Abstract
Purpose :
Mutations in rhodopsin, the light sensitive protein of rod photoreceptor cells, are the most common cause of autosomal dominant retinitis pigmentosa. Most rhodopsin mutations, such as P23H, cause rhodopsin misfolding and ER retention and are classified as class II mutations. Enhancement of the cell stress response through arimoclomol or Hsp90 inhibitors can protect photoreceptors in P23H transgenic rats and in cells overexpressing P23H rod opsin. Here we have characterised a range of rhodopsin mutations to investigate their effects on the localisation of heterologously expressed rod opsin and whether these mutants respond to manipulation of the stress response. Additionally, we investigated whether arimoclomol could protect the P23H knock-in (KI) mouse model, which better mimics the gene dosage and rhodopsin expression levels seen in humans.
Methods :
Cells were transfected with a range of GFP-tagged rod opsin mutants (including P23H, G106R, C110R, P171L, E181K, C185R, G188R) and treated with 1 μM arimoclomol or 1 μM of the Hsp90 inhibitor 17-AAG. Mutant rod opsin localisation and inclusion formation was assessed by immunofluorescence and confocal microscopy. P23H KI mice were treated from P21 to P43 with 10 mg/kg arimoclomol or vehicle administered daily via intraperitoneal injection. Mice were then subjected to electroretinogram (ERG) and tissue was taken for biochemical studies and histology.
Results :
The cellular localisation of the variants was different to wild-type rod opsin, which traffics to the plasma membrane, as they were ER retained and formed intracellular inclusions similar to those formed by P23H rod opsin, suggesting that they behave like misfloded class II mutants on heterologous over-expression. Pharmacological manipulation with arimoclomol or 17-AAG led to significant reduction of the inclusion incidence, indicating that both drugs can reduce mutant rod opsin aggregation. Preliminary data from the treatment of the P23H KI mice with arimoclomol showed improved photoreceptor function since the ERG responses of treated mice were significantly enhanced.
Conclusions :
Pharmacological stimulation of the cell stress responses can be applicable to other rhodopsin mutants that behave similar to P23H rod opsin and appears to be a promising therapeutic strategy for class II rhodopsin RP.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.