The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy

Abstract

Inherited mutations in the rod visual pigment, rhodopsin, cause the degenerative blinding condition, retinitis pigmentosa (RP). Over 150 different mutations in rhodopsin have been identified and, collectively, they are the most common cause of autosomal dominant RP (adRP). Mutations in rhodopsin are also associated with dominant congenital stationary night blindness (adCSNB) and, less frequently, recessive RP (arRP). Recessive RP is usually associated with loss of rhodopsin function, whereas the dominant conditions are a consequence of gain of function and/or dominant negative activity. The in-depth characterisation of many rhodopsin mutations has revealed that there are distinct consequences on the protein structure and function associated with different mutations. Here we categorise rhodopsin mutations into seven discrete classes; with defects ranging from misfolding and disruption of proteostasis, through mislocalisation and disrupted intracellular traffic to instability and altered function. Rhodopsin adRP offers a unique paradigm to understand how disturbances in photoreceptor homeostasis can lead to neuronal cell death. Furthermore, a wide range of therapies have been tested in rhodopsin RP, from gene therapy and gene editing to pharmacological interventions. The understanding of the disease mechanisms associated with rhodopsin RP and the development of targeted therapies offer the potential of treatment for this currently untreatable neurodegeneration.

Keywords: CRISPR; Endocytosis; GPCR; Mutation; Neurodegeneration; Protein misfolding; Protein traffic; Proteostasis; Retinal dystrophy; Rhodopsin; Therapy.

Figure 1. The structure of rhodopsin.

The tertiary structure of bovine rhodopsin from the intradiscal N-terminus (dark blue) to the cytoplasmic C-terminus (red) containing 7 transmembrane motifs (α-helix I-VII) and cytosplamic helix VIII shown in a 3D model. 11-cis retinal (black), T4, P23, E113, R135, K296 and the disulphide bond site C110-C187 are highlighted. The 3D image was made using PyMol (PDB 1U19).

Figure 2. The position of rhodopsin mutations.

The primary amino acid sequence of human rhodopsin is shown as a secondary structure schematic to show the position of different classes of mutation. Class 1, post-Golgi trafficking and outer segment targeting (blue). Class 2 misfolding, ER retention and instability (green). Class 3, disrupted vesicular trafficking and endocytosis (violet). Class 4, altered post-translational modifications and reduced stability (yellow). Class 5 altered transducin activation (pink). Class 6 constitutive activation (brown). Class 7 dimerization deficiency (orange). Where there is evidence for more than one type of class it is shown with a vertical colour split. Uncategorised mutations, or those with no biochemical or cellular defects are shown in grey. Those with predicted effects on folding or binding from FoldX (Rakoczy et al., 2011) are shown with a horizontal colour split. The 8 alpha helix motifs of rhodopsin are highlighted by blue boxes.

Figure 3. Schematic illustration of the potential pathogenic consequences caused by rhodopsin mutations.

A rod photoreceptor has distinct regions; including, the outer segments (OS) and inner segments (IS), cell body and synaptic terminals. Pathogenic mutations in rhodopsin disturb several cellular pathways, including endocytosis dysfunction, structural instability of the OS, mistrafficking, and misfolding. Top left, class 3, hyperphosyphorylated rhodopsin is bound by arrestin (green), the Rho-arrestin complex is endocytosed and disrupts vesicular traffic. Top right, upon illumination, unstable rhodopsin mutants (class 2, 4 and 7) could aggregate in the disk of OS, thus causing damage of the plasma membrane homeostasis. Bottom left, class 1 mutations that affect the ciliary targeting signal, VXPX, are mislocalised, including at the synapse, where they could inhibit synaptic vesicle fusion or be abnormally activate transducin. Misfolding class 2 rhodopsin mutants in the ER might induce ER stress, degraded by ERAD and potentially aggregate, leading to the activation of pro-apoptotic pathways.

Figure 4. 4-PBA does not affect photoreceptor function and survival in P23H-1 rats.

P23H-1 rats were either left untreated or treated from P21 to P49 with 400 mg/kg 4-PBA (n = 4 for each condition) via IP injection. (A) Scotopic ERG’s were performed as previously described (Aguila et al., 2014). No significant differences were observed in P23H-1 rats that had received the treatment at any time point and at any intensity (-6 to 2.7 log cds/m²) (data not shown). Average at 0 log cds/m² for a-wave and b-wave of P23H-1 rats at 3, 5 and 7 weeks of age. Values are means ± SEM. n.s non-significant values, 2 way ANOVA. (B) Retinal histology for measuring the outer nuclear layer (ONL) thickness was made on digital images of stained cryosections, every 500 microns from the optic nerve outwards for both the inferior and superior hemisphere. The ONL thickness across the whole from 4 animals at each time point was averaged and analysed using Graphpad Prism (Sigmastat). (C) Representative images of the ONL in treated and untreated P23H-1 rats at 3. 5 and 7 weeks stained with DAPI (blue), anti-rhodopsin antibody 1D4 (green) and cone marker peanut agglutinin (PNA) (red). Scale bar = 50 microns.

Figure 5. TUDCA improves photoreceptor function and survival in P23H-1 rats but has no effect in P23H KI mice.

P23H-1 rats were received 500 mg/kg TUDCA (n=9) or vehicle-PBS (n=7) from P21-P35 and P23H KI mice were received 500 mg/kg TUDCA (n=8) or vehicle-PBS (n=7) from P21-P43. (A, C) Scotopic ERG’s were performed as previously described (Athanasiou et al., 2017) at -5 to 1-log intensities (data not shown). (A) Average at 0 log cds/m² for a-wave and b-wave of P23H-1 rats showing significant improvement of a-wave in TUDCA-treated compared to vehicle-treated P23H-1 rats (*p<0.05). The b-wave is not significantly affected (n.s) by TUDCA treatment. (B, D) OCT analysis was performed as previously described across the nasal-temporal meridian (Athanasiou et al., 2017a). The mean ONL thickness across the whole retina is significantly enhanced in TUDCA-treated P23H-1 rats (**p<0.01). (C) Average at 0 log cds/m² for a-wave and b-wave of P23H-KI mice showing no significant changes in both the a-wave and b-wave in TUDCA-treated compared to vehicle-treated P23H-KI mice (n.s). (D) The mean ONL thickness across the whole retina does not alter in TUDCA-treated mice (n.s). (A-D) Values are mean ± SEM. Unpaired two-sided Student's t test.

Figure 6. Valproic acid worsens photoreceptor function and survival in P23H-1 rats.

P23H-1 rats were received VPA (7.1 gm/Liter) in drinking water (n=9) or water (n=8) from P21-P35. (A) Scotopic ERG’s were performed as previously described (Athanasiou et al., 2017a) at -5 to 1-log intensities (data not shown). Average at 0 log cds/m² for a-wave and b-wave of P23H-1 rats showing significant reduction of a-wave in VPA-treated animals (**p<0.01). The b-wave is not significantly affected (n.s) by VPA treatment. Values are mean ± SEM. Unpaired two-sided Student's t test. (B) OCT analysis was performed as previously described (Athanasiou et al., 2017a) across the nasal-temporal meridian. The mean ONL thickness across the whole retina is significantly reduced in VPA-treated animals (**p<0.001). Values are mean ± SEM. Unpaired two-sided Student's t test.