Caspase-7 ablation modulates UPR,reprograms TRAF2-JNK apoptosis and protects T17M rhodopsin mice from severe retinal degeneration

The UPR is activated in the mouse retina expressing misfolded T17M rhodopsin (RHO) during autosomal dominant retinitis pigmentosa (ADRP) progression. Therefore, the goal of this study is to validate the UPR-induced caspase-7 as a new therapeutic target that modulates the UPR, reduces the level of apoptosis and protects the ADRP retina from retinal degeneration and light-induced damage. Mice were analyzed using ERG, SD-OCT and histology to determine the role of caspase-7 ablation. The results of these experiments demonstrate the significant preservation of photoreceptors and their function in T17M RHO CASP-7 retinas from P30 to P90 compared with control mice. These mice were also protected from the light-induced decline in the ERG responses and apoptosis. The RNA and protein analyses of T17M RHO+Csp7-siRNA, Tn+Csp7-siRNA 661W cells and T17M RHO CASP-7 retinas revealed that caspase-7 ablation reprograms the UPR and reduces JNK-induced apoptosis. This reduction is believed to occur through the downregulation of the mTOR and Hif1a proteins. In addition, decline in activated PARP1 was detected in T17M RHO CASP-7 retina. Altogether, our findings indicate that the targeting of caspase-7 in T17M RHO mice could be a feasible therapeutic strategy for advanced stages of ADRP.     

Modulation of cellular signaling pathways in P23H rhodopsin photoreceptors

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.     

Calpain and PARP Activation during Photoreceptor Cell Death in P23H and S334ter Rhodopsin Mutant Rats

Retinitis pigmentosa (RP) is a heterogeneous group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness. Many human cases are caused by mutations in the rhodopsin gene. An important question regarding RP pathology is whether different genetic defects trigger the same or different cell death mechanisms. To answer this question, we analysed photoreceptor degeneration in P23H and S334ter transgenic rats carrying rhodopsin mutations that affect protein folding and sorting respectively. We found strong activation of calpain and poly(ADP-ribose) polymerase (PARP) in both mutants, concomitant with calpastatin down-regulation, increased oxidative DNA damage and accumulation of PAR polymers. These parameters were strictly correlated with the temporal progression of photoreceptor degeneration, mirroring earlier findings in the phosphodiesterase-6 mutant rd1 mouse, and suggesting execution of non-apoptotic cell death mechanisms. Interestingly, activation of caspases-3 and -9 and cytochrome c leakage—key events in apoptotic cell death—were observed only in the S334ter mutant, which also showed increased expression of PARP-1. The identification of the same metabolic markers triggered by different mutations in two different species suggests the existence of common cell death mechanisms, which is a major consideration for any mutation independent treatment.     

Inhibitory Peptide of Mitochondrial μ-Calpain Protects against Photoreceptor Degeneration in Rhodopsin Transgenic S334ter and P23H Rats

Mitochondrial μ-calpain and apoptosis-inducing factor (AIF)-dependent photoreceptor cell death has been seen in several rat and mouse models of retinitis pigmentosa (RP). Previously, we demonstrated that the specific peptide inhibitor of mitochondrial μ-calpain, Tat-µCL, protected against retinal degeneration following intravitreal injection or topical eye-drop application in Mertk gene-mutated Royal College of Surgeons rats, one of the animal models of RP. Because of the high rate of rhodopsin mutations in RP patients, the present study was performed to confirm the protective effects of Tat-µCL against retinal degeneration in rhodopsin transgenic S334ter and P23H rats. We examined the effects of intravitreal injection or topical application of the peptide on retinal degeneration in S334ter and P23H rats by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electroretinogram (ERG), immunohistochemistry for AIF, and histological staining. In S334ter rats, we found that intravitreal injection or topical application of the peptide prevented photoreceptor cell death from postnatal (PN) 15 to 18 days, the time of early-stage retinal degeneration. Topical application of the peptide also delayed attenuation of ERG responses from PN 28 to 56 days. In P23H rats, topical application of the peptide protected against photoreceptor cell death and nuclear translocation of AIF on PN 30, 40, and 50 days, as the primary stages of degeneration. We observed that topical application of the peptide inhibited the thinning of the outer nuclear layer and delayed ERG attenuations from PN 30 to 90 days. Our results demonstrate that the mitochondrial μ-calpain and AIF pathway is involved in early-stage retinal degeneration in rhodopsin transgenic S334ter and P23H rats, and inhibition of this pathway shows curative potential for rhodopsin mutation-caused RP.     

Morphological alterations in retinal neurons in the S334ter-line3 transgenic rat

The S334ter-line-3 rat is a transgenic model of retinal degeneration developed to express a rhodopsin mutation similar to that found in human retinitis pigmentosa (RP) patients. Previous studies have focused on physiological changes in retinal cells and higher centers of the visual system with this model of retinal degeneration. However, little is known about the morphological changes in retinal cells during the development of the S334ter-line-3 rat. In order to understand and aid vision-rescue strategies, our aim has been to describe the retinal degeneration pattern in this model. We focus on changes in the morphologies of horizontal, bipolar, and amacrine cells in developing S334ter-line-3 rat retinas. Degeneration of photoreceptors begins in the central retina and progresses toward the periphery. In retinas at post-natal day 15 (P15), horizontal and rod bipolar cells show normal morphology. However, at P21, horizontal and rod bipolar cells exhibit abnormal processes at the outer plexiform layer, whereas the outer nuclear layer is significantly thinner. A glial reaction occurs concomitantly. In contrast, modifications in cone-bipolar and amacrine cells are much slower and do not occur until P90 and P180, respectively. The density of horizontal and rod-bipolar cells significantly drops after P60. Overall, the S334ter-line-3 model exhibits the hallmarks of cellular remodeling caused by photoreceptor degeneration. Its moderately fast time course makes the S334ter-line-3 a good model for studying vision-rescue strategies.     

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.     

Enhanced Retinal Insulin Receptor-activated Neuroprotective Survival Signal in Mice Lacking the Protein-tyrosine Phosphatase-1B Gene

Protein-tyrosine phosphatase 1B (PTP1B) has been implicated in the negative regulation of insulin signaling. We previously demonstrated that light-induced tyrosine phosphorylation of the retinal insulin receptor (IR) results in the activation of phosphoinositide 3-kinase/Akt survival pathway in rod photoreceptor cells. The molecular mechanism behind light-induced activation of IR is not known. We investigated the in vivo mechanism of IR activation and found that PTP1B activity in dark-adapted retinas was significantly higher than in light-adapted retinas. We made a novel finding in this study that the light-dependent regulation of PTP1B activity is signaled through photobleaching of rhodopsin. Conditional deletion of PTP1B in rod photoreceptors by the Cre-loxP system resulted in enhanced IR signaling. Further PTP1B activity negatively regulated the neuroprotective survival signaling in the retina. One of the challenging questions in the retina research is how mutations in human rhodopsin gene slowly disable and eventually disrupt photoreceptor functions. Our studies suggest that a defect in the photobleaching of rhodopsin and mutation in rhodopsin gene enhances the activity of PTP1B, and this activated activity could down-regulate the IR survival signaling. Our studies suggest that PTP1B antagonists could be potential therapeutic agents to treat stress-induced photoreceptor degenerations and provide further evidence that rhodopsin photoexcitation may trigger signaling events alternative to the classic phototransduction.     

Immunohistochemical detection of phosphorylated rhodopsin in light-exposed retina of living mouse with in vivo cryotechnique.

The purpose of this study is to analyze the time-dependent molecular states of rhodopsin (Rho) phosphorylation in the specimens originating from eyeballs cryoimmobilized in situ in living animals. Whole eyeballs of living mice under various dark- and light-exposure conditions were quickly frozen using the in vivo cryotechnique with isopentane-propane cryogen cooled down in liquid nitrogen (-196C). The frozen whole-mount eyeballs were freeze substituted in acetone containing paraformaldehyde and embedded in paraffin wax. Deparaffinized sections were immunostained with anti-phosphorylated (334)Ser Rho (P-Rho334) antibody. Immunoreactivity of P-Rho334 was specifically recognized in the outer segments of mouse retinas exposed to daylight. In the 12-h dark-adapted retinas, P-Rho334 immunoreactivity was completely eliminated. Moreover, in other retinas dark adapted for 12 or 36 hr and then exposed under the safety red light for 2 min, it was still barely recognized. Even in the eyeballs exposed to strong visible light for 10 sec, it was not detected. However, after 30, 60, and 180 sec of visible light exposure, P-Rho334 immunoreactivity was definitely recovered, similar to that under daylight condition. This is a new immunohistochemical approach to visualize the time-dependent Rho phosphorylation of living mice using the in vivo cryotechnique, in which changes could be detected within seconds following exposure to light.     

A 3-bp deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa.

Autosomal dominant retinitis pigmentosa (ADRP) has recently been linked to locus D3S47 (probe C17), with no recombination, in a single large Irish family. Other ADRP pedigrees have shown linkage at zero recombination, linkage with recombination, and no linkage, demonstrating genetic heterogeneity. The gene encoding rhodopsin, the rod photoreceptor pigment, is closely linked to locus D3S47 on chromosome 3q. A point mutation changing a conserved proline to histidine in the 23d codon of the gene has been demonstrated in affected members of one ADRP family and in 17 of 148 unrelated ADRP patients. We have sequenced the rhodopsin gene in a C17-linked ADRP family and have identified in the 4th exon and in-frame 3-bp deletion which deletes one of the two isoleucine monomers at codons 255 and 256. This mutation was not found in 30 other unrelated ADRP families. The deletion has arisen in the sequence TCATCATCAT, deleting one of a run of three x 3-bp repeats. The mechanism by which this occurred may be similar to that which creates length variation in so-called mini- and microsatellites. Thus ADRP is an extremely heterogeneous disorder which can result from a range of defects in rhodopsin and which can have a locus or loci elsewhere in the genome.     

Autosomal dominant retinitis pigmentosa: linkage to rhodopsin and evidence for genetic heterogeneity

Retinitis pigmentosa (RP) is the most prevalent human retinopathy of genetic origin. Chromosomal locations for X-linked RP and autosomal dominant RP genes have recently been established. Multipoint analyses with ADRP and seven markers on the long arm of chromosome 3 demonstrate that the gene for rhodopsin, the pigment of the rod photoreceptors, cosegregates with the disease locus with a maximum lod score of approximately 19, implicating rhodopsin as a causative gene. Recent studies have indicated the presence of a point mutation at codon 23 in exon 1 of rhodopsin which results in the substitution of histidine for the highly conserved amino acid proline, suggesting that this mutation is a cause of rhodopsin-linked ADRP. This mutation is not present in the Irish pedigree in which ADRP has been mapped close to rhodopsin. Another mutation in the rhodopsin gene or in a gene closely linked to rhodopsin may be involved. Moreover, the gene in a second ADRP pedigree, with Type II late onset ADRP, does not segregate with chromosome 3q markers, indicating that nonallelic as well as perhaps allelic genetic heterogeneity exists in the autosomal dominant form of this disease.     

A rod-dominated visual system in leptocephalus larvae of elopomorph fishes (Elopomorpha: Teleostei)

The nature and distributions of photoreceptor cell types were investigated in the retinas of 12 species (5 families) of elopomorph anguilliform leptocephalus larvae. Anti-opsin immunofluorescence, light microscopy and transmission electron microscopy (TEM) were used to assess opsin distribution across the retinas and to associate photoreceptor morphology and opsin content. Retinas of all species were immunoreactive with anti-rhodopsin throughout, while anti-cone opsin immunoreactivity was restricted only to the ventral region of the retina in all specimens. Rod and cone photoreceptors were morphologically indistinguishable at low magnifications; TEM revealed that nearly all photoreceptors had rod-like ultrastructure, with only rare examples of cone-like cells identified in the ventral retina. These results indicate a rhodopsin/rod-dominated retina in leptocephalus larvae of anguilliform eels in the teleost subdivision Elopomorpha, contrasting with the cone-dominated retinas of nearly all other species of teleost larvae. This distinctive developmental pattern shared among elopomorph larvae has important evolutionary and ecological implications, indicating a shared ancestor and/or ecological characteristics that are very different from most other teleost larvae.     

Light-mediated activation of Rac-1 in photoreceptor outer segments

Small GTP binding proteins regulate diverse biological processes including gene expression, cytoskeleton reorganization, and protein and vesicular transport. While small GTPases have been investigated in a wide variety of cells, few studies have addressed their role in photoreceptors. In vertebrate retinal rods, the light stimulus is transmitted from rhodopsin via the pathway mediated by the heterotrimeric G protein transducin. To increase their sensitivity to light, photoreceptors accumulate remarkably high concentrations of rhodopsin and transducin in specialized cellular compartments, the outer segments (OS). Transport of these proteins from the inner segments is regulated by the small GTPases Rab6 and Rab8, which do not enter OS. Here, we asked if small G proteins have other functions in photoreceptors. We show that OS contain the small GTPase Rac-1, a member of the Rho family. In contrast to other cells, Rac-1 in OS is exclusively associated with the membranes and resides in lipid rafts. Most importantly, Rac-1 is activated by light. This activation is specifically blocked by a synthetic peptide corresponding to the Asn-Pro-X-X-Tyr motif found in rhodopsin, and Rac-1 coprecipitates with rhodopsin on Concanavalin A Sepharose. These data provide the first direct evidence for the existence of a novel pathway activated by rhodopsin.     

Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells

Purpose

Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins.

The role of subunit assembly in peripherin-2 targeting to rod photoreceptor disk membranes and retinitis pigmentosa

Peripherin-2 is a member of the tetraspanin family of membrane proteins that plays a critical role in photoreceptor outer segment disk morphogenesis. Mutations in peripherin-2 are responsible for various retinal degenerative diseases including autosomal dominant retinitis pigmentosa (ADRP). To identify determinants required for peripherin-2 targeting to disk membranes and elucidate mechanisms underlying ADRP, we have generated transgenic Xenopus tadpoles expressing wild-type and ADRP-linked peripherin-2 mutants as green fluorescent fusion proteins in rod photoreceptors. Wild-type peripherin-2 and P216L and C150S mutants, which assemble as tetramers, targeted to disk membranes as visualized by confocal and electron microscopy. In contrast the C214S and L185P mutants, which form homodimers, but not tetramers, were retained in the rod inner segment. Only the P216L disease mutant induced photoreceptor degeneration. These results indicate that tetramerization is required for peripherin-2 targeting and incorporation into disk membranes. Tetramerization-defective mutants cause ADRP through a deficiency in wild-type peripherin-2, whereas tetramerization-competent P216L peripherin-2 causes ADRP through a dominant negative effect, possibly arising from the introduction of a new oligosaccharide chain that destabilizes disks. Our results further indicate that a checkpoint between the photoreceptor inner and outer segments allows only correctly assembled peripherin-2 tetramers to be incorporated into nascent disk membranes.     

Molecular genetics of retinal degeneration: A Drosophila perspective

Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca²⁺ influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP₂, or mediate the Ca²⁺-dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca²⁺ following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.     

ATF6alpha Promotes Astroglial Activation and Neuronal Survival in a Chronic Mouse Model of Parkinson’s Disease

Accumulating evidence suggests a crucial role for the unfolded protein response (UPR) in Parkinson’s disease (PD). In this study, we investigated the relevance of the UPR in a mouse model of chronic MPTP/probenecid (MPTP/P) injection, which causes severe and persistent degeneration of dopaminergic neurons. Enhanced activation of the UPR branches, including ATF6α and PERK/eIF2α/ATF4, was observed after MPTP/P injections into mice. Deletion of the ATF6α gene accelerated neuronal degeneration and ubiquitin accumulation relatively early in the MPTP/P injection course. Surprisingly, astroglial activation was strongly suppressed, and production of the brain-derived neurotrophic factor (BDNF) and anti-oxidative genes, such as heme oxygenase-1 (HO-1) and xCT, in astrocytes were reduced in ATF6α −/− mice after MPTP/P injections. Decreased BDNF expression in ATF6α −/− mice was associated with decreased expression of GRP78, an ATF6α-dependent molecular chaperone in the ER. Decreased HO-1 and xCT levels were associated with decreased expression of the ATF4-dependent pro-apoptotic gene CHOP. Consistent with these results, administration of the UPR-activating reagent tangeretin (5,6,7,8,4′-pentamethoxyflavone; IN19) into mice enhanced the expression of UPR-target genes in both dopaminergic neurons and astrocytes, and promoted neuronal survival after MPTP/P injections. These results suggest that the UPR is activated in a mouse model of chronic MPTP/P injection, and contributes to the survival of nigrostriatal dopaminergic neurons, in part, through activated astrocytes.     

Q344ter Mutation Causes Mislocalization of Rhodopsin Molecules That Are Catalytically Active: A Mouse Model of Q344ter-Induced Retinal Degeneration

Q344ter is a naturally occurring rhodopsin mutation in humans that causes autosomal dominant retinal degeneration through mechanisms that are not fully understood, but are thought to involve an early termination that removed the trafficking signal, QVAPA, leading to its mislocalization in the rod photoreceptor cell. To better understand the disease mechanism(s), transgenic mice that express Q344ter were generated and crossed with rhodopsin knockout mice. Dark-reared Q344ter^rho+/− mice exhibited retinal degeneration, demonstrating that rhodopsin mislocalization caused photoreceptor cell death. This degeneration is exacerbated by light-exposure and is correlated with the activation of transducin as well as other G-protein signaling pathways. We observed numerous sub-micrometer sized vesicles in the inter-photoreceptor space of Q344ter^rho+/− and Q344ter^rho−/− retinas, similar to that seen in another rhodopsin mutant, P347S. Whereas light microscopy failed to reveal outer segment structures in Q344ter^rho−/− rods, shortened and disorganized rod outer segment structures were visible using electron microscopy. Thus, some Q344ter molecules trafficked to the outer segment and formed disc structures, albeit inefficiently, in the absence of full length wildtype rhodopsin. These findings helped to establish the in vivo role of the QVAPA domain as well as the pathways leading to Q344ter-induced retinal degeneration.