The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation

Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with DeltaF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the-protein within the ER, suggesting that the proteasome is required for the efficient retrotranslocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER-associated degradation of the mutant protein but is not essential for the quality control of opsin folding. The addition of 9-cis-retinal to the media increased the amount of P23H, but not K296E, that was soluble and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance

Correlative light and immuno-electron microscopy of retinal tissue cryostat sections

Correlative light-electron microscopy (CLEM) is a powerful technique allowing localisation of specific macromolecules within fluorescence microscopy (FM) images to be mapped onto corresponding high-resolution electron microscopy (EM) images. Existing methods are applicable to limited sample types and are technically challenging. Here we describe novel methods to perform CLEM and immuno-electron microscopy (iEM) on cryostat sections utilising the popular FM embedding solution, optimal cutting temperature (OCT) compound. Utilising these approaches, we have (i) identified the same phagosomes by FM and EM in the retinal pigment epithelium (RPE) of retinal tissue (ii) shown the correct localisation of rhodopsin on photoreceptor outer segment disc like-structures in iPSC derived optic cups and (iii) identified a novel interaction between peroxisomes and melanosomes as well as phagosomes in the RPE. These data show that cryostat sections allow easy characterisation of target macromolecule localisation within tissue samples, thus providing a substantial improvement over many conventional methods that are limited to cultured cells. As OCT embedding is routinely used for FM this provides an easily accessible and robust method for further analysis of existing samples by high resolution EM

The role of the ER stress response protein PERK in rhodopsin retinitis pigmentosa

Mutations in rhodopsin, the light sensitive protein of rod cells, are the most common cause of dominant retinitis pigmentosa (RP), a type of inherited blindness caused by the dysfunction and death of photoreceptor cells. The P23H mutation, the most frequent single cause of RP in the USA, causes rhodopsin misfolding and induction of the unfolded protein response (UPR), an adaptive ER stress response and signalling network that aims to enhance the folding and degradation of misfolded proteins to restore proteostasis. Prolonged UPR activation, and in particular the PERK branch, can reduce protein synthesis and initiate cell death through induction of pro-apoptotic pathways. Here, we investigated the effect of pharmacological PERK inhibition on retinal disease process in the P23H-1 transgenic rat model of retinal degeneration. PERK inhibition with GSK2606414A led to an inhibition of eIF2α phosphorylation, which correlated with reduced ERG function and decreased photoreceptor survival at both high and low doses of PERK inhibitor. Additionally, PERK inhibition increased the incidence of inclusion formation in cultured cells overexpressing P23H rod opsin, and increased rhodopsin aggregation in the P23H-1 rat retina, suggesting enhanced P23H misfolding and aggregation. In contrast, treatment of P23H-1 rats with an inhibitor of eIF2α phosphatase, salubrinal, led to improved photoreceptor survival. Collectively, these data suggest the activation of PERK is part of a protective response to mutant rhodopsin that ultimately limits photoreceptor cell death

The X-linked Retinitis Pigmentosa protein RP2 facilitates traffic of cilia target proteins

Photoreceptors are specialized ciliated sensory neurons and aberrant traffic of proteins to the outer segment causes photoreceptor cell death. RP2 is a GTPase activating protein (GAP) for the small GTPase Arl3 and both proteins facilitate protein trafficking to primary cilia. We used GST-RP2 pull down from retinal lysates and identified the Gβ subunit of transducin (Gβ1) as a novel RP2 interacting protein. RP2 competes with Gγ1 for Gβ1 binding and does not interact with the Gβ:Gγ heterodimer. In SK-N-SH cells, overexpression of Gβ1 resulted in the cytoplasmic accumulation of the protein, whereas co-expression of Gβ1 with either RP2 or Gγ1 restored membrane association of Gβ1. Depletion of RP2 in ARPE19 cells by siRNA resulted in a shift of Gβ1 from the membrane to the cytosol, confirming that RP2 facilitates the membrane association of Gβ1. This shift in Gβ1 localization was rescued by Gγ1 overexpression. Membrane targeting of Gβ1 required RP2 N-terminal myristoylation and occurs via the co-factor C (TBCC) homology domain. The interaction was disrupted by the pathogenic RP2 mutation R118H, which blocks Arl3 GAP activity. Arl3-Q71L competed with Gβ1 for RP2 binding suggesting that RP2 GAP activity on Arl3 would release Gβ1. RP2 stimulated the association of Gβ1 with Rab11, an important GTPase for post-Golgi vesicle trafficking of photoreceptor proteins. Collectively our data support a role for RP2 in facilitating membrane association and traffic of Gβ1. Combined with other recent evidence, this suggests that RP2 may co-operate with Arl3 and its effectors in cilia associated trafficking of G proteins

Using induced pluripotent stem cells to understand retinal ciliopathy disease mechanisms and develop therapies

The photoreceptor cells in the retina have a highly specialised sensory cilium, the outer segment (OS), which is important for detecting light. Mutations in cilia-related genes often result in retinal degeneration. The ability to reprogramme human cells into induced pluripotent stem cells and then differentiate them into a wide range of different cell types has revolutionised our ability to study human disease. To date, however, the challenge of producing fully differentiated photoreceptors in vitro has limited the application of this technology in studying retinal degeneration. In this review, we will discuss recent advances in stem cell technology and photoreceptor differentiation. In particular, the development of photoreceptors with rudimentary OS that can be used to understand disease mechanisms and as an important model to test potential new therapies for inherited retinal ciliopathies

REEP6 Deficiency Leads to Retinal Degeneration through Disruption of ER Homeostasis and Protein Trafficking

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. We recently identified mutations in REEP6, which encodes the receptor expression enhancing protein 6, in several families with autosomal recessive RP. REEP6 is related to the REEP and Yop1p family of ER shaping proteins and potential receptor accessory proteins, but the role of REEP6 in the retina is unknown. Here we characterise the disease mechanisms associated with loss of REEP6 function using a Reep6 knockout mouse generated by CRISPR/Cas9 gene editing. In control mice REEP6 was localised to the inner segment and outer plexiform layer of rod photoreceptors. The Reep6-/- mice exhibited progressive photoreceptor degeneration from P20 onwards. Ultrastructural analyses at P20 by transmission electron microscopy and 3View serial block face scanning EM revealed an expansion of the distal ER in the Reep6-/- rods and an increase in their number of mitochondria. Electroretinograms revealed photoreceptor dysfunction preceded degeneration, suggesting potential defects in phototransduction. There was no effect on the traffic of rhodopsin, Rom1 or peripherin/rds; however, the retinal guanylate cyclases GC1 and GC2 were severely affected in the Reep6 knockout animals, with almost undetectable expression. These changes correlated with an increase in C/EBP homologous protein (CHOP) expression and the activation of caspase 12, suggesting that ER stress contributes to cell death. Collectively, these data suggest that REEP6 plays an essential role in maintaining cGMP homeostasis though facilitating the stability and/or trafficking of guanylate cyclases and maintaining ER and mitochondrial homeostasis

Identification and Correction of Mechanisms Underlying Inherited Blindness in Human iPSC-Derived Optic Cups

Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290, which causes missplicing and premature termination, but the basis of this sensitivity is unclear. Here, we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups, despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups, explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290, restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention

Induced Pluripotent Stem Cells for Inherited Optic Neuropathies—Disease Modeling and Therapeutic Development

Background: Inherited optic neuropathies (IONs) cause progressive irreversible visual loss in children and young adults. There are limited disease-modifying treatments, and most patients progress to become severely visually impaired, fulfilling the legal criteria for blind registration. The seminal discovery of the technique for reprogramming somatic nondividing cells into induced pluripotent stem cells (iPSCs) has opened several exciting opportunities in the field of ION research and treatment. / Evidence Acquisition: A systematic review of the literature was conducted with PubMed using the following search terms: autosomal dominant optic atrophy, ADOA, dominant optic atrophy, DOA, Leber hereditary optic neuropathy, LHON, optic atrophy, induced pluripotent stem cell, iPSC, iPSC derived, iPS, stem cell, retinal ganglion cell, and RGC. Clinical trials were identified on the ClinicalTrials.gov website. / Results: This review article is focused on disease modeling and the therapeutic strategies being explored with iPSC technologies for the 2 most common IONs, namely, dominant optic atrophy and Leber hereditary optic neuropathy. The rationale and translational advances for cell-based and gene-based therapies are explored, as well as opportunities for neuroprotection and drug screening. / Conclusions: iPSCs offer an elegant, patient-focused solution to the investigation of the genetic defects and disease mechanisms underpinning IONs. Furthermore, this group of disorders is uniquely amenable to both the disease modeling capability and the therapeutic potential that iPSCs offer. This fast-moving area will remain at the forefront of both basic and translational ION research in the coming years, with the potential to accelerate the development of effective therapies for patients affected with these blinding diseases