Modification of mutant bestrophin-1 processing to prevent retinal degeneration

Name of the University: The University of ManchesterCandidate?s full name: Carolina UggentiDegree Title: PhD in MedicineThesis Title:Modification of mutant bestrophin-1 processing to prevent retinal degenerationDate: 2015Bestrophin-1 is a homopentameric Ca2+-gated anion channel which localises to the basolateral plasma membrane of retinal pigment epithelium (RPE) cells. Homozygous and compound heterozygous mutations in the BEST1 gene are associated with autosomal recessive bestrophinopathy (ARB), a retinopathy characterised by altered electrooculogram (EOG), deposits in the retina, and is often associated with the risk of developing angle-closure glaucoma. The mechanism by which mutations in bestrophin-1 cause disease remains unknown. Expression of four ARB-causing bestrophin-1 proteins in polarised MDCKII cells, a cell model for RPE, results in mutant proteins mislocalisation and degradation. Furthermore, when the ability of the mutant proteins to conduct Cl- ions was investigated in HEK293 cells by whole-cell patch-clamp, a reduction in the Cl- current was observed in all mutants compared to the WT.The use of a combination of the small molecules bortezomib and 4-phenylbutyrate (4PBA) successfully restored the expression and trafficking of all four ARB-causing bestrophin-1 proteins. Importantly, 4PBA was also able to restore the ability of the mutant channel to conduct Cl- ions. Biotinylation of cell surface proteins shows that the number of active channels at the plasma membrane of HEK293 cells increases following 4PBA treatment. The functional rescue achieved with 4PBA supports the hypothesis that ARB-associated missense mutations reduce the number of functional channels that reach the cell membrane rather than altering other aspects of channel function.The results presented in this thesis suggest that 4PBA may be a promising therapy for the treatment of ARB and the other bestrophinopathies resulting from missense mutations in BEST1, particularly as 4PBA is already approved for long-term use in infants and adults. These finding also pave the way for the use of small molecule therapies to treat conformational diseases caused by mutation in other protein expressed in the RPE.