Nuclear NAD+-biosynthetic enzyme NMNAT1 facilitates development and early survival of retinal neurons.

Despite mounting evidence that the mammalian retina is exceptionally reliant on proper NAD+ homeostasis for health and function, the specific roles of subcellular NAD+ pools in retinal development, maintenance, and disease remain obscure. Here, we show that deletion of the nuclear-localized NAD+ synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) in the developing murine retina causes early and severe degeneration of photoreceptors and select inner retinal neurons via multiple distinct cell death pathways. This severe phenotype is associated with disruptions to retinal central carbon metabolism, purine nucleotide synthesis, and amino acid pathways. Furthermore, transcriptomic and immunostaining approaches reveal dysregulation of a collection of photoreceptor and synapse-specific genes in NMNAT1 knockout retinas prior to detectable morphological or metabolic alterations. Collectively, our study reveals previously unrecognized complexity in NMNAT1-associated retinal degeneration and suggests a yet-undescribed role for NMNAT1 in gene regulation during photoreceptor terminal differentiation.

Tribbles homolog 3-mediated targeting the AKT/mTOR axis in mice with retinal degeneration.

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.

Involvement of c-Jun N-terminal kinase 2 (JNK2) in Endothelin-1 (ET-1) Mediated Neurodegeneration of Retinal Ganglion Cells.

Purpose: The goal of this study was to determine whether JNK2 played a causative role in endothelin-mediated loss of RGCs in mice. Methods: JNK2-/- and wild type (C57BL/6) mice were intravitreally injected in one eye with 1 nmole of ET-1, whereas the contralateral eye was injected with the vehicle. At two time points (two hours and 24 hours) after the intravitreal injections, mice were euthanized, and phosphorylated c-Jun was assessed in retinal sections. In a separate set of experiments, JNK2-/- and wild type mice were intravitreally injected with either 1 nmole of ET-1 or its vehicle and euthanized seven days after injection. Retinal flat mounts were stained with antibodies to the RGC marker, Brn3a, and surviving RGCs were quantified. Axonal degeneration was assessed in paraphenylenediamine stained optic nerve sections. Results: Intravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice, which was appreciably lower in the JNK2 -/- mice. A significant (P < 0.05) 26% loss of RGCs was found in wild type mice, seven days after injection with ET-1. JNK2-/- mice showed a significant protection from RGC loss following ET-1 administration, compared to wild type mice injected with ET-1. A significant decrease in axonal counts and an increase in the collapsed axons was found in ET-1 injected wild type mice eyes. Conclusions: JNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice. Neuroprotective effects in JNK2-/- mice following ET-1 administration occur mainly in the soma and not in the axons of RGCs.

Selective Ablation of Dehydrodolichyl Diphosphate Synthase in Murine Retinal Pigment Epithelium (RPE) Causes RPE Atrophy and Retinal Degeneration.

Patients with certain defects in the dehydrodolichyl diphosphate synthase (DHDDS) gene (RP59; OMIM #613861) exhibit classic symptoms of retinitis pigmentosa, as well as macular changes, suggestive of retinal pigment epithelium (RPE) involvement. The DHDDS enzyme is ubiquitously required for several pathways of protein glycosylation. We wish to understand the basis for selective ocular pathology associated with certain DHDDS mutations and the contribution of specific ocular cell types to the pathology of mutant Dhdds-mediated retinal degeneration. To circumvent embryonic lethality associated with Dhdds knockout, we generated a Cre-dependent knockout allele of murine Dhdds (Dhddsflx/flx). We used targeted Cre expression to study the importance of the enzyme in the RPE. Structural alterations of the RPE and retina including reduction in outer retinal thickness, cell layer disruption, and increased RPE hyper-reflectivity were apparent at one postnatal month. At three months, RPE and photoreceptor disruption was observed non-uniformly across the retina as well as RPE transmigration into the photoreceptor layer, external limiting membrane descent towards the RPE, and patchy loss of photoreceptors. Functional loss measured by electroretinography was consistent with structural loss showing scotopic a- and b-wave reductions of 83% and 77%, respectively, at three months. These results indicate that RPE dysfunction contributes to DHDDS mutation-mediated pathology and suggests a more complicated disease mechanism than simply disruption of glycosylation.

MicroRNA-24 protects retina from degeneration in rats by down-regulating chitinase-3-like protein 1.

MicroRNAs (miRNAs) have been shown to play critical roles in the pathogenesis and progression of degenerative retinal diseases like age-related macular degeneration (AMD). In this study, we first demonstrated that miR-24 plays an important role in maintaining retinal structure and visual function of rats by targeting chitinase-3-like protein 1 (CHI3L1). In the retinal pigment epithelial (RPE) cells of Royal College of Surgeons (RCS) rats, an animal model of genetic retinal degeneration (RD), miR-24 was found lower and CHI3L1 level was higher in comparison with those in Sprague-Dawley (SD) rats. Other changes in the eyes of RCS rats include activated AKT/mTOR and ERK pathways and abnormal autophagy in the RPE cells. Such roles of miR-24 and CHI3L1 were further confirmed in RCS rats by subretinal injection of agomiR-24, which decreased CHI3L1 level and preserved retinal structure and function. Upstream, NF-κB was identified as the regulator of miR-24 in the RPE cells of these rats. On the other hand, in SD rats, intraocular treatment of antagomiR-24 induced pathological changes similar to those in RCS rats. The results revealed the protective roles for miR-24 to RPE cells and a mechanism for RD in RCS rats was proposed: extracellular stress stimuli first activate the NF-κB signaling pathway, which lowers miR-24 expression so that CHI3L1 increased. CHI3L1 sequentially results in aberrant autophagy and RPE dysfunction by activating AKT/mTOR and ERK pathways. Taken together, although the possibility, that the therapeutic effects in RCS rats are caused by other transcriptional changes regulated by miR-24, cannot be excluded, these findings indicate that miR-24 protects rat retina by targeting CHI3L1. Thus, miR-24 and CHI3L1 might be the targets for developing more effective therapy for degenerative retinal diseases like AMD.

Deletion of Arginase 2 Ameliorates Retinal Neurodegeneration in a Mouse Model of Multiple Sclerosis.

Optic neuritis is a major clinical feature of multiple sclerosis (MS) and can lead to temporary or permanent vision loss. Previous studies from our laboratory have demonstrated the critical involvement of arginase 2 (A2) in retinal neurodegeneration in models of ischemic retinopathy. The current study was undertaken to investigate the role of A2 in MS-mediated retinal neuronal damage and degeneration. Experimental autoimmune encephalomyelitis (EAE) was induced in wild-type (WT) and A2 knockout (A2-/-) mice. EAE-induced motor deficits, loss of retinal ganglion cells, retinal thinning, inflammatory signaling, and glial activation were studied in EAE-treated WT and A2-/- mice and their respective controls. Increased expression of A2 was observed in WT retinas in response to EAE induction. EAE-induced motor deficits were markedly reduced in A2-/- mice compared with WT controls. Retinal flat mount studies demonstrated a significant reduction in the number of RGCs in WT EAE retinas in comparison with normal control mice. A significant improvement in neuronal survival was evident in retinas of EAE-induced A2-/- mice compared with WT. RNA levels of the proinflammatory molecules CCL2, COX2, IL-1α, and IL-12α were significantly reduced in the A2-/- EAE retinas compared with WT EAE. EAE-induced activation of glia (microglia and Müller cells) was markedly reduced in A2-/- retinas compared with WT. Western blot analyses showed increased levels of phospho-ERK1/2 and reduced levels of phospho-BAD in the WT EAE retina, while these changes were prevented in A2-/- mice. In conclusion, our studies establish EAE as an excellent model to study MS-mediated retinal neuronal damage and suggest the potential value of targeting A2 as a therapy to prevent MS-mediated retinal neuronal injury.

Histone Deacetylases Contribute to Excitotoxicity-Triggered Degeneration of Retinal Ganglion Cells In Vivo.

Excitotoxicity is known to modulate the nuclear accumulation, and thus activity state, of histone deacetylases (HDACs) in pyramidal neurons. In the retina, deregulation in activity and expression of different HDACs has been linked to pathological conditions such as retinitis pigmentosa, retinal ischemia, glaucoma, and acute optic nerve injury. Up to now, however, the effects of in vivo excitotoxicity on the different HDACs in retinal ganglion cells (RGCs) have not been thoroughly investigated. Here, we injected adult mice intravitreally with N-methyl-D-aspartate (NMDA) as a mean to trigger excitotoxicity-mediated RGC degeneration and we detected time-dependent loss of RGCs at 1 and 7 days after the insult. Further, we characterized the subcellular localization of HDACs belonging to class I (HDAC1, HDAC3), IIa (HDAC4, HDAC5, HDAC7, HDAC9), IIb (HDAC6, HDAC10), and IV (HDAC11) in RGCs. Our analyses revealed a differential pattern of HDACs nuclear distribution in RGCs following excitotoxicity. After 1 day, HDAC3, HDAC5, HDAC6, HDAC7, and HDAC11 showed altered subcellular localization in RGCs while 7 days after the excitotoxic insult, HDAC4 and HDAC9 were the only HDACs displaying changes in their subcellular distribution. Moreover, we found that in vivo selective inhibition of HDAC1/3 or HDAC4/5 via MS-275 (entinostat) or LMK-235, respectively, could prevent ongoing RGC degeneration. In conclusion, our results point towards a role of HDACs in RGC degeneration and identify HDAC1/3 and HDAC4/5 as potential therapeutic targets to treat degenerative retinal diseases.

Involvement of matrix metalloproteinases in capillary degeneration following NMDA-induced neurotoxicity in the neonatal rat retina.

Interactions between neuronal cells and vascular cells in the retina are critical for maintaining retinal tissue homeostasis. Impairment of cellular interactions contributes to development and progression of retinal diseases. Previous studies demonstrated that neuronal cell damage leads to capillary degeneration in an N-methyl-D-aspartic acid (NMDA)-induced retinal degeneration model. However, the mechanisms underlying this phenomenon are not fully understood. In this study, we examined the possible role of matrix metalloproteinase (MMP)-9 in neuronal cell loss and capillary degeneration in NMDA-treated retinas of neonatal rats. Intravitreal injection of NMDA (50 or 200 nmol) was performed on postnatal day (P) 7 and morphological changes in retinal neurons and vasculature were examined on P14. The MMP inhibitor CP101537 (100 nmol) or vehicle (dimethyl sulfoxide) was intravitreally injected simultaneously with, or 2 days after, NMDA injection. CP101537 protected against neurovascular degeneration in a time-dependent manner as follows: 1) simultaneous injection of CP101537 with NMDA prevented morphological changes in retinal neurons induced by NMDA (50 nmol); and 2) reduction in capillary density and number of vertical sprouts induced by NMDA (200 nmol) was prevented when CP101537 was injected 2 days after NMDA injection. Gelatin zymography and western blot analyses indicated that activity and protein levels of MMP-9 were enhanced from 4 h to 2 days after NMDA injection. Increased activity and protein levels of MMP-9 were suppressed by MMP inhibitors (CP101537 and GM6001). In situ zymography revealed that MMP activity was enhanced throughout the retinal vasculature in NMDA-treated retinas. These results indicate that MMP-9 plays an important role in neurovascular degeneration in the injured retina. Inhibition of MMP-9 may be an effective strategy for preventing and reducing neurovascular degeneration.

Archaeal Unfoldase Counteracts Protein Misfolding Retinopathy in Mice.

Deregulation of cellular proteostasis due to the failure of the ubiquitin proteasome system to dispose of misfolded aggregation-prone proteins is a hallmark of various neurodegenerative diseases in humans. Microorganisms have evolved to survive massive protein misfolding and aggregation triggered by heat shock using their protein-unfolding ATPases (unfoldases) from the Hsp100 family. Because the Hsp100 chaperones are absent in homoeothermic mammals, we hypothesized that the vulnerability of mammalian neurons to misfolded proteins could be mitigated by expressing a xenogeneic unfoldase. To test this idea, we expressed proteasome-activating nucleotidase (PAN), a protein-unfolding ATPase from thermophilic Archaea, which is homologous to the 19S eukaryotic proteasome and similar to the Hsp100 family chaperones in rod photoreceptors of mice. We found that PAN had no obvious effect in healthy rods; however, it effectively counteracted protein-misfolding retinopathy in Gγ1 knock-out mice. We conclude that archaeal PAN can rescue a protein-misfolding neurodegenerative disease, likely by recognizing misfolded mammalian proteins.SIGNIFICANCE STATEMENT This study demonstrates successful therapeutic application of an archaeal molecular chaperone in an animal model of neurodegenerative disease. Introducing the archaeal protein-unfolding ATPase proteasome-activating nucleotidase (PAN) into the retinal photoreceptors of mice protected these neurons from the cytotoxic effect of misfolded proteins. We propose that xenogeneic protein-unfolding chaperones could be equally effective against other types of neurodegenerative diseases of protein-misfolding etiology.

Trehalose reduces retinal degeneration, neuroinflammation and storage burden caused by a lysosomal hydrolase deficiency.

The accumulation of undegraded molecular material leads to progressive neurodegeneration in a number of lysosomal storage disorders (LSDs) that are caused by functional deficiencies of lysosomal hydrolases. To determine whether inducing macroautophagy/autophagy via small-molecule therapy would be effective for neuropathic LSDs due to enzyme deficiency, we treated a mouse model of mucopolysaccharidosis IIIB (MPS IIIB), a storage disorder caused by deficiency of the enzyme NAGLU (alpha-N-acetylglucosaminidase [Sanfilippo disease IIIB]), with the autophagy-inducing compound trehalose. Treated naglu-/ - mice lived longer, displayed less hyperactivity and anxiety, retained their vision (and retinal photoreceptors), and showed reduced inflammation in the brain and retina. Treated mice also showed improved clearance of autophagic vacuoles in neuronal and glial cells, accompanied by activation of the TFEB transcriptional network that controls lysosomal biogenesis and autophagic flux. Therefore, small-molecule-induced autophagy enhancement can improve the neurological symptoms associated with a lysosomal enzyme deficiency and could provide a viable therapeutic approach to neuropathic LSDs. ABBREVIATIONS: ANOVA: analysis of variance; Atg7: autophagy related 7; AV: autophagic vacuoles; CD68: cd68 antigen; ERG: electroretinogram; ERT: enzyme replacement therapy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GNAT2: guanine nucleotide binding protein, alpha transducing 2; HSCT: hematopoietic stem cell transplantation; INL: inner nuclear layer; LC3: microtubule-associated protein 1 light chain 3 alpha; MPS: mucopolysaccharidoses; NAGLU: alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); ONL: outer nuclear layer; PBS: phosphate-buffered saline; PRKCA/PKCα: protein kinase C, alpha; S1BF: somatosensory cortex; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; VMP/VPL: ventral posterior nuclei of the thalamus.

The Role of c-Jun N-Terminal Kinase (JNK) in Retinal Degeneration and Vision Loss.

c-Jun N-terminal kinase (JNK), a member of stress-induced mitogen-activated protein (MAP) kinase family, has been shown to modulate a variety of biological processes associated with neurodegenerative pathology of the retina. In particular, various retinal cell culture and animal models related to glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa indicate that JNK signaling may contribute to disease pathogenesis. This mini-review discusses the impact of JNK signaling in retinal disease, with a focus on retinal ganglion cells (RGCs), photoreceptor cells, retinal pigment epithelial (RPE) cells, and animal studies, with particular attention to modulation of JNK signaling as a potential therapeutic target for the treatment of retinal disease.

The Absence of Indoleamine 2,3-Dioxygenase Inhibits Retinal Capillary Degeneration in Diabetic Mice.

Purpose: Loss of retinal capillary endothelial cells and pericytes through apoptosis is an early event in diabetic retinopathy (DR). Inflammatory pathways play a role in early DR, yet the biochemical mechanisms are poorly understood. In this study, we investigated the role of indoleamine 2,3-dioxygenase (IDO), an inflammatory cytokine-inducible enzyme, on retinal endothelial apoptosis and capillary degeneration in the diabetic retina. Methods: IDO was detected in human and mouse retinas by immunohistochemistry or Western blotting. Interferon-γ (IFN-γ) levels were measured by ELISA. IDO levels were measured in human retinal capillary endothelial cells (HREC) cultured in the presence of IFN-γ ± 25 mM D-glucose. Reactive oxygen species (ROS) were measured using CM-H2DCFDA dye and apoptosis was measured by cleaved caspase-3. The role of IDO in DR was determined in IDO knockout (IDO-/-) mice with streptozotocin-induced diabetes. Results: The IDO and IFN-γ levels were higher in human diabetic retinas with retinopathy relative to nondiabetic retinas. Immunohistochemical data showed that IDO is present in capillary endothelial cells. IFN-γ upregulated the IDO and ROS levels in HREC. The blockade of either IDO or kynurenine monooxygenase led to inhibition of ROS in HREC. Apoptosis through this pathway was inhibited by an ROS scavenger, TEMPOL. Capillary degeneration was significantly reduced in diabetic IDO-/- mice compared to diabetic wild-type mice. Conclusions: The results suggest that the kynurenine pathway plays an important role in the inflammatory damage in the diabetic retina and could be a new therapeutic target for the treatment of DR.

A novel RPE65 inhibitor CU239 suppresses visual cycle and prevents retinal degeneration.

The retinoid visual cycle is an ocular retinoid metabolism specifically dedicated to support vertebrate vision. The visual cycle serves not only to generate light-sensitive visual chromophore 11-cis-retinal, but also to clear toxic byproducts of normal visual cycle (i.e. all-trans-retinal and its condensation products) from the retina, ensuring both the visual function and the retinal health. Unfortunately, various conditions including genetic predisposition, environment and aging may attribute to a functional decline of the all-trans-retinal clearance. To combat all-trans-retinal mediated retinal degeneration, we sought to slow down the retinoid influx from the RPE by inhibiting the visual cycle with a small molecule. The present study describes identification of CU239, a novel non-retinoid inhibitor of RPE65, a key enzyme in the visual cycle. Our data demonstrated that CU239 selectively inhibited isomerase activity of RPE65, with IC50 of 6 µM. Further, our results indicated that CU239 inhibited RPE65 via competition with its substrate all-trans-retinyl ester. Mice with systemic injection of CU239 exhibited delayed chromophore regeneration after light bleach, and conferred a partial protection of the retina against injury from high intensity light. Taken together, CU239 is a potent visual cycle modulator and may have a therapeutic potential for retinal degeneration.

A homozygous founder missense variant in arylsulfatase G abolishes its enzymatic activity causing atypical Usher syndrome in humans.

PURPOSE: We aimed to identify the cause of disease in patients suffering from a distinctive, atypical form of Usher syndrome. METHODS: Whole-exome and genome sequencing were performed in five patients from three families of Yemenite Jewish origin, suffering from distinctive retinal degeneration phenotype and sensorineural hearing loss. Functional analysis of the wild-type and mutant proteins was performed in human fibrosarcoma cells. RESULTS: We identified a homozygous founder missense variant, c.133G>T (p.D45Y) in arylsulfatase G (ARSG). All patients shared a distinctive retinal phenotype with ring-shaped atrophy along the arcades engirdling the fovea, resulting in ring scotoma. In addition, patients developed moderate to severe sensorineural hearing loss. Both vision and hearing loss appeared around the age of 40 years. The identified variant affected a fully conserved amino acid that is part of the catalytic site of the enzyme. Functional analysis of the wild-type and mutant proteins showed no basal activity of p.D45Y. CONCLUSION: Homozygosity for ARSG-p.D45Y in humans leads to protein dysfunction, causing an atypical combination of late-onset Usher syndrome. Although there is no evidence for generalized clinical manifestations of lysosomal storage diseases in this set of patients, we cannot rule out the possibility that mild and late-onset symptoms may appear.

Resveratrol Ameliorates Retinal Ischemia/Reperfusion Injury in C57BL/6J Mice via Downregulation of Caspase-3.

PURPOSE: Ischemia/reperfusion (I/R) injury induces apoptosis in retinal ganglion cells (RGCs). Resveratrol (Res) is a potent natural antioxidant with beneficial effects in many ocular diseases, such as age-related macular degeneration, diabetic retinopathy, and glaucoma. Because caspase-3 expression is highly correlated with activation of the apoptotic pathway, the present study aimed to determine whether Res regulates the expression of caspase-3 using an I/R retinal injury mouse model. METHODS: Male C57BL/6J mice were injected with Res for 2 consecutive days before I/R retinal injury. I/R retinal injury was induced by increasing the intraocular pressure for 1 h. Res was then injected for 3 consecutive days. Changes in retinal morphology were monitored for 3 days after injury by histochemistry using hematoxylin and eosin staining. mRNAs and proteins were extracted 2 days after injury. The expression levels of caspase-8 and caspase-3 mRNA and protein were determined using reverse-transcriptase polymerase chain reaction (RT-PCR) and western blot analyses. RESULTS: I/R injury induced declines in retinal thickness and number of RGCs during 5 days after injury. Caspase-8 and caspase-3 mRNA and protein activation increased. Res treatment reduced the significant loss of retinal morphology and downregulated the expression of mRNA and activation of caspase-8 and caspase-3 protein. CONCLUSIONS: The observed changes in retinal morphology suggest that I/R injury promotes retinal degeneration. Increased expression of caspase-8 and caspase-3 mRNA indicates apoptosis activation. Res, however, suppresses apoptosis via downregulation of caspase-8 and caspase-3 expression.

Impact of LCA-Associated E14L LRAT Mutation on Protein Stability and Retinoid Homeostasis.

Vitamin A (all-trans-retinol) is metabolized to the visual chromophore (11-cis-retinal) in the eyes and to all-trans-retinoic acid, a hormone like compound, in most tissues. A key enzyme in retinoid metabolism is lecithin:retinol acyltransferase (LRAT), which catalyzes the esterification of vitamin A. The importance of LRAT is indicated by pathogenic missense and nonsense mutations, which cause devastating blinding diseases. Retinoid-based chromophore replacement therapy has been proposed as treatment for these types of blindness based on studies in LRAT null mice. Here, we analyzed the structural and biochemical basis for retinal pathology caused by mutations in the human LRAT gene. Most LRAT missense mutations associated with retinal degeneration are localized within the catalytic domain, whereas E14L substitution is localized in an N-terminal α-helix, which has been implicated in interaction with the phospholipid bilayer. To elucidate the biochemical consequences of this mutation, we determined LRAT(E14L)'s enzymatic properties, protein stability, and impact on ocular retinoid metabolism. Bicistronic expression of LRAT(E14L) and enhanced green fluorescence protein revealed instability and accelerated proteosomal degradation of this mutant isoform. Surprisingly, instability of LRAT(E14L) did not abrogate the production of the visual chromophore in a cell-based assay. Instead, expression of LRAT(E14L) led to a rapid increase in cellular levels of retinoic acid upon retinoid supplementation. Thus, our study unveils the potential role of retinoic acid in the pathology of a degenerative retinal disease with important implications for the use of retinoid-based therapeutics in affected patients.

Inhibition of Matrix Metalloproteinase 9 Enhances Rod Survival in the S334ter-line3 Retinitis Pigmentosa Model.

Retinitis Pigmentosa (RP) is one of the most common forms of inherited visual loss with the initial degeneration of rod photoreceptors, followed by a progressive cone photoreceptor deterioration. Coinciding with this visual loss, the extracellular matrix (ECM) is reorganized, which alters matrix metalloproteinase (MMP) activity levels. A potential pathological role of MMPs, MMP-9 in particular, involves an excitotoxicity-mediated physiological response. In the current study, we examine the MMP-9 and MMP-2 expression levels in the rhodopsin S334ter-line3 RP rat model and investigate the impact of treatment with SB-3CT, a specific MMP-9 and MMP-2 inhibitor, on rod cell survival was tested. Retinal MMP-9 and MMP-2 expression levels were quantified by immunoblot analysis from S334ter-line3 rats compared to controls. Gelatinolytic activities of MMP-9 and MMP-2 by zymography were examined. The geometry of rod death was further evaluated using Voronoi analysis. Our results revealed that MMP-9 was elevated while MMP-2 was relatively unchanged when S334ter-line 3 retinas were compared to controls. With SB-3CT treatment, we observed gelatinolytic activity of both MMPs was decreased and diminished clustering associated with rod death, in addition to a robust preservation of rod photoreceptors. These results demonstrate that up-regulation of MMP-9 in retinas of S334ter-line3 are associated with rod death. The application of SB-3CT dramatically interferes with mechanisms leading to apoptosis in an MMP-9-dependent manner. Future studies will determine the feasibility of using SB-3CT as a potential therapeutic strategy to slow progression of vision loss in genetic inherited forms of human RP.

Lycium barbarum polysaccharides attenuates N-methy-N-nitrosourea-induced photoreceptor cell apoptosis in rats through regulation of poly (ADP-ribose) polymerase and caspase expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Lycium barbarum L., popularly known as "Goji berry", a classic of Traditional Chinese Medicine has long been used to treat ocular diseases and cardiovascular diseases. Recently, the photoreceptor cell protection of Lycium barbarum polysaccharides (LBP), a water extract from Lycium barbarum L. has received more attention. The present study was designed to investigate the effect of LBP on N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell apoptosis, and the involvement of the poly (ADP-ribose) polymerase (PARP) and caspase. MATERIALS AND METHODS: Photoreceptor cell injury was induced in male Sprague-Dawley rats by an intraperitoneal injection of MNU 60mg/kg. Seven days prior to MNU injection, LBP were intragastrical administered daily, rats were sacrificed at 24h and 7 days after MNU injection. Retinal morphologies, photoreceptor cells apoptosis, and protein expression were evaluated at 24h and 7 days after MNU injection. RESULTS: Morphologically, the outer nuclear layer was well preserved in the LBP-treated rat retinas throughout the experimental period. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) assays showed that LBP could significantly suppress the loss of photoreceptor cells, as determined by the photoreceptor cell ratio at the central retina 24h and 7 days after MNU administration. Western-blot analysis demonstrated the expression levels of procaspase-9, -7, -3 and cleaved caspase-9, -7, -3 were upregulated, and PARP were downregulated both 24h and 7 days after MNU injection. LBP treatment significantly decreased protein levels of procaspase and cleaved caspase, increased the level of PARP and cleaved PARP on 24h and 7 days. CONCLUSIONS: LBP inhibits MNU-induced rat photoreceptor cell apoptosis and protects retinal structure via the regulation of the expressions of PARP and caspase.

Alterations in glutamate cysteine ligase content in the retina of two retinitis pigmentosa animal models.

Retinitis Pigmentosa (RP) comprises a group of rare genetic retinal disorders in which one of several different mutations induces photoreceptor death. Oxidative stress and glutathione (GSH) alterations may be related to the pathogenesis of RP. GSH has been shown to be present in high concentrations in the retina. In addition, the retina has the capability to synthesize GSH. In this study, we tested whether the two subunits of glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis, and the concentrations of retinal GSH, oxidized glutathione (GSSG), cysteine (Cys) and glutamate are altered in the retina of two different RP mice models. Retinas from C3H and rd1 mice at different postnatal days (P7, P11, P15, P19, P21 and P28) and from C57BL/6 and rd10 mice at P21 were obtained. Western blot analysis was performed to determine the protein content of catalytic and modulatory subunits from glutamate cysteine ligase (GCLC and GCLM, respectively). In another set of experiments, control and rd1 mice were administered buthinine sulfoximine, a glutathione synthase inhibitor, or paraquat. GSH, GSSG, glutamate and Cys concentrations were determined, by HPLC. A decrease in retinal GCLC content was observed in C3H and rd1 mice with age, nevertheless, there was an increase in retinal GCLC in rd1 mice compared to control retinas at P19. No modifications in GCLM content with age and no difference between GCLM content in rd1 and control retinas were observed. The GSH concentration decreased in the rd1 retinas compared with control ones at P15, it increased at P19, and was again similar at P21 and P28. No changes in GSSG concentration in control retinas with age were observed; the GSSG levels in rd1 retinas were similar from P7 to P19 and then increased significantly at P21 and P28. Glutamate concentration was increased in the rd1 retinas compared to control mice from P7 to P15 and were comparable at P21 and P28. The Cys concentrations was measured in control and rd1 retinas, but no significant changes were observed between them. BSO administration decreases GSH retinal concentration in control and rd1 mice, while paraquat administration induced an increase in GSH retinal concentration in control mice and a decrease in GSH in rd1 mice retina. Retinal GCLC was significantly increased in rd10 mice at P21 as well as GSSG. Our results suggest alterations in retinal GCLC content and GSH and/or its precursors in these two RP animal models. Regulation of the enzymes related to GSH metabolism and the retinal concentration of glutamate may be a possible target to delay especially cone death in RP.

Degeneration of Photoreceptor Cells in Arylsulfatase G-Deficient Mice.

PURPOSE: Retinal degeneration is a common feature of several lysosomal storage disorders, including the mucopolysaccharidoses, a group of metabolic disorders that is characterized by widespread accumulation of glycosaminoglycans due to lysosomal enzyme dysfunction. We used a new mouse model of mucopolysaccharidosis IIIE to study the effect of Arylsulfatase G (ARSG) deficiency on retina integrity. METHODS: The retina of Arsg knockout mice aged 1 to 24 months was studied by immunohistochemistry and Western blot analysis. Electron microscopic analyses were performed on retinas from 15- and 22-month-old animals. Photoreceptor and microglia cell numbers and retina thickness were determined to quantitatively characterize retinal degeneration in ARSG-deficient mice. RESULTS: Arsg knockout mice showed a progressive degeneration of photoreceptor cells starting between 1 and 6 months of age, resulting in the loss of more than 50% of photoreceptor cells in 24-month-old mice. Photoreceptor loss was accompanied by reactive astrogliosis, reactive microgliosis that was evident in the outer but not inner retina, and elevated expression levels of some lysosomal proteins. Electron microscopic analyses of retinas revealed no evidence for the presence of storage vacuoles. Of note, expression of ARSG protein in wild-type mice was detectable only in the RPE which, however, appeared morphologically unaffected in knockout mice at the electron microscopic level. CONCLUSIONS: To our knowledge, this is the first study demonstrating that ARSG deficiency results in progressive photoreceptor degeneration and dysregulation of various lysosomal proteins.