Haoyu Mao

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.

AAV Delivery of Wild-Type Rhodopsin Preserves Retinal Function in a Mouse Model of Autosomal Dominant Retinitis Pigmentosa

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.

Long-Term Rescue of Retinal Structure and Function by Rhodopsin RNA Replacement with a Single Adeno-Associated Viral Vector in P23H RHO Transgenic Mice

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.

Mitochondrial oxidative stress in the retinal pigment epithelium leads to localized retinal degeneration.

PURPOSE Oxidative stress in the RPE is widely accepted as a contributing factor to AMD. We have previously shown that ribozyme-mediated reduction in the antioxidant enzyme manganese superoxide dismutase (MnSOD) leads to some of the features of geographic atrophy in mice. To develop a mouse model independent of viral injection, we used a conditional knockout of the Sod2 gene in the RPE to elevate mitochondrial oxidative stress in that cell layer. METHODS Experimental mice in which exon 3 of Sod2 was flanked by loxP sites were also transgenic for PVMD2-rtTA and tetO-PhCMV cre, so that cre recombinase was expressed only in the RPE. Pups of this genotype (Sod2(flox/flox)VMD2cre) were induced to express cre recombinase by feeding doxycycline-laced chow to nursing dams. Controls included mice of this genotype not treated with doxycycline and doxycycline-treated Sod2(flox/flox) mice lacking the cre transgene. Expression of cre in the RPE was verified by immunohistochemistry, and deletion of Sod2 exon 3 in the RPE was confirmed by PCR. Mice were followed up over a period of 9 months by spectral-domain optical coherence tomography (SD-OCT), digital fundus imaging, and full-field ERG. Following euthanasia, retinas were examined by light and electron microscopy or by immunohistochemistry. Contour length of rod outer segments and thickness of the RPE layer were measured by unbiased stereology. RESULTS Following doxycycline induction of cre, Sod2(flox/flox) cre mice demonstrated increased signs of oxidative stress in the RPE and accumulation of autofluorescent material by age 2 months. They showed a gradual decline in the ERG response and thinning of the outer nuclear layer (by SD-OCT), which were statistically significant by 6 months. In addition, OCT and electron microscopy revealed increased porosity of the choroid. At the same interval, hypopigmented foci appeared in fundus micrographs, and vascular abnormalities were detected by fluorescein angiography. By 9 months, the RPE layer in Sod2(flox/flox) cre mice was thicker than in nontransgenic littermates, and the rod outer segments were significantly longer over most of the retina, although localized atrophy of photoreceptors was also obvious in some eyes. CONCLUSIONS Conditional tissue-specific reduction in MnSOD induced oxidative stress in mouse RPE, leading to RPE dysfunction, damage to the choroid, and death of photoreceptor cells. The RPE oxidative stress did not cause drusen-like deposits, but the model recapitulated certain key aspects of the pathology of dry AMD and may be useful in testing therapies.

The 5HT1a Receptor Agonist 8-Oh DPAT Induces Protection from Lipofuscin Accumulation and Oxidative Stress in the Retinal Pigment Epithelium

Age-related macular degeneration (AMD), a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT1A receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagy-derived and photoreceptor outer segment (POS)-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE) cells in the presence or absence of the 5-HT1A receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 µM H2O2 was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD) levels and the ratio of reduced glutathione to the oxidized form of glutathione in H2O2-treated cells compared to controls and protected against H2O2-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in the ONL thickness in mice treated with 8-OH DPAT at 4 months past the time of MnSOD knockdown compared to untreated controls together with a 60% reduction in RPE lipofuscin. The data indicate that 5-HT1A agonists can reduce lipofuscin accumulation and protect the retina from oxidative damage and mitochondrial dysfunction. 5-HT1A receptor agonists may have potential as therapeutic agents in the treatment of retinal degenerative disease.

Pathological consequences of long-term mitochondrial oxidative stress in the mouse retinal pigment epithelium.

Oxidative stress in the retinal pigment epithelium (RPE) is hypothesized to be a major contributor to the development of age-related macular degeneration (AMD). Mitochondrial manganese superoxide dismutase (MnSOD) is a critical antioxidant protein that scavenges the highly reactive superoxide radical. We speculated that specific reduction of MnSOD in the RPE will increase the level of reactive oxygen species in the retina/RPE/choroid complex leading to pathogenesis similar to geographic atrophy. To test this hypothesis, an Sod2-specific hammerhead ribozyme (Rz), delivered by AAV2/1 and driven by the human VMD2 promoter was injected subretinally into C57BL/6J mice. Dark-adapted full field electroretinogram (ERG) detected a decrease in the response to light. We investigated the age-dependent phenotypic and morphological changes of the outer retina using digital fundus imaging and SD-OCT measurement of ONL thickness. Fundus microscopy revealed pigmentary abnormalities in the retina and these corresponded to sub-retinal and sub-RPE deposits seen in SD-OCT B-scans. Light and electron microscopy documented the localization of apical deposits and thickening of the RPE. In RPE flat-mounts we observed abnormally displaced nuclei and regions of apparent fibrosis in the central retina of the oldest mice. This region was surrounded by enlarged and irregular RPE cells that have been observed in eyes donated by AMD patients and in other mouse models of AMD.

Gene Augmentation for adRP Mutations in RHO

Mutations in the gene for rhodopsin, RHO, cause autosomal dominant retinitis pigmentosa, a disease characterized by death of rod photoreceptor cells. At the end stage, when most rods are gone, cones die too, taking central vision with them. One goal of gene therapy, therefore, is to preserve central vision by promoting rod survival in the vicinity of the macula. Dominance in RHO mutations is associated with two phenomena: interference with the function of normal rhodopsin and intrinsic toxicity of the mutant protein. In the case of interference, increased production of the wild-type protein may be therapeutic, but in the case of toxicity, suppression of the mutant protein may also be needed. RHO augmentation has made use of advances in gene delivery to the retina using adeno-associated virus (AAV). Several strategies have been developed for suppression of rhodopsin expression, but because of the heterogeneity of RHO mutations they are not specific for the mutant allele: They suppress both mutant and wild-type RHO. Experiments in autosomal dominant retinitis pigmentosa (adRP) mouse models suggest that both RHO augmentation and supplementation plus suppression preserve the survival of rod cells.

Conditional Induction of Oxidative Stress in RPE: A Mouse Model of Progressive Retinal Degeneration

An appropriate animal model is essential to screening drugs or designing a treatment strategy for geographic atrophy. Since oxidative stress contributes to the pathological changes of the retinal pigment epithelium (RPE), we are reporting a new mouse AMD model of retinal degeneration by inducing mitochondrial oxidative stress in RPE. Sod2 the gene for manganese superoxide dismutase (MnSOD) was deleted in RPE layer using conditional knockout strategy. Fundus microscopy, SD-OCT and electroretinography were used to monitor retinal structure and function in living animals and microscopy was used to assess pathology post mortem. Tissue specific deletion of Sod2 caused elevated signs of oxidative stress, RPE dysfunction and showed some key features of AMD. Due to induction of oxidative stress, the conditional knockout mice show progressive reduction in ERG responses and thinning of outer nuclear layer (ONL) compared to non-induced littermates.

Gene Delivery of Wild-Type Rhodopsin Rescues Retinal Function in an Autosomal Dominant Retinitis Pigmentosa Mouse Model

Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations within the gene for the opsin of rod photoreceptor cells. Studies on transgenic mice, carrying mutated rhodopsin (RHO) transgene on different genetic backgrounds, suggested that an increased amount of wild-type RHO in ADRP photoreceptors attenuated the impact of the mutant transgene. Therefore, we employed a gene therapy approach with the help of Adeno-associated virus (AAV) to treat mice expressing a P23H mutant human RHO transgene. Knowing that AAV5 primarily transduces photoreceptor cells, we designed “hardened” form of the rhodopsin gene (RHO301) that expressed normal rhodopsin and was specifically resistant to degradation by the previously tested siRNA301. AAV5 RHO301 was subretinaly injected into the right eyes of P23H RHO mice at postnatal day 15. Animals were analyzed monthly by electroretinography (ERG) for 6 months. Analysis of the full-field scotopic electroretinogram (ERG) demonstrated that increased expression of opsin slowed the rate of retinal degeneration in P23H mice with increased amplitudes in both a-wave and b-wave amplitudes compared to control eyes. An increase in the ERG amplitudes was correlated with improvement of retinal structure. The thickness of the outer nuclear layer in AAV-RHO301-injected eyes was increased by 80% compared to control eyes. This finding indicates that wild-type RHO could rescue the retinal degeneration in transgenic mice carrying a dominant RHO mutation and that increased production of normal rhodopsin could suppress the effect of the mutant protein. These findings suggest that wild-type RHO can be used as a therapeutic agent to retard retinal degeneration in ADRP caused by different mutations of RHO via increased production of normal rhodopsin protein.

393. Effectiveness of Antioxidant Gene Therapy in Delaying Retinal Degeneration

We established a mouse model of RPE(retinal pigment epithelium) oxidative stress by Cre-lox mediated deletion of the Sod2 gene, that codes for the protective enzyme manganese superoxide dismutase (MnSOD), leading to some of the features of geographic atrophy. It is the purpose of these experiments to determine whether early delivery of Sod2 using adeno-associated virus (AAV) can prevent retinal degeneration seen in these mice and whether gene therapy can prevent degeneration once it has begun. Deletion of Sod2 was induced by doxycycline treatment of mice with a “floxed” allele of Sod2 and an RPE-specific tet-transactivator controlling expression of Cre (Sod2-cre mice). Retinal degeneration was monitored by electroretinography (ERG) and spectral domain optical coherence tomography over a period of 9 months. Mouse Sod2 with a Myc epitope under the control of a small chicken beta- actin promoter (smCBA) was packaged into self-complementary AAV serotype 1 vector (ScAAV1). Ten Sod2-cre adult mice were injected subretinally with ScAAV1-smCBA-Sod2-Myc or ScAAV1-smCBA-GFP into right and left eye respectively. Three mice were sacrificed a month following injection to collect retina and RPE separately. Using an anti-myc antibody, western blotting was performed to detect MnSOD expression. For rest of the mice ERG and SD-OCT were measured at different time points. Following doxycycline induction of Cre, mice demonstrated increased signs of oxidative stress in RPE and accumulation of autofluorescent material by 2 months of age. They showed a gradual decline in the ERG response and thinning of the outer nuclear layer (by SD-OCT) which were statistically significant by 6 months. Myc tagged MnSOD expression was detected in RPE of mice injected with vector driven by smCBA promoter and negligible expression was seen in the neural retina. ERG and OCT data suggests no adverse functional and structural integrity due to increased expression of MnSOD and sub retinal injection. ERG response and thinning retinal thickness was significantly delayed in Sod2-vector injected eyes compared to control eye injected with GFP vector. Deletion of Sod2 in the RPE leads to some of the salient features of dry AMD. ScAAV1 delivery of Sod2 led to expression in RPE. Delivery of ScAAV1-smCBA-SOD2-Myc vector can be used as a tool to reverse oxidative stress in this mouse model of dry AMD.