The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy
Introduction
The outer retina of the mammalian eye possesses two types of ciliary photoreceptor, the rods and cones. Rods, with a peak absorption (λmax) of ∼500 nm, contain the rod-specific rod opsin and are responsible for dim-light (scotopic) photoreception. Cones, with a λmax for visible light ranging from ∼350 to 560 nm, contain one of up to three different cone opsins (ultraviolet/violet, middle- and long-wave sensitive). Image-forming photoreception is initiated by the absorption of a photon by the vitamin A derived chromophore 11-cis-retinal within rhodopsin, causing it to isomerise to the all-trans conformation. Whilst any opsin +11-cis-retinal moiety is correctly termed a ‘rhodopsin’, the term rhodopsin is most commonly applied to the rod opsin +11-cis-retinaldehyde photopigment complex (Lythgoe, 1979), and this convention will be followed in this review. Rhodopsin is the archetypal G-protein-coupled receptor (GPCR) with seven transmembrane (TM) α-helices (Palczewski et al., 2000). The 11-cis-retinal acts an inverse agonist suppressing constitutive activity of the receptor, is located within the pocket formed by the α-helices and is coordinated through a protonated Schiff-base (PSB) linkage to a lysine residue in the 7th TM domain (K296) (Smith, 2010) (Fig. 1).
In the mammalian genome, the ciliary opsin family is composed of the rhodopsin gene (RHO); an ultraviolet/violet sensitive opsin gene (OPN1SW); middle/long-wave sensitive opsin genes (OPN1MW/LW); and the less well characterised opsin 3 (encephalopsin) gene (OPN3). These genes share at least three perfectly conserved intronic sites (Bellingham et al., 2003b) and appear to have arisen through duplications early in vertebrate evolution (Nördstrom et al., 2004). Subsequent to the cloning and characterisation of the bovine rhodopsin gene (Nathans and Hogness, 1983), the same authors published the structure and sequence of the human orthologue, RHO (Nathans and Hogness, 1984). With the exception of the intronless rod opsin gene of the Actinopterygii (ray-finned fish) (Bellingham et al., 2003a, Fitzgibbon et al., 1995), the RHO gene like all other currently characterised vertebrate rod opsin genes is composed of 5 exons, and the open reading frame predicts a 348 amino acid protein with a molecular weight of ∼39 kDa.
The RHO gene was initially mapped to the long arm of chromosome 3 at 3q21-qter (Nathans et al., 1986), and is currently designated at 3q22.1 at coordinates 3:129,528,640–129,535,169 (build GRCh38.p10; http://www.ensembl.org/). Methodologies used to identify inherited retinal dystrophy (IRD) genes have recently been reviewed (Broadgate et al., 2017) and causative mutations in the RHO gene were identified by Dryja et al., in 1990 (Dryja et al., 1990b), as the first genetic cause of retinitis pigmentosa (RP). The identified nucleotide substitution, c.68C > A, results in the following codon change CCC > CAC such that the normally observed proline (CCC) at position 23 of the rod opsin protein is substituted by a histidine (CAC) residue (p.P23H). Since this discovery, many different RHO mutations have been identified and many other genes for RP and other IRDs have been discovered, but P23H remains one of the most intensively studied causes of IRD. Further information regarding the genetic basis of IRDs can be accessed at the RetNet™ Retinal Information Network (https://sph.uth.edu/retnet/).
Mutations in RHO are associated with a classical form of RP, which is a rod-cone dystrophy caused by the dysfunction and death of rod cells, followed by the dysfunction and death of cone cells. A detailed clinical description of RP is beyond the scope of this review and readers are directed to this book chapter for more information (Gregory-Evans et al., 2013). Briefly, classical RP is characterised by a progressive loss of peripheral vision leading to so-called “tunnel vision”. The initial symptom is typically impaired dark adaptation and the development of “night blindness” (nyctalopia) through the loss of rod function starting in the 1st or 2nd decade of life (Hartong et al., 2006). As photoreceptor loss proceeds, there is a loss of pigmentation from the retinal pigment epithelium (RPE), and a build-up of intraretinal melanin deposits that can assume a “bone spicule” conformation. Central visual acuity is often preserved until the end stages of RP. Studies on the prevalence of non-syndromic RP in different populations indicate a frequency of between 1:3000 to 1:5000 individuals (Berson, 1993, Hamel, 2006, Hartong et al., 2006, Sharon et al., 2016), with between 15 and 35% of cases being reported as being of an autosomal dominant inheritance, depending on the population studied (Bravo-Gil et al., 2017, Hartong et al., 2006). In excess of 150 missense/nonsense RHO mutations are catalogued by the professional 2017.1 release of the Human Gene Mutation Database (Stenson et al., 2014) (http://www.hgmd.cf.ac.uk/).
Retinal pathologies resulting from mutations in the RHO gene can be inherited in either an autosomal dominant (ad) or autosomal recessive (ar) manner. Two disease states are associated with RHO mutations, RP and congenital stationary night blindness (CSNB). Whilst CSNB associated with a RHO mutation is inherited in an autosomal dominant manner (adCSNB), RP associated with a RHO mutation can be inherited in either an autosomal dominant (adRP) or autosomal recessive (arRP) form. Inherited variants in RHO are most often associated with adRP, while the recessive mode of inheritance is relatively uncommon.
RHO associated adCSNB (MIM#610445; CSNBAD1; https://www.omim.org/entry/610445) is characterised by a lack of scotopic vision typified by an absence of detectable rod function by electroretinogram (ERG). Cone function typically appears normal. There are currently five RHO missense mutations associated with adCSNB: p.G90D (Rao et al., 1994, Sieving et al., 1995); p.T94I (al-Jandal et al., 1999); p.E113K (Reiff et al., 2016); p.A292E (Dryja et al., 1993) and p.A295V (Zeitz et al., 2008) (Table 1). Whilst normally non-progressive in nature, peripheral pigmentary changes and retinal degeneration have been noted as a feature in older patients (Sieving et al., 1995), suggesting potential overlap with an adRP phenotype later in life. The E113K mutation is striking in that both CSNB and adRP are present in the same pedigree with phenotype seemingly not linked to age of patient (Reiff et al., 2016). With the exception of the E113K mutation, constitutive activation of rhodopsin due to these mutations is believed to be the mechanism behind this form of adCSNB (Gross et al., 2003), see section 2 and classification (Table 1).
Two missense RHO mutations, p.E150K (Azam et al., 2009, Kumaramanickavel et al., 1994, Saqib et al., 2015, Van Schil et al., 2016), and M253I (Collin et al., 2011); and two nonsense (premature stop codon) mutations, p.W161ter (Kartasasmita et al., 2011) and p.E249ter (Rosenfeld et al., 1992), are associated with arRP (Table 2). The observed homozygous recessive inheritance of loss-of-function RHO mutations (compound heterozygous RHO mutations have yet to be identified), suggests that scope for innocuous loss-of-function mutations in the RHO molecule is very restricted. This may account for the high number of adRP mutations. Interestingly, research on a knock-in (KI) mouse model of E150K suggests that this is actually a mild dominant mutation, which progresses more quickly in the homozygous state (Zhang et al., 2013). M253I was identified by homozygosity mapping and has not been studied in detail biochemically, but was also suggested to potentially be a mild mutation that is only pathogenic if present on both alleles (Collin et al., 2011). In contrast, it would appear that the p.W161ter and p.E249ter mutations might potentially cause a true null phenotype since the locations of the nucleotide substitutions, c.482G > A (TGG > TAG) and c.745G > T (GAG > TAG) in exon 2 and exon 4 respectively, suggest that nonsense mediated decay is likely to clear the premature stop codon containing mRNA transcript to an unknown degree (Hernan et al., 2011, Roman-Sanchez et al., 2016). Rhodopsin is essential for rod cell function and rod cell survival, as homozygous rhodopsin (Rho) knock-out (KO) mice do not develop outer segments (OS) and show a progressive retinal degeneration (Humphries et al., 1997, Lem et al., 1999). By contrast, the heterozygous Rho KO mice do not display overt retinal degeneration, but show alterations in their photoresponses, OS morphology and have smaller disk membranes within their OS (Makino et al., 2012, Rakshit and Park, 2015).
Currently more than 150 documented missense/nonsense RHO mutations are associated with an adRP phenotype (Table 1). Collectively, they are the most common cause of adRP, accounting for 20–30% of all cases (Sullivan et al., 2006). Whilst the P23H rod opsin mutation is arguably the most studied, it also likely represents a founder effect (Farrar et al., 1990), appearing to be confined to North America and seemingly accounting for ∼10% of adRP cases in the USA in patients with a western European origin (Sullivan et al., 2006). The null alleles for arRP and lack of aggressive retinal degeneration in heterozygous Rho KO mouse models (Humphries et al., 1997, Lem et al., 1999), suggest that haploinsufficiency is not the disease mechanism and that dominant mutations are typically gain-of-function or dominant-negative mutations. Several attempts have been made to classify RHO mutations on the basis of (a) their clinical manifestation e.g. (Cideciyan et al., 1998, Gal et al., 1997, Krebs et al., 2010), and (b) their biochemical and cellular behaviour e.g. (Kaushal and Khorana, 1994, Krebs et al., 2010, Mendes et al., 2005, Rakoczy et al., 2011, Sung et al., 1991). Cideciyan et al. (1998) developed a broad phenotype-genotype correlation of adRP RHO mutations consisting of two classes (Table 3). Clinical classifications typically have two major classes: class A that has early onset severe rod dysfunction; whilst class B is normally associated with a later onset, less severe phenotype with a slower progression (Table 3). The difference in rates of presentation are likely related to the underlying biochemical and cellular defects associated with the mutation, but other factors such as genetic modifiers and environment can also affect the presentation of a condition and/or lead to interfamilial variability (Iannaccone et al., 2006). Examples of known modifiers include different alleles of Rpe65 and light exposure, which are discussed in depth later. Here we have collated the available biochemical and cellular information to suggest potential classes for each dominant mutation (Table 1, Fig. 2); however, given that RHO mutations cause RP through multiple potential mechanisms, this classification is not mutually exclusive, with some mutants potentially leading to consequences that are applicable to more than one class.
Given that RHO was the first gene in which RP causing mutations were identified (Dryja et al., 1990a, Dryja et al., 1990b), it received intensive screening in RP cohorts across the globe, and it was thus tempting to assume that all variants in RHO identified in individuals with RP were likely pathogenic. Caution needs to be used, however, as some changes could be benign, especially where evidence of familial transmission is absent, such as in isolated patients, where there is no family history, or familial cases where other individuals in a pedigree have not been assessed and genotyped. The collection of data on human genetic variation has enabled researchers to investigate the frequency of nucleotide changes on an as yet unprecedented global scale (Lek et al., 2016). We used the Genome Aggregation Database (gnomAD; http://gnomad.broadinstitute.org/), which contains 123,136 exome sequences and 15,496 whole-genome sequences from unrelated individuals and excludes patients with severe paediatric disease, to determine the allele frequencies for potential missense/nonsense RHO mutations and compared against the currently catalogued RHO mutations. Whilst presence in the gnomAD dataset does not exclude the possibility of late onset disease causing alleles being present, based on the prevalence of adRP associated with RHO mutations in the range between 1:20,000 and 1:150,000, we estimate that a fully penetrant adRP causing RHO variant cannot be more common than 1:20,000 (0.0005) and would more likely have a minor allele frequency of <1:80,000 (<0.0000125), otherwise they would be very common disease associated alleles. These analyses revealed that missense changes that had previously been reported as potential RHO ‘mutations’ such as G51A (Cideciyan et al., 1998, Macke et al., 1993), V104I (Macke et al., 1993), A333V (Eisenberger et al., 2013), and T340M (Clark et al., 2010, Stone, 2003), are almost certainly benign single nucleotide polymorphisms (SNPs) with allele frequencies of between 1:1000 (G51A) and 1:10,000 (A333V). As a control, the T320N substitution that has been shown not to segregate with adRP (Mandal et al., 2005) exhibits a gnomAD frequency of 1:10,000. Therefore, we have excluded these five substitutions from our classifications. Whilst the presence of rare RHO variants in the general population (italicised variants in Table 1) questions their potential to cause fully penetrant aggressive adRP, they could still be associated with very mild late onset disease, arRP, or potentially act as disease modifiers. Nevertheless, these data reinforce the need for thorough genetics, such as segregation analyses, and in depth functional analyses to confirm pathogenicity.
Section snippets
Structural and biochemical basis of rhodopsin RP mutants
Rhodopsin is a member of the class A family of GPCRs. Upon activation by light, rhodopsin couples with the G protein transducin to initiate the first step in vision. Rhodopsin is synthesised in the rod cell inner segments (IS) and transported to rod OS, where it is densely packed into stacks of membranous discs. Fully folded rhodopsin adopts a seven TM helical bundle configuration in these disc membranes (Fig. 1). The N-terminus resides in the interior of the rod discs, while the C-terminus is
Rhodopsin biogenesis, post-translational modifications and trafficking
Rhodopsin is co-translationally translocated into the rod photoreceptor ER and trafficked to the OS, via the Golgi apparatus on membranous vesicle carriers (Fig. 3). Early in its biogenesis, the N-terminus of rhodopsin becomes glycosylated at two asparagine residues: N2 and N15 (Fig. 1, Fig. 2). For many proteins, glycosylation is necessary for the binding of ER chaperones (typically lectins) to assist protein folding and targeting misfolded species for ER-associated degradation (ERAD) (
Photoreceptor cell death
Rhodopsin RP mutations lead to photoreceptor cell death by a two-stage process. Firstly, the rod photoreceptors degenerate and this leads to the second stage, which is death of cone photoreceptors. Despite the availability of various animal models, the mechanisms underlying the eventual loss of cone photoreceptors and the loss of daylight vision remain largely unknown.
Classical apoptosis depends on activity of caspase-type proteases, with caspase-3 as the prototypic mediator and executioner of
Therapeutic approaches to rhodopsin RP
Photoreceptors require a stringent regulation of proteostasis for their function and viability. Therefore, their vulnerability to the toxic gain-of-function effects of mutant rhodopsin could reflect their long-term inability to maintain proteostasis in the presence of this additional stress. Proteostasis mechanisms have been a therapeutic target in several studies which aimed to correct misfolding, reduce rhodopsin aggregation, enhance the degradation of mutant rhodopsin, stimulate autophagy or
Concluding remarks
Immense progress has been made in the understanding of molecular pathways that lead to retinal degeneration associated with rhodopsin mutations. Conflicting therapeutic outcomes from different animal models reflect the complexity of this type of retinal degeneration. They also highlight the importance of understanding the molecular consequences of the many different types of mutation and developing mutation mechanism-dependent therapeutic strategies, regardless of whether these are dealing with
Acknowledgements
We are grateful to the reviewers for their considered and constructive comments that have improved the depth and content of this review. We are grateful to Professor Molday (Department of Biochemistry and Molecular Biology, University of British Columbia, Canada) for the gift of the Rho-1D4 antibody, Professor Matt LaVail (University of California San Francisco, USA) for providing P23H-1 transgenic rats and to Dalila Bevilacqua for assisting with the drug treatments. This work was supported by
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