Identifying links between mutations in Adiponectin Receptor 1 (ADIPOR1) and Retinitis Pigmentosa (RP) as potential targets for gene therapy
Creators: name: Author, Unknown
Publisher: name: University of Lincoln
Year: 2023

description: Retinitis pigmentosa (RP) is a hereditary disease characterised by progressive destruction of photoreceptors in the retina. Recently, mutations in the gene encoding one of the two adiponectin receptors, AdipoR1, have been reported to cause RP. Several functions of AdipoR1, and AdipoR2, may protect the retina and prevent RP, via ceramidase activity and regulation of membrane fluidity. The aim was to investigate the effects of targeted and naturally occurring mutations in AdipoR1 to gain understanding of structure/function and predict if they would cause RP. Mutants (S187A/T, D208A/N, H341A) were designed targeting zinc coordinating residues within the receptor as the zinc ion stabilises the receptor and participates in ceramidase activity. Naturally occurring mutations predicted to cause functional effects (R40W, L143P, C183S, M309R, Y310C, R320stop, R324L, H341Y, A348G, G367R) were identified from the genome aggregation database, gnomAD. The effect of these targeted and naturally occurring mutations on AdipoR1 expression and cell surface expression (CSE) was investigated by transient transfection, western blot and confocal microscopy. Targeted mutants showed significantly reduced protein levels and CSE except for the D208A/N mutants, which had increased protein and CSE. All naturally occurring mutants showed significantly reduced protein levels with a subset showing relatively unaffected CSE and another showing significantly reduced CSE. To determine functional effects, a mass spectrometry assay measuring cellular ceramide concentration was developed to investigate ceramidase activity; alongside an MTT assay and qPCR measuring gene expression as an indirect measure of effects on membrane fluidity and metabolic health. Ceramide concentration was significantly increased by palmitate but was unaffected by overexpression of wildtype or mutant adiponectin receptors in control or palmitate treated cells. siRNA-mediated knockdown of AdipoR1, or AdipoR2 was used to investigate changes of indices associated with membrane fluidity in cells treated with palmitate. Results from MTT assays showed that cell viability/metabolism was significantly reduced by palmitate in a dose-dependent manner however there was no detectable effect of knockdown of AdipoR1, AdipoR2 or both. Similar results were observed measuring CHOP and FADS1 gene expression, markers of ER stress and desaturase gene expression. This is the first study showing that AdipoR1 mutants with mutations at zinc coordinating sites significantly reduce protein expression and abolish cell surface expression, further highlighting the importance of these sites and of zinc coordination. Characteristics of the D208A/N mutant raise the intriguing possibility of a gain of function mutation. This is also the first characterisation of a series of naturally occurring SNPs, showing that all exhibit reduced protein expression that can be subdivided into two sub-groups, with unaltered or compromised CSE. Interpretation of the functional studies is limited by technical limitations: Measurement of ceramide levels, and S1P, should be optimised to provide greater confidence in the negative findings (which are consistent with some independent groups) whilst the efficiency of AdipoR1/R2 knockdown (?60%) needs to be optimised. Future work should then focus on further elaboration of the properties of the mutants and determining the downstream effects on function.