Background: Triple A Syndrome is a rare, autosomal recessive cause of adrenal failure that usually manifests in the first decade. Most cases have isolated glucocorticoid deficiency, but this is accompanied by mineralocorticoid deficiency in approximately 10% of cases. Additional features include alacrima (~90%), achalasia of the oesophageal cardia (~75%), and a progressive neurodegenerative process (~60%). The AAAS gene product is the nuclear pore complex protein ALADIN of unknown function. Previous studies have described dermal fibroblasts from AAAS patients as being more sensitive to oxidative stress than wild-type fibroblasts.
Methods: To provide a better disease model we have successfully achieved greater than 80% knockdown of AAAS-gene expression in H295R adrenocortical tumour cells (chosen as representative of the cell type affected by AAAS) using synthetic shRNA lentiviral transduction. This was confirmed by quantifying mRNA expression of AAAS by real time qPCR and western blotting for protein expression of ALADIN. To assess the adverse effects of oxidative stress, cells were exposed to hydrogen peroxide.
Results: Without hydrogen peroxide treatment there was significantly reduced cell growth, both by cell counting (P<0.0001, n=4) and the use of MTS assays (P<0.05, n=3), of AAAS-knockdown cells in comparison with controls. These cells, when treated, show significantly reduced cell survival in comparison with controls (P<0.01, n=3). An increase in apoptosis was observed, assessed by cleavage of PARP (Poly ADP ribose polymerase), of AAAS-knockdown cells after treatment compared with control cells (P<0.05, n=3).
Conclusion: Using AAAS-knockdown cells we provide further evidence that oxidative stress is involved in the progression of Triple A syndrome. As the steroidogenic activity of the adrenal cortex induces a highly oxidative environment this may explain the susceptibility of the tissue in the absence of functional ALADIN.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: Declaration of Funding: This work was supported by the Barts and the London Charity and the Wellcome Trust