A rhodopsin mutant linked to autosomal dominant retinitis pigmentosa is prone to aggregate and interacts with the ubiquitin proteasome system

The inherited retinal degenerations are typified by retinitis pigmentosa (RP), a heterogeneous group of inherited disorders that causes the destruction of photoreceptor cells, the retinal pigmented epithelium, and choroid. This group of blinding conditions affects over 1.5 million persons worldwide. Approximately 30-40% of human autosomal dominant (AD) RP is caused by dominantly inherited missense mutations in the rhodopsin gene. Here we show that P23H, the most frequent RP mutation in American patients, renders rhodopsin extremely prone to form high molecular weight oligomeric species in the cytoplasm of transfected cells. Aggregated P23H accumulates in aggresomes, which are pericentriolar inclusion bodies that require an intact microtubule cytoskeleton to form. Using fluorescence resonance energy transfer (FRET), we observe that P23H aggregates in the cytoplasm even at extremely low expression levels. Our data show that the P23H mutation destabilizes the protein and targets it for degradation by the ubiquitin proteasome system. P23H is stabilized by proteasome inhibitors and by co-expression of a dominant negative form of ubiquitin. We show that expression of P23H, but not wild-type rhodopsin, results in a generalized impairment of the ubiquitin proteasome system, suggesting a mechanism for photoreceptor degeneration that links RP to a broad class of neurodegenerative diseases.     

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.     

Rhodopsin Gene Expression Determines Rod Outer Segment Size and Rod Cell Resistance to a Dominant-Negative Neurodegeneration Mutant

Two outstanding unknowns in the biology of photoreceptors are the molecular determinants of cell size, which is remarkably uniform among mammalian species, and the mechanisms of rod cell death associated with inherited neurodegenerative blinding diseases such as retinitis pigmentosa. We have addressed both questions by performing an in vivo titration with rhodopsin gene copies in genetically engineered mice that express only normal rhodopsin or an autosomal dominant allele, encoding rhodopsin with a disease-causing P23H substitution. The results reveal that the volume of the rod outer segment is proportional to rhodopsin gene expression; that P23H-rhodopsin, the most common rhodopsin gene disease allele, causes cell death via a dominant-negative mechanism; and that long term survival of rod cells carrying P23H-rhodopsin can be achieved by increasing the levels of wild type rhodopsin. These results point to promising directions in gene therapy for autosomal dominant neurodegenerative diseases caused by dominant-negative mutations.     

Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa.

We inserted into the germline of mice either a mutant or wild-type allele from a patient with retinitis pigmentosa and a missense mutation (P23H) in the rhodopsin gene. All three lines of transgenic mice with the mutant allele developed photoreceptor degeneration; the one with the least severe retinal photoreceptor degeneration had the lowest transgene expression, which was one-sixth the level of endogenous murine rod opsin. Of two lines of mice with the wild-type allele, one expressed approximately equal amounts of transgenic and murine opsin and maintained normal retinal function and structure. The other expressed approximately 5 times more transgenic than murine opsin and developed a retinal degeneration similar to that found in mice carrying a mutant allele, presumably due to the overexpression of this protein. Our findings help to establish the pathogenicity of mutant human P23H rod opsin and suggest that overexpression of wild-type human rod opsin leads to a remarkably similar photoreceptor degeneration.     

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.     

Retinoids assist the cellular folding of the autosomal dominant retinitis pigmentosa opsin mutant P23H

The clinically common mutant opsin P23H, associated with autosomal dominant retinitis pigmentosa, yields low levels of rhodopsin when retinal is added following induction of the protein in stably transfected HEK-293 cells. We previously showed that P23H rhodopsin levels could be increased by providing a 7-membered ring, locked analog of 11-cis-retinal during expression of P23H opsin in vivo. Here we demonstrate that the mutant opsin is effectively rescued by 9- or 11-cis-retinal, the native chromophore. When retinal was added during expression, P23H rhodopsin levels were 5-fold (9-cis) and 6-fold (11-cis) higher than when retinal was added after opsin was expressed and cells were harvested. Levels of P23H opsin were increased approximately 3.5-fold with both compounds, but wild-type protein levels were only slightly increased. Addition of retinal during induction promoted the Golgi-specific glycosylation of P23H opsin and transport of the protein to the cell surface. P23H rhodopsins containing 9- or 11-cis-retinal had blue-shifted absorption maxima and altered photo-bleaching properties compared with the corresponding wild-type proteins. Significantly, P23H rhodopsins were more thermally unstable than the wild-type proteins and more rapidly bleached by hydroxylamine in the dark. We suggest that P23H opsin is similarly unstable and that retinal binds and stabilizes the protein early in its biogenesis to promote its cellular folding and trafficking. The implications of this study for treating retinitis pigmentosa and other protein conformational disorders are discussed.     

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.     

Rhodopsin mutant P23H destabilizes rod photoreceptor disk membranes

Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus) to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and Rho(P23H)-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS), accumulating in the OS to a concentration of ～0.1% compared to endogenous opsin. Rho(P23H)-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with Rho(P23H)-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The Rho(P23H)-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing Rho(P23H), suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss.     

A diffusible factor from normal retinal cells promotes rod photoreceptor survival in an in vitro model of retinitis pigmentosa

Transgenic mice expressing a dominant mutation in the gene for the phototransduction molecule rhodopsin undergo retinal degeneration similar to that experienced by patients with the retinal degenerative disease, retinitis pigmentosa (RP). Although the mutation is thought to cause photoreceptor degeneration in a cell‐autonomous manner, the fact that rod photoreceptor degeneration is slowed in chimeric wild‐type/mutant mice suggests that cellular interactions are also important for maintaining photoreceptor survival. To more fully characterize the nature of the cellular interactions important for rod degeneration in the RP mutant mice, we have used an in vitro approach. We found that when the retinas of the transgenic mice were isolated from the pigmented epithelium and cultured as explants, the rod photoreceptors underwent selective degeneration with a similar time course to that observed in vivo. This selective rod degeneration also occurred when the cells were dissociated and cultured as monolayers. These data indicate that the mutant rod photoreceptors degenerate when removed from their normal cellular relationships and without contact with the pigmented epithelium, thus confirming the relative cell autonomy of the mutant phenotype. We next tested whether normal retinal cells could rescue the mutant photoreceptors in a coculture paradigm. Coculture of transgenic mouse with wild‐type mouse or rat retinal cells significantly enhanced transgenic rod photoreceptor survival; this survival‐promoting activity was diffusible through a filter, was heat labile, and not present in transgenic retinal cells. Several peptide growth factors known to be present in the retina were tested as the potential survival‐promoting molecule responsible for the effects of the conditioned medium; however, none of them promoted survival of the photoreceptors expressing the Pro23His mutant rhodopsin. Nevertheless, we were able to demonstrate that the mutant photoreceptors could be rescued by an antagonist to a retinoic acid receptor, suggesting that the endogeneous survival‐promoting activity may function through this pathway. These data thus confirm and extend the findings of previous work that local trophic interactions are important in regulating rod photoreceptor degeneration in retinitis pigmentosa. A diffusible factor found in normal but not transgenic retinal cells has a protective effect on the survival of rod photoreceptors from Pro23His mutant rhodopsin mice. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 475–490, 1999     

LEDGF(1-326) decreases P23H and wild type rhodopsin aggregates and P23H rhodopsin mediated cell damage in human retinal pigment epithelial cells

Background

P23H rhodopsin, a mutant rhodopsin, is known to aggregate and cause retinal degeneration. However, its effects on retinal pigment epithelial (RPE) cells are unknown. The purpose of this study was to determine the effect of P23H rhodopsin in RPE cells and further assess whether LEDGF_1-326, a protein devoid of heat shock elements of LEDGF, a cell survival factor, reduces P23H rhodopsin aggregates and any associated cellular damage.

Methods

ARPE-19 cells were transiently transfected/cotransfected with pLEDGF_1-326 and/or pWT-Rho (wild type)/pP23H-Rho. Rhodopsin mediated cellular damage and rescue by LEDGF_1-326 was assessed using cell viability, cell proliferation, and confocal microscopy assays. Rhodopsin monomers, oligomers, and their reduction in the presence of LEDGF_1-326 were quantified by western blot analysis. P23H rhodopsin mRNA levels in the presence and absence of LEDGF_1-326 was determined by real time quantitative PCR.

Principal Findings

P23H rhodopsin reduced RPE cell viability and cell proliferation in a dose dependent manner, and disrupted the nuclear material. LEDGF_1-326 did not alter P23H rhodopsin mRNA levels, reduced its oligomers, and significantly increased RPE cell viability as well as proliferation, while reducing nuclear damage. WT rhodopsin formed oligomers, although to a smaller extent than P23H rhodopsin. Further, LEDGF_1-326 decreased WT rhodopsin aggregates.

Conclusions

P23H rhodopsin as well as WT rhodopsin form aggregates in RPE cells and LEDGF_1-326 decreases these aggregates. Further, LEDGF_1-326 reduces the RPE cell damage caused by P23H rhodopsin. LEDGF_1-326 might be useful in treating cellular damage associated with protein aggregation diseases such as retinitis pigmentosa.     

Molecular Mechanisms of Rhodopsin Retinitis Pigmentosa and the Efficacy of Pharmacological Rescue

Variants of rhodopsin, a complex of 11-cis retinal and opsin, cause retinitis pigmentosa (RP), a degenerative disease of the retina. Trafficking defects due to rhodopsin misfolding have been proposed as the most likely basis of the disease, but other potentially overlapping mechanisms may also apply. Pharmacological therapies for RP must target the major disease mechanism and contend with overlap, if it occurs. To this end, we have explored the molecular basis of rhodopsin RP in the context of pharmacological rescue with 11-cis retinal. Stable inducible cell lines were constructed to express wild-type opsin; the pathogenic variants T4R, T17M, P23A, P23H, P23L, and C110Y; or the nonpathogenic variants F220L and A299S. Pharmacological rescue was measured as the fold increase in rhodopsin or opsin levels upon addition of 11-cis retinal during opsin expression. Only Pro23 and T17M variants were rescued significantly. C110Y opsin was produced at low levels and did not yield rhodopsin, whereas the T4R, F220L, and A299S proteins reached near-wild-type levels and changed little with 11-cis retinal. All of the mutant rhodopsins exhibited misfolding, which increased over a broad range in the order F220L, A299S, T4R, T17M, P23A, P23H, P23L, as determined by decreased thermal stability in the dark and increased hydroxylamine sensitivity. Pharmacological rescue increased as misfolding decreased, but was limited for the least misfolded variants. Significantly, pathogenic variants also showed abnormal photobleaching behavior, including an increased ratio of metarhodopsin-I-like species to metarhodopsin-II-like species and aberrant photoproduct accumulation with prolonged illumination. These results, combined with an analysis of published biochemical and clinical studies, suggest that many rhodopsin variants cause disease by affecting both biosynthesis and photoactivity. We conclude that pharmacological rescue is promising as a broadly effective therapy for rhodopsin RP, particularly if implemented in a way that minimizes the photoactivity of the mutant proteins.     

Retinobenzaldehydes as proper-trafficking inducers of folding-defective P23H rhodopsin mutant responsible for Retinitis Pigmentosa

The Retinitis pigmentosa (RP)-causing mutant of rhodopsin, P23H rhodopsin, is folding-defective and unable to traffic beyond the endoplasmic reticulum (ER). This ER retention, and in some cases aggregation, are proposed to result in ER-stress and eventually cell death. The endogenous rhodopsin ligand 11-cis-retinal and its isomer 9-cis-retinal have been shown to act as pharmacological chaperones, promoting proper folding and trafficking of the P23H rhodopsin. In spite of this promising effect, the development of retinals and related polyenealdehydes as pharmacological agents has been hampered by their undesirable properties, which include chemical instability, photolability, and potential retinoidal actions. Here, we report the design and synthesis of a class of more stable nonpolyene-type rhodopsin ligands, structurally distinct from, and with lower toxicity than, retinals. A structure–activity relationship study was conducted using cell-surface expression assay to quantify folding/trafficking efficiency of P23H rhodopsin.     

Effects of a Metabotropic Glutamate 1 Receptor Antagonist on Light Responses of Retinal Ganglion Cells in a Rat Model of Retinitis Pigmentosa

Background

Retinitis pigmentosa (RP) is a progressive retinal degenerative disease that causes deterioration of rod and cone photoreceptors. A well-studied animal model of RP is the transgenic P23H rat, which carries a mutation in the rhodopsin gene. Previously, I reported that blocking retinal GABA_C receptors in the P23H rat increases light responsiveness of retinal ganglion cells (RGCs). Because activation of metabotropic glutamate 1 (mGlu1) receptors may enhance the release of GABA onto GABA_C receptors, I examined the possibility that blocking retinal mGlu1 receptors might in itself increase light responsiveness of RGCs in the P23H rat.

Methodology/Principal Findings

Electrical recordings were made from RGCs in isolated P23H rat retinas. Spike activity of RGCs was measured in response to brief flashes of light over a range of light intensities. Intensity-response curves were evaluated prior to and during bath application of the mGlu1 receptor antagonist JNJ16259685. I found that JNJ16259685 increased light sensitivity of all ON-center RGCs and most OFF-center RGCs studied. RGCs that were least sensitive to light showed the greatest JNJ16259685-induced increase in light sensitivity. On average, light sensitivity increased in ON-center RGCs by 0.58 log unit and in OFF-center RGCs by 0.13 log unit. JNJ16259685 increased the maximum peak response of ON-center RGCs by 7% but had no significant effect on the maximum peak response of OFF-center RGCs. The effects of JNJ16259685 on ON-center RGCs were occluded by a GABA_C receptor antagonist.

Conclusions

The results of this study indicate that blocking retinal mGlu1 receptors in a rodent model of human RP potentiates transmission of any, weak signals originating from photoreceptors. This augmentation of photoreceptor-mediated signals to RGCs occurs presumably through a reduction in GABA_C-mediated inhibition.     

Opsin stability and folding: the role of Cys185 and abnormal disulfide bond formation in the intradiscal domain

The structure in the extracellular, intradiscal domain of rhodopsin surrounding the Cys110–Cys187 disulfide bond has been shown to be important for correct folding of this receptor in vivo. Retinitis pigmentosa misfolding mutants of the apoprotein opsin (such as P23H) misfold, as defined by a deficiency in ability to bind 11-cis retinal and form rhodopsin. These mutants also possess an abnormal Cys185–Cys187 disulfide bond in the intradiscal domain. Here, by mutating Cys185 to alanine, we eliminate the possibility of forming this abnormal disulfide bond and investigate the effect of combining the C185A mutation with the retinitis pigmentosa mutation P23H. Both the P23H and P23H/C185A double mutant suffer from low expression and poor 11-cis retinal binding. Our data suggest that misfolding events occur that do not have an absolute requirement for abnormal Cys185–Cys187 disulfide bond formation. In the detergent-solubilised, purified state, the C185A mutation allows formation of rhodopsin at wild-type (WT) levels, but has interesting effects on protein stability. C185A rhodopsin is less thermally stable than WT, whereas C185A opsin shows the same ability to regenerate rhodopsin in detergent as WT. Purified C185A and WT opsins, however, have contrasting 11-cis retinal binding kinetics. A high proportion of C185A opsin binds 11-cis retinal with a slow rate that reflects a denatured state of opsin reverting to a fast-binding, open-pocket conformation. This slower rate is not observed in a stabilising lipid/detergent system, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Chaps, in which C185A exhibits WT (fast) retinal binding. We propose that the C185A mutation destabilises the open-pocket conformation of opsin in detergent resulting in an equilibrium between correctly folded and denatured states of the protein. This equilibrium can be driven towards the correctly folded rhodopsin state by the binding of 11-cis retinal.     

Inherent Instability of the Retinitis Pigmentosa P23H Mutant Opsin

The P23H opsin mutation is the most common cause of autosomal dominant retinitis pigmentosa. Even though the pathobiology of the resulting retinal degeneration has been characterized in several animal models, its complex molecular mechanism is not well understood. Here, we expressed P23H bovine rod opsin in the nervous system of Caenorhabditis elegans. Expression was low due to enhanced protein degradation. The mutant opsin was glycosylated, but the polysaccharide size differed from that of the normal protein. Although P23H opsin aggregated in the nervous system of C. elegans, the pharmacological chaperone 9-cis-retinal stabilized it during biogenesis, producing a variant of rhodopsin called P23H isorhodopsin. In vitro, P23H isorhodopsin folded correctly, formed the appropriate disulfide bond, could be photoactivated but with reduced sensitivity, and underwent Meta II decay at a rate similar to wild type isorhodopsin. In worm neurons, P23H isorhodopsin initiated phototransduction by coupling with the endogenous G_i/o signaling cascade that induced loss of locomotion. Using pharmacological interventions affecting protein synthesis and degradation, we showed that the chromophore could be incorporated either during or after mutant protein translation. However, regeneration of P23H isorhodopsin with chromophore was significantly slower than that of wild type isorhodopsin. This effect, combined with the inherent instability of P23H rhodopsin, could lead to the structural cellular changes and photoreceptor death found in autosomal dominant retinitis pigmentosa. These results also suggest that slow regeneration of P23H rhodopsin could prevent endogenous chromophore-mediated stabilization of rhodopsin in the retina.     

Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model

Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3’ UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.     

Misfolded rhodopsin mutants display variable aggregation properties

The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental.     

Protein Aggregation in Retinal Cells and Approaches to Cell Protection

1. Retinal dystrophies (RD) comprise a group of clinically and genetically heterogeneous retinal disorders, which typically result in the degeneration of photoreceptors followed by the impairment or loss of vision. Although age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are among the most common forms of RD, currently, there is no effective treatment for either disorder. 2. Recently, abnormal protein accumulation and aggregation due to protein misfolding and proteasome inhibition have been implicated in the pathogenesis of RD. In this paper we describe effects of several factors on protein aggregation and survival of photoreceptor cells. 3. Expression of rhodopsin carrying P23H mutation causes its accumulation in intracellular inclusion bodies in a perinuclear area of photoreceptor cells. beta- and gamma-synucleins and heat shock protein Hsp-70, but not alpha-synuclein, protect cultured ocular cells from mutant opsin accumulation. This effect might be explained by their chaperonic activity. 4. Knock-out of alpha- and gamma-synucleins does not affect gross retinal morphology, but induces tyrosine hydroxylase in the inner prexiform layer of the retina. Selegiline-a monoamine oxidase inhibitor used for the treatment of Parkinson's disease, reduces apoptosis and increases viability in cultured retinal pigment epithelium cells (APRE-19). 5. These results suggest that chaperones and selegiline may be considered promising candidates for the protection of ocular cells from the accumulation of misfolded and aggregated proteins.     

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.     

Inhibiting autophagy reduces retinal degeneration caused by protein misfolding

Mutations in the genes necessary for the structure and function of vertebrate photoreceptor cells are associated with multiple forms of inherited retinal degeneration. Mutations in the gene encoding RHO (rhodopsin) are a common cause of autosomal dominant retinitis pigmentosa (adRP), with the Pro23His variant of RHO resulting in a misfolded protein that activates endoplasmic reticulum stress and the unfolded protein response. Stimulating macroautophagy/autophagy has been proposed as a strategy for clearing misfolded RHO and reducing photoreceptor death. We found that retinas from mice heterozygous for the gene encoding the RHO^P23H variant (hereafter called P23H) exhibited elevated levels of autophagy flux, and that pharmacological stimulation of autophagy accelerated retinal degeneration. In contrast, reducing autophagy flux pharmacologically or by rod-specific deletion of the autophagy-activating gene Atg5, improved photoreceptor structure and function. Furthermore, proteasome levels and activity were reduced in the P23H retina, and increased when Atg5 was deleted. Our findings suggest that autophagy contributes to photoreceptor cell death in P23H mice, and that decreasing autophagy shifts the degradation of misfolded RHO protein to the proteasome and is protective. These observations suggest that modulating the flux of misfolded proteins from autophagy to the proteasome may represent an important therapeutic strategy for reducing proteotoxicity in adRP and other diseases caused by protein folding defects.