RETINITIS PIGMENTOSA

Retinitis pigmentosa (RP) is a collection of inherited neurodegenerative disorders in which rod cell apoptosis spreads throughout the retina, resulting in progressive vision loss and the characteristic pigmented retinal appearance. RP genetics demonstrate extreme heterogeneity in severity, progression rate, and mode of inheritance, covering autosomal dominant, autosomal recessive, X-linked, and sporadic mutations. Over 30 different genes have been identified to produce RP, with additional loci also implicated. Of at least 15 autosomal dominant RP (adRP) inherited disease genes, some have obvious correlations to RP biochemistry, while others have uncertain or indeterminate relationship to the disease. By definition, all RP mutants result in photoreceptor degeneration. Degenerative effects trigger multiple cell-death pathways, including caspase-dependent apoptosis, complement activation, and autophagy [76], with phase profiles reflecting the initial cause of cell death, be it calcium overload, structural defects, or oxidative damage. Uniform retinal degeneration was demonstrated to be independent of cellular genotype in chimeric retinas [77], indicating that transcellular factors or interactions are responsible for the final global retinal degeneration.

Rhodopsin mutations account for an estimated 40% of autosomal dominant gain-of- function mutations, with over 100 distinct mutations within the receptor (Fig. 3A). Initial association of RP with single-point mutations in rhodopsin stemmed from identification of the predominant rhodopsin RP mutant, P23H [5]. Clinical visual parameters of P23H RP suggest increased ROS shedding and impaired ROS renewal [78], findings consistent with the notion of misfolded opsin impairing the disk integrity. With an astounding number of different single-point mutations in rhodopsin leading to autosomal dominant RP [79], it becomes useful to categorize the resulting effects when evaluating disease mechanism and therapeutic approaches. Although the final pathology may appear to be similar, rhodopsin mutations are a heterogeneous group at the molecular level, with differing structural perturbations. Potential for pharmacological and molecular therapy may ultimately depend on the location of the mutation and amino acid change.

Classification of RP mutants in terms of structural loci and mechanistic impact can be reduced to three critical structural regions [50]. Most severely, modifications in the cytoplasmic tail interfere with rhodopsin trafficking to the ROS. Mutants interfering with the normal disulfide bond formation, between C187 on the second intradiskal loop and C110 at the end of the first intradiskal loop, lead to receptor misfolding. Finally, mutations among the transmembraneand chromaphore-binding regions can effect protein folding and receptor activation. Associating cytoplasmic, intradiskal, and transmembrane mutations with trafficking, misfolding, and activation defects provides a useful means to discuss major routes of rhodopsin-RP development, but also provides drastic overgeneralization of a highly heterogeneous problem.

An extended classification scheme is based on inactivating mutations of GPCRs in general [79]: class I, defective biosynthesis; class II, defective trafficking; class III, defective ligand binding; class IV, defective activation; and class V, unknown defects. Defective biosynthesis predominantly occurs through premature termination, often through frameshift, but may also entail accelerated degradation. Defective surface trafficking, in this scheme, covers the majority of GPCR mutations, where normally produced receptors demonstrate intracellular retention. Receptors with ligand-binding defects,

despite proper production and surface expression, are incompetent for ligand binding. Activation-defective receptors, it follows, demonstrate proper production, localization, and ligand binding, failing only to carry out the final activation step, resulting in reduced maximal response or sensitivity (manifest as an increased half maximal response, EC50). The final category of unknown defects refers to disease-associated mutants with no apparent mechanistic deficiency, functioning normally in model systems and suggesting a case for which, despite association, the mutant may not be the cause of disease.

While categorizing mutation effects may facilitate discussion of general mechanisms, the differences in location and physicochemical properties of replacement residues likely result in a broad range of subtle changes among each major class. Mutation of the arginine in the conserved [E/D]RY sequence associated with G protein signaling results in impaired activation in the melanocortin MC1R receptor (R142H) through G protein decoupling. The corresponding mutation in the vasopressin V2R receptor, R147H, also causes constitutive internalization and desensitization [80], resulting in nephrogenic diabetes insipidus. Similar RP mutants have been found, R135L and R135W, that are unable to activate transducin despite normal folding and ligand binding [81], a phenotype that could fall into the class IV activation-impaired mutant group. However, further research into these mutants has identified the defect to be caused by constitutive phosphorylation and arrestin binding, leading to constitutive internalization [82]. One could also argue that this may fall into the category of defective trafficking. However, the cellular outcome of this defect remains distinctly different from that of traditional mistrafficking.

Transmembrane RP Rhodopsin Mutants

The transmembrane region of rhodopsin, consisting of half of the protein, is of obvious structural importance. This region also forms the retinal-binding pocket and coordinates G protein activation through conformational movements of the transmembrane (TM) domains. As such, rhodopsin mutations within the transmembrane region can be further subdivided by those in proximity of the retinal β-ionone ring or carbon chain and those found in remaining regions of the transmembrane domains (Fig. 4). Evaluation of RP mutants across the transmembrane domains (G51A,V in TM1, G89D in TM2, L125R,A,F in TM3, A164V in TM4, H211P in TM5, P267L,R in TM6, and T297R in TM7) demonstrated that mutations in each TM segment can lead to abnormal bleaching and MII photointermediates [25], indicators of protein misfolding. These mutations appear to result in nonnative packing of the transmembrane helices, which relay misfolding to the intradiskal domain, where they may cause abnormal disulfide formation.

The L125 residue is in TM3, within the ligand-binding pocket, close to the retinal β-ionone ring. A comprehensive list of mutations at this site (G, N, I, H, P, T, D, E, Y, and W) decreased 11-cis retinal binding, causing a red-shift of λmax, increased solvent exposure, and decreased thermostability [24, 83]. These findings support the importance of ligand binding in maintaining the structural integrity of rhodopsin, highlighting an interaction between L125 and the β-ionone ring as critical in maintaining the structure of the chromaphore-binding pocket. Further use of mutagenesis in evaluating the structural role of L125 demonstrated the role of this residue in maintaining additional interhelical interactions [29]. Rescue of the L125R RP mutant through compensatory mutations of W126E,D or E122L eliminated steric hindrance caused by L125R, restoring

Fig. 4. Regional clusters of autosomal dominant retinitis pigmentosa (adRP) rhodopsin mutations. Organization of adRP mutations according to structural features demonstrates clusters in relation to (A) the retinal-binding site, (B) the transmembrane scaffold, (C) the cytoplasmic region, and (D) the intradiskal region.

the TM3–TM5 interaction formed by the salt bridge between E122 and H211, by reconstructing the hydrogen bond between W126 and E122. Similarly, RP mutant A164 in TM4 interfered with residues L119 and I123 in TM3, disrupting the same salt bridge.

Congenital stationary night blindness (CSNB; nyctalopia) is a condition characterized by inability to see in conditions of low illumination. The most common cause of nyctalopia