Retinal Degeneration In Mice Research Articles

Tissue inhibitor of metalloproteinases 1 enhances rod survival in the rd1 mouse retina.

Retinitis pigmentosa (RP), an inherited retinal degenerative disease, is characterized by a progressive loss of rod photoreceptors followed by loss of cone photoreceptors. Previously, when tissue inhibitor of metalloproteinase 1 (TIMP1), a key extracellular matrix (ECM) regulator that binds to and inhibits activation of Matrix metallopeptidase 9 (MMP9) was intravitreal injected into eyes of a transgenic rhodopsin rat model of RP, S334ter-line3, we discovered cone outer segments are partially protected. In parallel, we reported that a specific MMP9 and MMP2 inhibitor, SB-3CT, interferes with mechanisms leading to rod photoreceptor cell death in an MMP9 dependent manner. Here, we extend our initial rat studies to examine the potential of TIMP1 as a treatment in retinal degeneration by investigating neuroprotective effects in a classic mouse retinal degeneration model, rdPde6b-/- (rd1). The results clearly demonstrate that intravitreal injections of TIMP1 produce extended protection to delay rod photoreceptor cell death. The mean total number of rods in whole-mount retinas was significantly greater in TIMP-treated rd1 retinas (postnatal (P) 30, P35 (P<0.0001) and P45 (P<0.05) than in saline-treated rd1 retinas. In contrast, SB-3CT did not delay rod cell death, leading us to further investigate alternative pathways that do not involve MMPs. In addition to inducing phosphorylated ERK1/2, TIMP1 significantly reduces BAX activity and delays attenuation of the outer nuclear layer (ONL). Physiological responses using scotopic electroretinograms (ERG) reveal b-wave amplitudes from TIMP1-treated retinas are significantly greater than from saline-treated rd1 retinas (P<0.05). In later degenerative stages of rd1 retinas, photopic b-wave amplitudes from TIMP1-treated rd1 retinas are significantly larger than from saline-treated rd1 retinas (P<0.05). Our findings demonstrate that TIMP1 delays photoreceptor cell death. Furthermore, this study provides new insights into how TIMP1 works in the mouse animal model of RP.

Increased proteasomal activity supports photoreceptor survival in inherited retinal degeneration

Inherited retinal degenerations, affecting more than 2 million people worldwide, are caused by mutations in over 200 genes. This suggests that the most efficient therapeutic strategies would be mutation independent, i.e., targeting common pathological conditions arising from many disease-causing mutations. Previous studies revealed that one such condition is an insufficiency of the ubiquitin–proteasome system to process misfolded or mistargeted proteins in affected photoreceptor cells. We now report that retinal degeneration in mice can be significantly delayed by increasing photoreceptor proteasomal activity. The largest effect is observed upon overexpression of the 11S proteasome cap subunit, PA28α, which enhanced ubiquitin-independent protein degradation in photoreceptors. Applying this strategy to mice bearing one copy of the P23H rhodopsin mutant, a mutation frequently encountered in human patients, quadruples the number of surviving photoreceptors in the inferior retina of 6-month-old mice. This striking therapeutic effect demonstrates that proteasomes are an attractive target for fighting inherited blindness.

Translational attenuation and retinal degeneration in mice with an active integrated stress response

An integrated stress response (ISR), identified in several different animal models of inherited retinal degeneration (IRD), is activated following various cellular stresses. The ISR results in the phosphorylation of eIF2α (p-eIF2α) and a consequent halt in protein synthesis. Although generally protective, persistent elevations in p-eIF2α could lead to cell demise. Therefore, we aimed to determine whether ISR activation is associated with diminished translation rates in mice with IRD. Retinal protein extracts from rd16 mice at different time points were analyzed and the retinal levels of protein synthesis were assessed using the SUnSET method. We found that rd16 mice experience persistent ISR activation: p-eIF2α, ATF4, and CHOP were significantly upregulated at P15 and P20. In agreement with ISR activation, we found that rd16 mice experience translational attenuation at P15. Similar to rd16, other IRD models, T17M RHO, and rd10 also demonstrated a decline in protein synthesis, correlating with p-eIF2α elevation. We then assessed the role of PERK and eIF2α in translational attenuation in rd16 using a PERK inhibitor, GSK2606414. We found that while the treatment significantly reduced p-eIF2α, it did not cause a complete recovery in translation. This suggests that eIF2α is not the only or even the primary point of translational control in IRD, and a second node of translational regulation comprising AKT and mTOR should be evaluated. Surprisingly, we found that AKT-mTOR signaling was diminished in rd16 and rd10 retinas, suggesting a potential link between AKT-mTOR and translational inhibition. Therefore, for the first time, this study shows translation attenuation in IRD models, and highlights the potential roles of eIF2α kinases and AKT-mTOR signaling that could grant valuable insight into the potential treatments for IRD.

All-optical recording and stimulation of retinal neurons in vivo in retinal degeneration mice.

Here we demonstrate the application of a method that could accelerate the development of novel therapies by allowing direct and repeatable visualization of cellular function in the living eye, to study loss of vision in animal models of retinal disease, as well as evaluate the time course of retinal function following therapeutic intervention. We use high-resolution adaptive optics scanning light ophthalmoscopy to image fluorescence from the calcium sensor GCaMP6s. In mice with photoreceptor degeneration (rd10), we measured restored visual responses in ganglion cell layer neurons expressing the red-shifted channelrhodopsin ChrimsonR over a six-week period following significant loss of visual responses. Combining a fluorescent calcium sensor, a channelrhodopsin, and adaptive optics enables all-optical stimulation and recording of retinal neurons in the living eye. Because the retina is an accessible portal to the central nervous system, our method also provides a novel non-invasive method of dissecting neuronal processing in the brain.

Leaves of Acer palmatum thumb. Rescues N-ethyl-N-nitrosourea (ENU)-Induced retinal degeneration in mice

BackgroundIn the East Asia, the genus Acer (Aceraceae) is a herbal medicine that is used to treat various diseases, including hemostasis, hepatic disorders, traumatic bleeding and poor eyesight. However, the effects of Acer palmatum thumb. on retinal degeneration are unknown. AimIn this study, we investigated whether Acer palmatum thumb.ethanol extract (KIOM-2015E) can protect eyes from retinal degeneration. Our research investigated whether KIOM-2015E could have a protective effect in the retinal degenerating mouse model induced by N-ethyl-N-nitrosourea (ENU). Materials and methodsRetinal degeneration was induced by a single intraperitoneal injection of ENU in ICR mice. KIOM-2015E (100, 200 mg/kg) was orally administered once per day. The eyeballs were embedded and lysed after drug administration to examine the histological changed and protein expression levels. ResultsThe ENU-induced retinal degeneration model exhibited increased photoreceptor cell death and a loss of the outer nuclear layer. Additionally, the expression of PKCα and OPN1SW was reduced, and that of GFAP and Nestin was increased in ENU-treated retinal tissues. ConclusionKIOM-2015E treatment ameliorated the ENU-induced retinal degeneration. KIOM-2015E prevents ENU-induced retinal degeneration by modulating protein expression and the thickness of the outer nuclear layer in the retina.

HIF-1α stabilization reduces retinal degeneration in a mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies characterized by progressive and irreversible loss of vision due to rod and cone degeneration. Evidence suggests that an inappropriate oxygen level could contribute to its pathogenesis. Rod cell death could increase oxygen concentration, reduce hypoxia-inducible factor 1 (HIF-1α) and contribute to cone cell death. The purposes of this study were: 1) to analyze the temporal profile of HIF-1α, its downstream effectors VEGF, endothelin-1 (ET-1), iNOS, and glucose transporter 1 (GLUT1), and neuroinflammation in retinas of the murine model of rd10 ( retinal degeneration 10) mice with RP; 2) to study oxygen bioavailability in these retinas; and 3) to investigate how stabilizing HIF-1α proteins with dimethyloxaloglycine (DMOG), a prolyl hydroxylase inhibitor, affects retinal degeneration, neuroinflammation, and antioxidant response in rd10 mice. A generalized down-regulation of HIF-1α and its downstream targets was detected in parallel with reactive gliosis, suggesting high oxygen levels during retinal degeneration. At postnatal d 18, DMOG treatment reduced photoreceptor cell death and glial activation. In summary, retinas of rd10 mice seem to be exposed to a hyperoxic environment even at early stages of degeneration. HIF-1α stabilization could have a temporal neuroprotective effect on photoreceptor cell survival, glial activation, and antioxidant response at early stages of RP.-Olivares-González, L., Martínez-Fernández de la Cámara, C., Hervás, D., Millán, J. M., Rodrigo, R. HIF-1α stabilization reduces retinal degeneration in a mouse model of retinitis pigmentosa.

Intravitreal Injection of Hydrogen Peroxide Induces Acute Retinal Degeneration, Apoptosis, and Oxidative Stress in Mice.

Purpose Oxidative stress is a common pathological condition for multiple retinal diseases. Hydrogen peroxide (H2O2) has been applied as an oxidative stress inducer for the in vitro studies. Here, we report the in vivo effect of H2O2 exposure to the mouse retina and its underlying mechanism. Methods The H2O2 or saline solution was intravitreally injected into the eyes of female C57BL/6J mice for two consecutive days. The retinal structure was evaluated by in vivo imaging using spectral domain optical coherence tomography (OCT) and validated by histological assessment as well as retinal marker expression. In addition, retinal stress, cell apoptosis, and antioxidant enzyme expression were also determined. Results Retinal and outer nuclear layer thickness thinning was observed at days 7 and 14 by OCT imaging with the treatment of 10 μg H2O2, which was confirmed by the histopathological analysis. The expressions of photoreceptor (Rho, Rora, Rorb, and Rcvrn), bipolar cell (Chat and Calb2), and retinal pigment epithelial (Rpe65) markers were reduced in the H2O2-treated group, whereas the expression of retinal ganglion cell marker (Tubb3) was increased. TUNEL-positive cells were obviously found in the outer nuclear layer and inner nuclear layer of H2O2-treated mice but sparely found in the ganglion cell layer. Coherently, apoptotic gene expressions (Casp3, Casp9, Bax, and Parp8) were significantly increased in the retina with increasing dosages of H2O2, while Bcl2 expression was mildly decreased. In addition, the expressions of Gfap and antioxidant enzyme genes (Txn2, Sod2, and Gpx4) were significantly upregulated in the retina after the H2O2 treatment, compared to the vehicle control group. Conclusions This study revealed that intravitreal injection of H2O2 induces acute retinal damage by increasing oxidative stress and cell apoptosis in the retina. This acute retinal degeneration mouse model could provide a platform for drug screening against oxidative stress and retinal diseases.

Intraperitoneal administration of adipose tissue-derived stem cells for the rescue of retinal degeneration in a mouse model via indigenous CNTF up-regulation by IL-6.

As the world's population begins to age, retinal degeneration is an increasing problem, and various treatment modalities are being developed. However, there have been no therapies for degenerative retinal conditions that are not characterized by neovascularization. We investigated whether transplantation of mouse adipose tissue-derived stem cells (mADSC) into the intraperitoneal space has a rescue effect on NaIO3 -induced retinal degeneration in mice. In this study, mADSC transplantation recovered visual function and preserved the retinal outer layer structure compared to the control group without any integration of mADSC into the retina. Moreover, endogenous ciliary neurotrophic factor (CNTF) was elevated in the retinas of mADSC-treated mice. We found that lipopolysaccharide (LPS) or LPS-stimulated monocyte supernatant induced the secretion of granulocyte colony stimulating factor (GCSF), CD54, CXCL10, interleukin-6 (IL-6), and CCL5 from the mADSC by cytokine array. Network inference was conducted to investigate signaling networks related to CNTF regulation. Based on bioinformatics data, the expression of IL-6 was related to the expression of CNTF. Additionally, intravitreal injection of IL-6 in rats produced up-regulation of endogenous CNTF in the retina. mADSC had a rescue effect on retinal degeneration through the up-regulation of endogenous CNTF by IL-6. Thus, transplantation of mADSC could be a potential treatment option for retinal degeneration.

Involvement of cannabinoid receptor type 2 in light-induced degeneration of cells from mouse retinal cell line in vitro and mouse photoreceptors in vivo

Earlier studies showed that the expressions of the agonists of the cannabinoid receptors are reduced in the vitreous humor of patients with age-related macular degeneration (AMD), and the cannabinoid type 2 receptor is present in the retinas of rats and monkeys. The purpose of this study was to determine whether the cannabinoid type 2 receptor is involved in the light-induced death of cultured 661W cells, an immortalized murine retinal cell line, and in the light-induced retinal degeneration in mice. Time-dependent changes in the expression and location of retinal cannabinoid type 2 receptor were determined by Western blot and immunostaining. The cannabinoid type 2 receptor was down-regulated in murine retinae and cone cells. In the in vitro studies, HU-308, a cannabinoid type 2 receptor agonist, had a protective effect on the light-induced death of 661W cells, and this effect was attenuated by SR144528, a cannabinoid type 2 receptor antagonist. Because the cannabinoid type 2 receptor is a G-protein coupled receptor and is coupled with Gi/o protein, we investigated the effects of the cAMP-dependent protein kinase (PKA). HU-308 and H89, a PKA inhibitor, deactivated PKA in retinal cone cells, and H89 also suppressed light-induced cell death. For the in vivo studies, a cannabinoid type 2 receptor agonist, HU-308, or an antagonist, SR144528, was injected intravitreally into mouse eyes before the light exposure. Electroretinography was used to determine the physiological status of the retinas. Injection of HU-308 improved the a- and b-waves of the ERGs and also the thickness of the outer nuclear layer of the murine retina after light exposure. These findings indicate that the cannabinoid type 2 receptor is involved in the light-induced retinal damage through PKA signaling. Thus, activation of cannabinoid type 2 receptor may be a therapeutic approach for light-associated retinal diseases.

Conditional loss of Spata7 in photoreceptors causes progressive retinal degeneration in mice

The mammalian retina consists of multiple cell layers including photoreceptor cells, which are light sensing neurons that play essential functions in the visual process. Previously, we identified mutations in SPATA7, encoding spermatogenesis associated protein 7, in families with Leber Congenital Amaurosis (LCA) and juvenile Retinitis Pigmentosa (RP), and showed that Spata7 null mice recapitulate the human disease phenotype of retinal degeneration. SPATA7 is expressed in the connecting cilium of photoreceptor (PR) cells in the mouse retina, as well as in retinal pigment epithelium (RPE) cells, but the functional role of Spata7 in the RPE remains unknown. To investigate whether Spata7 is required in PRs, the RPE, or both, we conditionally knocked out Spata7 in photoreceptors and RPE cells using Crx-Cre and Best1-Cre transgenic mouse lines, respectively. In Spata7 photoreceptor-specific conditional (cKO) mice, both rod and cone photoreceptor dysfunction and degeneration is observed, characterized by progressive thinning of the outer nuclear layer and reduced response to light; however, RPE-specific deletion of Spata7 does not impair retinal function or cell survival. Furthermore, our findings show that both Rhodopsin and RPGRIP1 are mislocalized in the Spata7Flox/-; Crx-Cre cKO mice, suggesting that loss of Spata7 in photoreceptors alone can result in altered trafficking of these proteins in the connecting cilium. Together, our findings suggest that loss of Spata7 in photoreceptors alone is sufficient to cause photoreceptor degeneration, but its function in the RPE is not required for photoreceptor survival; therefore, loss of Spata7 in photoreceptors alters both rod and cone function and survival, consistent with the clinical phenotypes observed in LCA and RP patients with mutations in SPATA7.

Dynamic changes of FasL protein expression in retinal tissue with aging in retinal degeneration mice

Background Retinal transplantation is a new approach to the treatment of retinal degeneration diseases, and how to avoid or reduce the immune rejection after transplantation is a problem.In experimental studies on retinal transplantation, C57BL/6 mice are often used as donors and rd mice serve as recipients.Studies showed that Fas ligand (FasL) protein induces the apoptosis of Fas+ inflammatory cells by FasL/Fas signal pathway, speculating that FasL protein is associated with immune rejection after transplantation. Objective The aim of this study was to investigate FasL protein expression change in retinas of C57BL/6 mice and rd mice with aging and reveal the immune characteristics of the mouse retinas. Methods The frozen sections of eyeball from C57BL/6 mice and rd mice at the age of postnatal (PN)-0 week, PN-1 week, PN-2 week, PN-3 week and PN-4 week were prepared.The expression of FasL protein in the mouse retinas was examined by immnofluorescense technique.Images were acquired by fluorescence microscope and analyzed semi-quantitively by software from laser scanning confocal microscope as the fluorescence intensity (FI). The results were compared among different strains of mice. Results The retina developed imperfectly in PN-1 week C57BL/6 mice and FasL protein was positively expressed in the whole retina.In PN-2, 3 and 4 week C57BL/6 mice, retinas finished the development with 10 layers, and retinal pigment epithelium (RPE), inner segment (IS), outer limiting membrane (OLM), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL). Retinal structure and expression of FasL protein in the whole retina of rd mice in the PN-1 week were similar with C57BL/6 mice, however, ONL cells of rd mice were evidently decreased with aging.The RPE, OPL, INL, IPL and GCL expressed the FasL protein in PN-2, PN-3 and PN-4 week rd mice.The mean FI of FasL protein in the RPE layer was 184.199±16.747, 186.797±7.904 and 184.319±18.795 in rd mice of PN-2 week, PN-3 week and PN-4 week, which were significantly higher than 160.402±22.851, 160.995±22.799 and 105.787±17.676 in C57BL/6 mice (t=-3.360, P=0.002; t'=-4.277, P=0.000; t=-12.175, P=0.000). There were not significant differences in the mean FI of FasL protein in IPL between C57BL/6 mice and rd mice at ages of PN-2 week, PN-3 week and PN-4 week (all at P>0.05). Conclusions The cells of retinal ONL are gradually decreased with the development of rd mice, and FasL protein expression intensity in RPE is evidently enhanced in rd mice compared with C57BL/6 mice. Key words: Fas ligand protein; Retina/immunology; Pigment epithelium of eye/immunology; Apoptosis; Immune privilege; Retinitis pigmentosa; Mice, inbred, C57BL; rd mice

A kind of rd1 mouse in C57BL/6J mice from crossing with a mutated Kunming mouse

We occasionally discovered a mouse with spontaneous retinitis pigmentosa (RP) from Kunming (KM) mouse breeding colony, with no obvious waveforms in ERG recordings. The aim of this study is to cross the spontaneously hereditary retinal degeneration mice (temporarily designated as KM/rd mice) derived from KM mice with C57BL/6J mice to establish a congenic inbred strain (temporarily designated as the B6/rd mice), and study the ocular phenotype and genotype of the mice. Fundus photography, tissue morphology, electroretinography (ERG), qRT-PCR, western blot and DNA sequence analysis were performed to observe the ocular phenotype and genotype of KM/rd and B6/rd mice. The fundus photography showed progressive retinal vascular degeneration and depigmentation in KM/rd and B6/rd mice. Compared to wild-type mice, the histological analysis revealed that the outer nuclear layer of the mutated mice was significantly reduced at 14days post born (P14), and almost disappeared by P21. No obvious waveforms were detected at P14 and P21 in the ERG from KM/rd and B6/rd mice. qRT-PCR results showed that the expression quantities of mRNA of pde6b gene in KM/rd and B6/rd mice were significantly lower compared with those of wild-type controls at P21. Western blot results confirmed an abnormal protein expression of pde6b gene in KM/rd and B6/rd mice with no protein products, while there was an obvious protein expression in wild-type mice. The nonsense mutation in exon 7 (a mutation that changes the codon 347 from TAC to TAA) in the pde6b gene of KM/rd and B6/rd mice was identified by genomic DNA sequence analysis. All these findings revealed that the ocular phenotype and genotype of KM/rd and B6/rd mice were similar to those of rd1 mice, which indicates that KM/rd and B6/rd mice can be used as an RP mouse model.

Grafted c-kit+/SSEA1- eye-wall progenitor cells delay retinal degeneration in mice by regulating neural plasticity and forming new graft-to-host synapses.

BackgroundDespite diverse pathogenesis, the common pathological change observed in age-related macular degeneration and in most hereditary retinal degeneration (RD) diseases is photoreceptor loss. Photoreceptor replacement by cell transplantation may be a feasible treatment for RD. The major obstacles to clinical translation of stem cell-based cell therapy in RD remain the difficulty of obtaining sufficient quantities of appropriate and safe donor cells and the poor integration of grafted stem cell-derived photoreceptors into the remaining retinal circuitry.MethodsEye-wall c-kit+/stage-specific embryonic antigen 1 (SSEA1)− cells were isolated via fluorescence-activated cell sorting, and their self-renewal and differentiation potential were detected by immunochemistry and flow cytometry in vitro. After labeling with quantum nanocrystal dots and transplantation into the subretinal space of rd1 RD mice, differentiation and synapse formation by daughter cells of the eye-wall c-kit+/SSEA1− cells were evaluated by immunochemistry and western blotting. Morphological changes of the inner retina of rd1 mice after cell transplantation were demonstrated by immunochemistry. Retinal function of rd1 mice that received cell grafts was tested via flash electroretinograms and the light/dark transition test.ResultsEye-wall c-kit+/SSEA1− cells were self-renewing and clonogenic, and they retained their proliferative potential through more than 20 passages. Additionally, eye-wall c-kit+/SSEA1− cells were capable of differentiating into multiple retinal cell types including photoreceptors, bipolar cells, horizontal cells, amacrine cells, Müller cells, and retinal pigment epithelium cells and of transdifferentiating into smooth muscle cells and endothelial cells in vitro. The levels of synaptophysin and postsynaptic density-95 in the retinas of eye-wall c-kit+/SSEA1− cell-transplanted rd1 mice were significantly increased at 4 weeks post transplantation. The c-kit+/SSEA1− cells were capable of differentiating into functional photoreceptors that formed new synaptic connections with recipient retinas in rd1 mice. Transplantation also partially corrected the abnormalities of inner retina of rd1 mice. At 4 and 8 weeks post transplantation, the rd1 mice that received c-kit+/SSEA1− cells showed significant increases in a-wave and b-wave amplitude and the percentage of time spent in the dark area.ConclusionsGrafted c-kit+/SSEA1− cells restored the retinal function of rd1 mice via regulating neural plasticity and forming new graft-to-host synapses.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0451-8) contains supplementary material, which is available to authorized users.

Fundus Camera-Delivered Light-Induced Retinal Degeneration in Mice With the RPE65 Leu450Met Variant is Associated With Oxidative Stress and Apoptosis.

PurposeOxidative stress, partly due to light, has an important role in many retinal diseases, including macular degeneration and retinal dystrophies. The Leu450Met variant of RPE65 is expressed in C57BL/6 and in many genetically modified mice. It confers significant resistance to light induced retinal degeneration (LIRD). Our goal was to develop an effective and efficient method to induce LIRD in resistant mice that would recapitulate mechanisms seen in known models of LIRD.MethodsThe retinas of C57BL/6J mice were exposed to light using a murine fundus camera. Two protocols (with and without intraperitoneal fluorescein) were used. Optical coherence tomography (OCT) helped determine the location and extent of retinal damage. Histology, TUNEL assay, quantitative (q) PCR, and immunohistochemistry were performed.ResultsBoth protocols consistently generated LIRD in C57BL/6J mice. Optical coherence tomography and histology demonstrated that retinal damage starts at the level of the photoreceptor/outer retina and is more prominent in the superior retina. Fundus camera-delivered light-induced retinal degeneration (FCD-LIRD) is associated with apoptosis, subretinal microglia/macrophages, increased expression of oxidative stress response genes, and C3d deposition.ConclusionsWe characterize two new models of light-induced retinal degeneration that are effective in C57BL/6J mice, and can be modulated in terms of severity. We expect FCD-LIRD to be useful in exploring mechanisms of LIRD in resistant mice, which will be important in increasing our understanding of the retinal response to light damage and oxidative stress.

Neuroprotective effect of NecroX-5 against retinal degeneration in rodents.

NecroX-5 is a derivative of cyclopentylamino carboxymethylthiazolylindole (NecroX), an inhibitor of necrosis/necroptosis. NecroX-5 has been shown to scavenge mitochondrial reactive oxygen and nitrogen species, and thus preventing necrotic cell death against various kinds of oxidative stress in several tissues, including the brain. To examine the effect of NecroX-5 on retinal degeneration (RD), RD was induced in Sprague-Dawley rats by an intraperitoneal injection of N-methyl-N-nitrosourea and in BALB/c mice by blue light-emitting diode exposure. Scotopic electroretinography recording was used to evaluate retinal function. For histological evaluation, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunohistochemistry were performed. Electroretinography recordings showed that a-waves and b-waves were significantly reduced in both RD rats and mice, whereas the amplitudes of both waves were significantly increased in both NecroX-5-treated RD rats and mice compared with untreated RD animals. In hematoxylin and eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, the outer nuclear layer where photoreceptors reside appeared to be more preserved, and there were fewer apoptotic cells in NecroX-5-treated RD retinas than in untreated RD retinas. In addition, immunohistochemistry with antiglial fibrillary acidic protein and anti-8-hydroxy-2'-deoxyguanosine showed lower levels of retinal injury and oxidative stress in NecroX-5-treated RD retinas than in untreated RD retinas. These results indicated that NecroX-5 protects retinal neurons from experimentally induced RD, suggesting that NecroX-5 may have a potential for the treatment of RD as a medication.

Retinal Degeneration in Mice Deficient in the Lysosomal Membrane Protein CLN7.

Neuronal ceroid lipofuscinoses comprise a genetically heterogeneous group of mainly childhood-onset neurodegenerative lysosomal storage disorders. Progressive loss of vision is among the typical clinical symptoms of these fatal disorders. Here, we performed a detailed analysis of retinal degeneration in mice deficient in the lysosomal membrane protein CLN7, a novel animal model of CLN7 disease. Immunohistochemical analyses of retinas at different ages were performed to qualitatively and quantitatively characterize retinal degeneration in CLN7-deficient mice. Storage material in mutant retinas was analyzed by electron microscopy, and expression levels of various lysosomal proteins were studied using immunohistochemistry, immunoblot analyses, and quantitative real-time PCR. We observed an early onset and rapidly progressing degeneration of photoreceptor cells in CLN7-deficient mice, resulting in the loss of more than 70% rod photoreceptors in 4-month-old animals. The number of cone photoreceptors was not detectably altered at this age. Loss of rod photoreceptors was accompanied by reactive astrogliosis and microgliosis. Immunohistochemical and immunoblot analyses revealed accumulation of subunit c of mitochondrial ATP synthase and saposin D in mutant retinas, and electron microscopic analyses demonstrated the presence of curvilinear bodies or fingerprint-like profiles in various cell types of CLN7-deficient retinas. We also found a marked dysregulation of various lysosomal proteins in mutant retinas. We conclude that the retina of CLN7-deficient mice represents a useful model to elucidate the pathomechanisms ultimately leading to neurodegeneration in CLN7 disease, and to evaluate the efficacy of strategies aimed at developing treatments for this fatal neurodegenerative lysosomal storage disorder.

A novel AhR ligand, 2AI, protects the retina from environmental stress.

Various retinal degenerative diseases including dry and neovascular age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy are associated with the degeneration of the retinal pigmented epithelial (RPE) layer of the retina. This consequently results in the death of rod and cone photoreceptors that they support, structurally and functionally leading to legal or complete blindness. Therefore, developing therapeutic strategies to preserve cellular homeostasis in the RPE would be a favorable asset in the clinic. The aryl hydrocarbon receptor (AhR) is a conserved, environmental ligand-dependent, per ARNT-sim (PAS) domain containing bHLH transcription factor that mediates adaptive response to stress via its downstream transcriptional targets. Using in silico, in vitro and in vivo assays, we identified 2,2′-aminophenyl indole (2AI) as a potent synthetic ligand of AhR that protects RPE cells in vitro from lipid peroxidation cytotoxicity mediated by 4-hydroxynonenal (4HNE) as well as the retina in vivo from light-damage. Additionally, metabolic characterization of this molecule by LC-MS suggests that 2AI alters the lipid metabolism of RPE cells, enhancing the intracellular levels of palmitoleic acid. Finally, we show that, as a downstream effector of 2AI-mediated AhR activation, palmitoleic acid protects RPE cells from 4HNE-mediated stress, and light mediated retinal degeneration in mice.

Depletion of BBS Protein LZTFL1 Affects Growth and Causes Retinal Degeneration in Mice

Bardet-Biedl syndrome (BBS) is a heterogeneous disease characterized by deficiencies in various organs that are caused by defects in genes involved in the genesis, structural maintenance, and protein trafficking of cilia. Leucine zipper transcription factor-like 1 (LZTFL1) has been identified as a BBS protein (BBS17), because patients with mutations in this gene exhibit the common BBS phenotypes. In this study, we generated a knockout mouse model to investigate the effects of LZTFL1 depletion. Lztfl1 knockout mice were born with low birth weight, reached similar weight to those of wild-type mice at 10 weeks of age, and later gained more weight than their wild-type counterparts. LZTFL1 was localized to the primary cilium of kidney cells, and the absence of LZTFL1 increased the ciliary localization of BBS9. Moreover, in the retinas of Lztfl1 knockout mice, the photoreceptor outer segment was shortened, the distal axoneme of photoreceptor connecting cilium was significantly enlarged, and rhodopsin was targeted to the outer nuclear layer. TUNEL assay showed that many of these abnormal photoreceptor cells in Lztfl1 knockout mice underwent apoptosis. Interestingly, the absence of LZTFL1 caused an abnormal increase of the adaptor protein complex 1 (AP1) in some photoreceptor cells. Based on these data, we conclude that LZTFL1 is a cilium protein and regulates animal weight and photoreceptor connecting cilium function probably by controlling microtubule assembly and protein trafficking in cilia.

270. Optogenetic Activation of Remnant Cone Cells Can Restore Ganglion Cell Light Responses in a Canine Model of Retinitis Pigmentosa

In retinitis pigmentosa, the rod photoreceptor cells are generally lost first followed by a slow loss of cone cells. Remnant cone cell bodies have been observed even after many years of vision loss in retinitis pigmentosa patients. Proof-of-principle studies in retinal degeneration mice have demonstrated vision restoration upon expression of the optogene halorhodopsin in remnant cone cells. However, whether this will work in a large animal model of retinal degeneration is unclear. Here we look at light responses and histology post-treatment with halorhodopsin in a dog model of autosomal recessive retinitis pigmentosa. Halorhodopsin was expressed in the cone cells unilaterally in rcd1 dogs (PDE6β mutation) with mid- to late stage retinal degeneration using recombinant adeno-associated viral vectors. The retinas were imaged using OCT and function was evaluated using ERG and pupillometry. The spiking activity of retinal ganglion cells (RGCs) in retinal patches adjacent to and distant from the point of injection was tested using multi electrode array recordings. Retinal patches were stimulated with 589 nm light of different durations/intensities. OCT revealed significant thinning of the photoreceptor layer in the retinas of the dogs. While the ERG and pupillometry data did not show a significant difference between the treated and untreated eyes, multi-electrode array recordings revealed a restoration of ganglion cell light responses (ON, OFF and ON/OFF-responses to discrete flashes and responses to 4Hz flicker, 589nm light) in the treated retinal patch compared to an untreated control patch of retina. The lowest intensities where responses become clearly detectable for two animals were 0.1 mW/cm2 and 2.8 mW/cm2 probably reflecting different levels of NpHR expression. We observed halorhodopsin expression by histology in the remnant cone cell bodies of the treated retinas. Restoration of ganglion cell responses in the treated retinal patches suggests that the retina can be sensitized to light even after major photoreceptor cell loss. Ongoing work will determine if this restoration of ganglion cell light responses in the dogs translate to being able to navigate in the light.

Retrograde Melanopsin Signaling Increases With Age in Retinal Degenerate Mice Lacking Rods and the Majority of Cones.

Following on from reports of retrograde retinal signaling in mice, we sought to investigate the influence of age and retinal location on this phenomenon using mice that lack rods and the majority of cones. We used functional anatomy for c-fos (Fos) and tyrosine hydroxylase (TH) to measure light-driven activation of dopamine neurons along a dorsal-ventral transect in C3H/He wild-type and rodless-coneless rd/rd cl (rdcl) mice aged 3, 5, and >14 months. A parallel series of retinae from 3-month-old mice was also stained for cone opsins and melanopsin. Analysis by confocal microscopy revealed light-driven Fos activation in TH cells residing in the middorsal retina of the youngest rdcl mice. This region was largely devoid of residual cones but contained a large number of intrinsically photosensitive retinal ganglion cells (ipRGCs) and the highest density of melanopsin neurites. With advancing age, there was a paradoxical increase in retrograde signaling from ∼3% Fos-positive (Fos+) TH cells at 3 months to ∼36% in rdcl mice >14 months. This increased activation occurred in more central and peripheral retinal regions. Our data provide new insights into the anatomy and plasticity of retrograde melanopsin signaling in mice with severe rod/cone dystrophy. The increased retrograde signaling we detect may result from either an increased potency of melanopsin signaling with advancing age and/or postsynaptic modification to dopaminergic neurons.