Chapter 64: Targeting the proteostasis network in rhodopsin retinitis pigmentosa

Abstract

Mutations in rhodopsin are one of the most common causes of retinitis pigmentosa (RP). Misfolding of rhodopsin can result in disruptions in cellular protein homeostasis, or proteostasis. There is currently no available treatment for RP. In this review, we discuss the different approaches currently being investigated for treatment of rhodopsin RP, focusing on the potential of manipulation of the proteostasis network as a therapeutic approach to combat retinal degeneration.

64.1. Introduction

Retinitis pigmentosa is a group of inherited disorders that cause retinal degeneration via progressive loss of the rod and cone photoreceptors (Hartong et al. 2006). The first RP gene identified was rhodopsin (Dryja et al. 1990). Rhodopsin is the prototypical G-protein coupled receptor (GPCR), responsible for detecting light in the rod photoreceptors, comprised of the protein rod opsin with its chromophore 11-cis-retinal. Rod opsin is produced in the endoplasmic reticulum (ER), where it undergoes multiple post-translational modifications, such as glycosylation and disulfide bond formation (Kosmaoglou et al. 2008). Correctly folded rhodopsin is then transported and packed into the disks in the outer segment (OS) of the photoreceptor (Pearring et al. 2013). Over 200 point mutations in rhodopsin have been identified so far (RetNet https://sph.uth.edu/retnet/), which can be classified according to their biochemical and cellular properties (Mendes et al. 2005). The majority of rhodopsin mutations are class II mutations, including P23H the most common mutation in North America, that cause protein misfolding, retention in the ER and degradation. Rhodopsin is the major protein of the rod OS, so there is a high demand on the photoreceptor ER to produce rhodopsin. Photoreceptors have multiple mechanisms to cope with high protein turnover and maintain protein homeostasis, or proteostasis, including the heat shock response (HSR), the unfolded protein response (UPR), ER-associated degradation (ERAD) and autophagy systems (Balch et al. 2008; Athanasiou et al. 2013). Misfolded proteins, such as P23H rhodopsin, can induce these adaptive networks to reduce protein production, enhance folding facilitators and stimulate degradation. Targeting these networks may, therefore, be beneficial in rhodopsin RP.

64.2. Potential treatments for rhodopsin RP

Pharmacological agents may be used to directly target the folding of misfolded proteins, as in the case of pharmacological and chemical chaperones, or by inducing the cell’s molecular chaperone machinery.

64.2.1. Pharmacological and chemical chaperones

Pharmacological chaperones are compounds that specifically bind and stabilize near-native states to improve the folding of misfolded proteins. For example, the retinoids 9-cis- and 11-cis-retinal have been shown to stabilize P23H rod opsin in the ER allowing it to traffic through the secretory pathway and improve the yield of folded rhodopsin (Saliba et al. 2002; Noorwez et al. 2004). Importantly, toxic gain-of-function effects, cell death and protein aggregation, of misfolded P23H rod opsin were reduced by retinoids in a cell model. Retinoids also counteracted the dominant-negative effect of misfolded rod opsin on wild-type rod opsin (Mendes and Cheetham 2008). Furthermore, transgenic mice with another rhodopsin misfolding mutation, T17M, had improved electroretinogram (ERG) responses and preservation of photoreceptor survival when treated with 11-cis-retinal (Li et al. 1998). Recent work suggests that 11-cis-retinal treatment can partially rescue the traffic and folding of a range of rhodopsin misfolding mutants in vitro (Krebs et al. 2010); however, the rescued mutant rhodopsin is still inherently unstable (Opefi et al. 2013; Chen et al. 2014) and is likely to misfold after leaving the ER, especially if the retinoid leaves the binding pocket following light exposure.

In contrast, chemical chaperones or kosmotropes are small molecules (e.g. 4-phenylbutyric acid (4-PBA)) that stabilize proteins in a non-specific manner. Kosmotropes have been shown to reduce P23H-mediated cell death and insoluble protein load in cells (Mendes and Cheetham 2008). Tauroursodeoxycholic acid (TUDCA) is another chemical chaperone with anti-apoptotic properties. P23H transgenic rats treated with TUDCA had improved ERG responses and preserved retinal architecture (Fernandez-Sanchez et al. 2011).

64.2.2. HSR inducers

The HSR is a transcriptional response to a wide variety of cell stress and induces the expression of many proteins, in particular heat shock proteins (Hsps). Many Hsps function as molecular chaperones to help proteins attain their correct conformation, regulate protein quality control and the degradation of misfolded client proteins. Therefore, this network is a potential target to treat protein-misfolding diseases, and upregulation of the HSR can protect against several models of neurodegeneration. One method of upregulating molecular chaperone expression is to inhibit Hsp90. Hsp90 is in a feedback loop with the HSR transcription factor, heat shock factor 1 (HSF-1), and Hsp90 inhibition results in the post-translational modification and traffic of HSF-1 to nucleus where it induces other heat shock proteins that act on misfolded proteins (Figure 64.1; (Morimoto 1998)). Treatment with Hsp90 inhibitors reduced aggregation of P23H rod opsin and associated cell death in a cell model (Mendes and Cheetham 2008). Furthermore, the Hsp90 inhibitor HSP990 can improve retinal function and architecture in vivo in models of rhodopsin RP (Aguila et al. 2014).

Figure 64.1.

Pharmacological manipulation of proteostasis networks in rhodopsin RP. Inducing molecular chaperone expression by manipulating (1) the HSR, (2) the UPR or (3) inhibiting Hsp90 can alleviate the effects of misfolded rhodopsin. (4) Inducing autophagy helps remove aggregated misfolded protein. (5) ER chaperones such as BiP, EDEM1 and ERdj5 can be directly manipulated to maintain solubility in the ER and promote ERAD (6) for the removal of misfolded rhodopsin.

Another method to induce the HSR is with hydroxylamine derivatives (HADs), such as bimoclomol and arimoclomol (Vigh et al. 1997). These compounds potentiate the induction of the HSR but rely upon a boosting a pre-existing stress; as such they are HSR co-inducers. We recently used arimoclomol in cell and animal models of P23H rhodopsin RP (Parfitt et al. 2014). Arimoclomol potentiated the HSR in the presence of P23H rhodopsin in cells, leading to enhanced Hsp expression. Interestingly, the HSR was already activated by the mutant rhodopsin expression in the retinae of P23H transgenic rats and this HSR was further enhanced by arimoclomol treatment. Furthermore, arimoclomol led to improved ERG responses and photoreceptor survival in lines of transgenic rats with fast (P23H-1) and medium (P23H-3) rates of degeneration. Arimoclomol treatment caused a reduction of rhodopsin immunoreactivity in the cell bodies of the ONL and decreased the amount of insoluble rhodopsin, but there was no change in the normalized levels of soluble rhodopsin, suggesting that arimoclomol was stimulating the degradation of aggregation-prone rhodopsin, rather than rescuing the folding of the mutant protein. These changes correlated with a preservation of the photoreceptor OS structure implying that the defects in OS structure seen in these models is due, at least in part, to a dominant gain of function potentially related to unstable rhodopsin, which can be suppressed by arimoclomol. Interestingly, in addition to the enhanced HSR, arimoclomol potentiated the UPR in the retina, suggesting that these two proteostasis pathways might co-operate in photoreceptors (Parfitt et al. 2014).

64.3. The UPR in rhodopsin RP

The UPR is activated in P23H and T17M animal models (Lin et al. 2007; Kunte et al. 2012). Chronic activation of the UPR is associated with cell death; however, arimoclomol treatment enhanced the activation of all three branches of the UPR, whilst still protecting against mutant rhodopsin (Parfitt et al. 2014). Furthermore, the ablation of CHOP, which is a downstream pro-apoptotic effector of PERK, in P23H or T17M rhodopsin mouse models did not alter retinal degeneration (Nashine et al. 2013; Adekeye et al. 2014). Collectively these data suggest that activation of the UPR by mutant rhodopsin per se is not toxic to photoreceptors and might be a protective adaptive response that stimulates factors that can help deal with the mutant rhodopsin.

64.4. ER chaperones and ERAD of rhodopsin

The ER-resident chaperones that interact with WT and mutant rhodopsin in the ER to facilitate rhodopsin folding or quality control and degradation are starting to be identified. BiP (HSPA5) has an important role in rod opsin biogenesis, as wild type rod opsin aggregates in the absence of BiP, whereas BiP overexpression improves P23H rhodopsin mobility and loss of BiP increases P23H rhodopsin aggregation (Athanasiou et al. 2012). BiP expression is increased in P23H transgenic rats (Lin et al. 2007; Parfitt et al. 2014), and overexpression of BiP in P23H rats improves ERG responses and ONL thickness (Gorbatyuk et al. 2010).

In ERAD, misfolded proteins are transported out of the ER where they are degraded by the ubiquitin-proteasome system (UPS) in the cytosol (Fig 64.1). The ERAD effector EDEM1 can stimulate the degradation of P23H mutant rhodopsin and promote the traffic of the remaining P23H protein by improving folding, although this is only transient as the protein is unstable once it leaves the ER (Kosmaoglou et al. 2009). The ER-resident reductase, ERdj5 (DNAJC10), forms a chaperone network with EDEM1 and BiP and also plays a role regulating the biogenesis of rhodopsin, maintaining solubility of mutant rhodopsin within the ER and stimulating ERAD (Athanasiou et al. 2014). The identity of the complex involved in translocation of P23H rhodopsin is unknown; however, the AAA-ATPase VCP/p97 promotes the retrotranslocation and degradation of P23H rhodopsin (Griciuc et al. 2010).

An alternative method for removing misfolded protein is autophagy, where substrates are enclosed in double-membrane autophagosomes before degradation by lysozymes. Rapamycin is an inhibitor of mTOR, which is a negative regulator of autophagy. Rapamycin treatment reduced inclusion formation in cells expressing P23H rod opsin (Mendes and Cheetham 2008). Recent work showed that rapamycin treatment in P23H-3 rats improved ERG responses (Sizova et al. 2014).

64.5. Conclusions

The proteostasis networks have varied roles in protecting cells against misfolded proteins, which is particularly important in photoreceptors. Manipulation of these pathways, through chemical or genetic means, has provided insights into the mechanisms behind this protection. The identification of compounds with low toxicity, like arimoclomol, that can restore proteostasis could be potentially beneficial for rhodopsin RP.