induced Parkinson-like neurodegeneration.

Purpose: The human rhodopsin (Rho) mutation T17M leads to autosomal dominant retinitis pigmentosa (adRP). The goal of our study was to elucidate the role of endoplasmic reticulum (ER) stress in retinal degeneration in hT17M Rho mice and identify potential candidates for adRP gene therapy., Methods: We used transgenic mice expressing the ER stress-activated indicator (ERAI) and hT17M Rho to evaluate the activation of ER stress responses. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze changes in the expression of 30 unfolded protein response (UPR)-associated genes at P12, 15, 18, 21, and 25. The cytosolic fraction of hT17M Rho retinal cells was used to measure the release of cytochrome C and apoptotic inducing factor-1 (AIF1) by Western blotting. Optical coherence tomography (OCT) analysis was performed for 1-month-old hT17M Rho mice., Results: hT17M Rho was localized in the outer nuclear layer (ONL) of T17M(+/-)ERAI(+/-) photoreceptors as well as C57BL/6 retinas injected with AAV-hT17M Rho-GFP. In P15 hT17M Rho retinas, we observed an up-regulation of UPR genes (Atf4, Eif2α, Xbp1, Bip, Canx, and Hsp90), autophagy genes and proapoptotic Bcl2 genes. OCT, and the downregulation of Nrl and Crx gene expression confirmed that cell death occurs in 55% of photoreceptors via the up-regulation of caspase-3 and caspase-12, and the release of AIF1 from the mitochondria., Conclusions: The ER stress response is involved in retinal degeneration in hT17M Rho mice. The final demise of photoreceptors occurs via apoptosis involving ER stress-associated and mitochondria-induced caspase activation. We identified Atg5, Atg7, Bax, Bid, Bik, and Noxa as potential therapeutic targets for adRP treatment.

Purpose: The absence of Mertk in RCS rats results in defective RPE phagocytosis, accumulation of outer segment (OS) debris in the subretinal space, and subsequent death of photoreceptors. Previous research utilizing Mertk gene replacement therapy in RCS rats provided proof of concept for treatment of this form of recessive retinitis pigmentosa (RP); however, the beneficial effects on retinal function were transient. In the present study, we evaluated whether delivery of a MERTK transgene using a tyrosine-mutant AAV8 capsid could lead to more robust and longer-term therapeutic outcomes than previously reported., Methods: An AAV8 Y733F vector expressing a human MERTK cDNA driven by a RPE-selective promoter was administrated subretinally at postnatal day 2. Functional and morphological analyses were performed at 4 months and 8 months post-treatment. Retinal vasculature and Müller cell activation were analyzed by quantifying acellular capillaries and glial fibrillary acidic protein immunostaining, respectively., Results: Electroretinographic responses from treated eyes were more than one-third of wild-type levels and OS were well preserved in the injection area even at 8 months. Rescue of RPE phagocytosis, prevention of retinal vasculature degeneration, and inhibition of Müller cell activation were demonstrated in the treated eyes for at least 8 months., Conclusions: This research describes a longer and much more robust functional and morphological rescue than previous studies. We also demonstrate for the first time that an AAV8 mutant capsid serotype vector has a substantial therapeutic potential for RPE-specific gene delivery. These results suggest that tyrosine-mutant AAV8 vectors hold promise for the treatment of individuals with MERTK-associated RP.

Many mutations in the human rhodopsin gene (RHO) cause autosomal dominant retinitis pigmentosa (ADRP). Our previous studies with a P23H (proline-23 substituted by histidine) RHO transgenic mouse model of ADRP demonstrated significant improvement of retinal function and preservation of retinal structure after transfer of wild-type rhodopsin by AAV. In this study we demonstrate long-term rescue of retinal structure and function by a single virus expressing both RHO replacement cDNA and small interfering RNA (siRNA) to digest mouse Rho and human P23H RHO mRNA. This combination should prevent overexpression of rhodopsin, which can be deleterious to photoreceptors. On the basis of the electroretinogram (ERG) response, degeneration of retinal function was arrested at 2 months postinjection, and the response was maintained at this level until termination at 9 months. Preservation of the ERG response in P23H RHO mice reflected survival of photoreceptors: both the outer nuclear layer (ONL) and outer segments of photoreceptor cells maintained the same thickness as in nontransgenic mice, whereas the control injected P23H eyes exhibited severe thinning of the ONL and outer segments. These findings suggest that delivery of both a modified cDNA and an siRNA by a single adeno-associated viral vector provided long-term rescue of ADRP in this model. Because the siRNA targets human as well as mouse rhodopsin mRNAs, the combination vector may be useful for the treatment of human disease.

The S334ter rhodopsin (Rho) rat (line 4) bears the rhodopsin gene with an early termination codon at residue 334 that is a model for several such mutations found in human patients with autosomal dominant retinitis pigmentosa (ADRP). The Unfolded Protein Response (UPR) is implicated in the pathophysiology of several retinal disorders including ADRP in P23H Rho rats. The aim of this study was to examine the onset of UPR gene expression in S334ter Rho retinas to determine if UPR is activated in ADRP animal models and to investigate how the activation of UPR molecules leads to the final demise of S334ter Rho photoreceptors. RT-PCR was performed to evaluate the gene expression profiles for the P10, P12, P15, and P21 stages of the development and progression of ADRP in S334ter Rho photoreceptors. We determined that during the P12-P15 period, ER stress-related genes are strongly upregulated in transgenic retinas, resulting in the activation of the UPR that was confirmed using western blot analysis and RT-PCR. The activation of UPR was associated with the increased expression of JNK, Bik, Bim, Bid, Noxa, and Puma genes and cleavage of caspase-12 that together with activated calpains presumably compromise the integrity of the mitochondrial MPTP, leading to the release of pro-apoptotic AIF1 into the cytosol of S334ter Rho photoreceptor cells. Therefore, two major cross-talking pathways, the UPR and mitochondrial MPTP occur in S334ter-4 Rho retina concomitantly and eventually promote the death of the photoreceptor cells.

Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in RHO, the gene for rod photoreceptor opsin. Earlier, a study on mice carrying mutated rhodopsin transgenes on either RHO + / +  or RHO + /- backgrounds suggested that the amount of wild-type rhodopsin affected survival of photoreceptors. Therefore, we treated P23H RHO transgenic mice with adeno-associated virus serotype 5 (AAV5) expressing a cDNA clone of the rhodopsin gene (RHO301) that expressed normal opsin from the mouse opsin promoter. Analysis of the electroretinogram (ERG) demonstrated that increased expression of RHO301 slowed the rate of retinal degeneration in P23H mice: at 6 months, a-wave amplitudes were increased by 100% and b-wave amplitudes by 79%. In contrast, nontransgenic mice injected with AAV5 RHO301 demonstrated a decrease in the ERG, confirming the damaging effect of rhodopsin overproduction in normal photoreceptors. In P23H mice, the increase in the ERG amplitudes was correlated with improvement of retinal structure: the thickness of the outer nuclear layer in RHO301-treated eyes was increased by 80% compared with control eyes. These findings suggest that the wild-type RHO gene can be delivered to rescue retinal degeneration in mice carrying a RHO mutation and that increased production of normal rhodopsin can suppress the effect of the mutated protein. These findings make it possible to treat ADRP caused by different mutations of RHO with the expression of wild-type RHO.

The P23H mutation within the rhodopsin gene (RHO) causes rhodopsin misfolding, endoplasmic reticulum (ER) stress, and activates the unfolded protein response (UPR), leading to rod photoreceptor degeneration and autosomal dominant retinitis pigmentosa (ADRP). Grp78/BiP is an ER-localized chaperone that is induced by UPR signaling in response to ER stress. We have previously demonstrated that BiP mRNA levels are selectively reduced in animal models of ADRP arising from P23H rhodopsin expression at ages that precede photoreceptor degeneration. We have now overexpressed BiP to test the hypothesis that this chaperone promotes the trafficking of P23H rhodopsin to the cell membrane, reprograms the UPR favoring the survival of photoreceptors, blocks apoptosis, and, ultimately, preserves vision in ADRP rats. In cell culture, increasing levels of BiP had no impact on the localization of P23H rhodopsin. However, BiP overexpression alleviated ER stress by reducing levels of cleaved pATF6 protein, phosphorylated eIF2alpha and the proapoptotic protein CHOP. In P23H rats, photoreceptor levels of cleaved ATF6, pEIF2alpha, CHOP, and caspase-7 were much higher than those of wild-type rats. Subretinal delivery of AAV5 expressing BiP to transgenic rats led to reduction in CHOP and photoreceptor apoptosis and to a sustained increase in electroretinogram amplitudes. We detected complexes between BiP, caspase-12, and the BH3-only protein BiK that may contribute to the antiapoptotic activity of BiP. Thus, the preservation of photoreceptor function resulting from elevated levels of BiP is due to suppression of apoptosis rather than to a promotion of rhodopsin folding.

Purpose: To develop a hammerhead ribozymes (Rz) that might be exploited in a "digest and replace" gene therapy strategy for autosomal dominant retinitis pigmentosa (ADRP) caused by mutations in the gene for rhodopsin (RHO)., Methods: A ribozyme (Rz397) was designed to hybridize with an accessible region in rhodopsin mRNA. It was tested in vitro to determine the kinetics of cleavage of a target oligonucleotide. Following transfection of cultured cells, reduction of rhodopsin mRNA in response to Rz397 was measured RT-PCR. The gene for the ribozyme (Rz397) was inserted in an adeno-associated virus (AAV2) vector and packaged in AAV2 capsids. The virus was injected subretinally in the eyes of C57BL/6J (RHO+/+) and rhodopsin knockout hemizygous (RHO+/-) mice at postnatal days 6 (P6) and 30 (P30). Mice were analyzed by full-field electroretinography (ERG). The reduction of opsin protein was measured by western blot analysis and visualized by immunocytochemistry. Reduction of rhodopsin mRNA was assessed using in situ hybridization. Morphometric microscopy of fluorescent antibody-antigen complexes and autoradiography of retinas were used to quantify levels of rhodopsin protein and mRNA, respectively., Results: Transient co-transfection of HEK 293 cells with a wild-type rhodopsin cDNA and Rz397 resulted in an approximately 60% reduction of RHO mRNA one day after transfection. RHO+/- -mice injected with AAV2-Rz397 at P6 showed a 50% reduction in b-wave amplitudes in injected eyes relative to saline injected contralateral eyes. However, injection of RHO+/- -animals at one month and of RHO+/+-animals at either age had no impact on ERG. Nevertheless, we detected an 80% reduction of opsin protein in ribozyme-injected eyes of hemizygous mice (by western blot) and a 50% reduction in opsin content in RHO+/+ mice (by morphometry). These reductions were confirmed by in situ hybridization., Conclusions: AAV2-Rz397 led to significant (greater than or equal to 50%) reduction of rhodopsin mRNA and protein in mice. It affected ERG amplitudes only when injected in hemizygous RHO knockout pups. This RNA inhibitor may prove useful in treating animal models of ADRP as part of an RNA replacement approach.