Deficiency of ALADIN impairs redox homeostasis in human adrenal cells and inhibits steroidogenesis

Abstract

Triple A syndrome is a rare, autosomal recessive cause of adrenal failure. Additional features include alacrima, achalasia of the esophageal cardia, and progressive neurodegenerative disease. The AAAS gene product is the nuclear pore complex protein alacrima-achalasia-adrenal insufficiency neurological disorder (ALADIN), of unknown function. Triple A syndrome patient dermal fibroblasts appear to be more sensitive to oxidative stress than wild-type fibroblasts. To provide an adrenal and neuronal-specific disease model, we established AAAS-gene knockdown in H295R human adrenocortical tumor cells and SH-SY5Y human neuroblastoma cells by lentiviral short hairpin RNA transduction. AAAS-knockdown significantly reduced cell viability in H295R cells. This effect was exacerbated by hydrogen peroxide treatment and improved by application of the antioxidant N-acetylcysteine. An imbalance in redox homeostasis after AAAS knockdown was further suggested in the H295R cells by a decrease in the ratio of reduced to oxidized glutathione. AAAS-knockdown SH-SY5Y cells were also hypersensitive to oxidative stress and responded to antioxidant treatment. A further impact on function was observed in the AAAS-knockdown H295R cells with reduced expression of key components of the steroidogenic pathway, including steroidogenic acute regulatory and P450c11β protein expression. Importantly a significant reduction in cortisol production was demonstrated with AAAS knockdown, which was partially reversed with N-acetylcysteine treatment.

Conclusion: Our in vitro data in AAAS-knockdown adrenal and neuronal cells not only corroborates previous studies implicating oxidative stress in this disorder but also provides further insights into the pathogenic mechanisms in triple A syndrome.

Figure 1.

Lentiviral shRNA knockdown of the AAAS gene in H295R (A) and SH-SY5Y (B) cells. AAAS mRNA expression was quantified using real-time qPCR and normalized to GAPDH (n = 3). Control represents transfection with scrambled shRNA. Reduced expression of the protein ALADIN is seen in AAAS-knockdown (AAAS-KD) H295R (C) and SH-SY5Y (D) cells. Data represents mean ± SD. **, P < .01; ***, P < .001.

Figure 2.

A reduction in cell viability of AAAS-KD cells. A, There was a significant difference in the total number of cells (by cell counting) on day 5 in the AAAS-KD H295R cells (n = 4) compared with controls after seeding at 2 × 10⁴ cells/well. B, MTS assays on days 2 and 5 with initial seeding density of 5 × 10³ cells/well on day 0. There was a significant reduction in absorbance readings of AAAS-KD H295R cells in comparison with controls on day 2 (n = 3) and day 5 (n = 3). After treatment with 100 μM H₂O₂, a significant reduction in absorbance readings is seen in AAAS-KD H295R cells (n = 3) (C) and AAAS-KD SH-SY5Y cells (n = 4) (D) using MTS assays. There was no reduction in absorbance in untreated AAAS-knockdown SH-SY5Y cells compared with controls. Data represent mean ± SD. *, P < .05; **, P < .01; ***, P < .001.

Figure 3.

Cell cycle arrest, increased apoptosis, and an imbalance of redox homeostasis is observed in AAAS-KD H295R adrenal cells. A, A significant reduction in the percentage cell population in the G₂/mitotic phase of the cell cycle is seen in AAAS-KD H295R cells compared with controls, suggesting cell cycle arrest (n = 3). B, An increase in cleaved relative to total PARP, measured by densitometric analysis after immunoblotting, is observed in H₂O₂-treated AAAS-KD cells compared with controls (n = 3). C, The GSH to GSSG ratio was lower in AAAS-KD cells relative to controls (n = 3). Oxidative stress induced by treatment with 40 μM menadione to control cells further reduces the ratio. RLU, relative light units. Data represent mean ± SD. *, P < .05.

Figure 4.

Treatment with the antioxidant NAC improves AAAS-KD cell viability. A, Treatment with 1 mM NAC for 48 hours significantly increases absorbance in both AAAS-KD and control H295R cells (n = 4). B, There is a significantly greater percentage increase in absorbance after treatment in the knockdown cells in comparison with the controls (n = 4). C, Treatment with 10 mM NAC significantly increases the absorbance in AAAS-KD SH-SY5Y cells but not controls (n = 4). Data represent mean ± SD. *, P < .05; **, P < .01.

Figure 5.

StAR and P450c11β (CYP11B1) expression are reduced in AAAS-KD H295R cells with a subsequent reduction in cortisol production. Immunoblot analysis (A) and densitometric analysis showing a reduction in StAR (B) (n = 4) and P450c11β protein expression (C) in AAAS-KD H295R cells compared with controls (n = 3), with no effect on P450scc levels (D) (n = 4). E, There is a significant reduction in StAR mRNA levels in AAAS-KD cells compared with controls, and hydrogen peroxide treatment of controls reduces levels to that seen with ALADIN deficiency (n = 3). F, There is a significant reduction in cortisol production by the untreated AAAS-KD H295R cells compared with controls (n = 9), with partial improvement after NAC treatment (n = 3). Data represent mean ± SD. *, P < .05; **, P < .01; ***, P < .001.