36.2Pharmacological Strategies for Misfolding Mutant Rod Opsin

strategies involved the use of pharmacological chaperones, such as the rod opsin chromophore (11-cis-retinal) and its retinoid analogues (e.g. 9-cis-retinal).

Several studies have suggested that these compounds have the potential to correct misfolding of mutant rod opsin by binding specifically to near-native forms and stabilising the protein structure. Li and colleagues (1998) showed that a vitamin A supplemented diet reduced the rate of decline of the ERG a-wave and b-wave in class II T17M transgenic mice. In vitro studies suggested that 9-cis-retinal increased the amount of P23H rod opsin reaching the plasma membrane (Saliba et al., 2002). Furthermore, the introduction of 11-cis-7-ring-retinal, a 7 membered ring analogue of 11-cis-retinal, to stable cell lines expressing P23H rod opsin also resulted in the folding of mutant protein (Noorwez et al., 2003). A similar effect was observed when 9-cis-retinal and 11-cis-retinal were added during rod opsin synthesis in cell culture (Noorwez et al., 2004).

Importantly, the improvement in folding promoted by these pharmacological chaperones also decreased the toxic gain of function and dominant-negative effects associated with P23H rod opsin expression in SK-N-SH neuroblastoma cells (Mendes and Cheetham 2008). Treatment with 9-cis-retinal and 11-cis-retinal resulted in a reduction in inclusion incidence and protein aggregation, increased the presence of mature glycosylated P23H rod opsin species, and protected against cell death and caspase activation suggesting a reduction of the gain of function effects of mutant rod opsin. Significantly, the dominant-negative effects of P23H rod opsin were also alleviated by these pharmacological chaperones, as the retention of wildtype rod opsin in the ER associated with the expression of the mutant protein was reduced. These data suggest that if 11-cis-retinal were present in the inner segment during rhodopsin biogenesis and could bind rod opsin, it would potentially stabilise class II mutant rod opsin folding, and decrease the gain of function and dominant negative effects. Furthermore, bright light exposure would isomerise the 11-cis-retinal and exacerbate the gain of function and dominant negative effects of the class II mutants and this may contribute to the acceleration in retinal degeneration caused by light in class II rhodospin animal models (Wenzel et al., 2005).

36.2.2 Kosmotropes

A group of low molecular weight compounds named kosmotropes, or chemical chaperones, have been reported to improve the folding and reduce aggregation of proteins involved in a variety of human diseases associated with protein misfolding, such as CFTR (Zeitlin et al., 2002). Kosmotropes include polyols such as glycerol; solvents such as dimethyl sulfoxide (DMSO); methylamines such as trimethylamine-N-oxide (TMAO); fatty acids such as 4-phenylbutyric acid (4-PBA); and sugars such as trehalose among others. Unlike pharmacological chaperones, the effects of kosmotropes are non-specific and might involve the hydration of proteins (Back et al. 1979) or the reduction of the free movement of proteins to prevent the aggregation of partially folded proteins (Singer and Lindquist 1998).

DMSO, TMAO, 4-PBA and trehalose were effective at reducing protein aggregation and inclusion incidence in SK-N-SH cells expressing P23H rod opsin (Mendes and Cheetham 2008). Furthermore, this correlated with protection against cell death and caspase activation induced by mutant rod opsin expression. However, unlike retinoids, kosmotropes did not appear to promote the trafficking of the mutant protein or alleviate the dominant-negative effects associated with P23H rod opsin. Therefore, it appeared that the gain of function effects could be alleviated by increasing the degradation of the aggregation prone species and reducing inclusion incidence rather than by assisting protein folding per se.

36.2.3 Molecular Chaperone Inducers

All secreted proteins are subject to ER quality control, the primary mediators of which are molecular chaperones that not only sample and help polypeptides to fold but also evaluate the conformations of their substrates. If folding is delayed or an illegitimate conformation arises, the substrate is either subjected to additional folding cycles or is selected for a process termed ER-associated degradation (ERAD) (Vembar and Brodsky 2008). Molecular chaperones recognise hydrophobic residues or unstructured backbone regions in misfolded proteins and promote the folding process through cycles of substrate binding and release regulated by their nucleotide binding, hydrolysis and facilitated by cofactor proteins (Kosmaoglou et al., 2008: Kosmaoglou and Cheetham 2008). There is now ample evidence in a variety of in vivo and in vitro models that the manipulation of the chaperone machinery can be used to alleviate toxicity associated with misfolded protein disorders. For example, Cummings and colleagues (1998) first showed that the overexpression of a chaperone decreased ataxin-1 aggregation in a model of spinocerebellar ataxia type 1 and several chaperones have now been shown to protect against polyglutamine-induced neurodegeneration (e.g. Westhoff et al., 2005; Howarth et al., 2007).

A pharmacological approach has been attempted in several models of neurodegenerative diseases using the Hsp90 inhibitors geldanamycin, radicicol, 17- allylamino-17-demethoxygeldanamycin (17-AAG). The rationale for using these Hsp90 inhibitors at low doses to induce molecular chaperones is based on the autoregulation of heat shock factor 1 (HSF1) by chaperones. HSF1 is usually bound to a molecular chaperone complex containing Hsp90 and other chaperones and is kept in an inactive state unless a stress signal is detected. Hsp90 inhibitors disrupt the chaperone: HSF1 complex and release the HSF1, which is then activated and increases the expression of chaperone proteins. This approach has been used in a cell model of Huntington’s disease where treatment of COS-1 cells with geldanamycin induced the expression of Hsp40, Hsp70 and Hsp90 and inhibited polyglutamine expanded Huntingtin exon 1 protein aggregation in a dose-dependent manner (Sittler et al., 2001).

In the rhodopsin RP cell model, geldanamycin, radicicol and 17-AAG alleviated the gain of function effects induced by P23H rod opsin. Treatment with these