Triple A syndrome (AAAS), a rare and debilitating autosomal recessive disorder. It is characterised by adrenal failure, alacrima and achalasia; ~70% patients develop a neurodegeneration. The AAAS gene encodes ALADIN, a nuclear pore complex (NPC) protein necessary for the selective nuclear import of DNA protective molecules and is important for cellular redox homeostasis. ALADINs role is not fully characterised: its discovery at the centrosome and the endoplasmic reticulum suggests a role outside the NPC. ALADIN deficiency has been linked to disruption of mitochondrial steroidogenic enzymes and increased mitochondrial superoxide species. We hypothesised that AAAS is a consequence of mitochondrial dysfunction.
Aim: To examine whether ALADIN has a role in mitochondrial physiology
Methods: A stable knockdown (KD) of AAAS-gene expression using synthetic shRNA lentiviral transduction was established in a neuroblastoma cell line (SHSY5Y) to model AAAS (AAAS-KD). Western blotting confirmed an 80% reduction of ALADIN expression. Microarray (Qiagen, PAHS-065) was used to profile the expression of 84 genes related to oxidative stress in AAAS-KD and wild-type (WT) SHSY5Y cells. Immunofluorescence was used to detect ALADIN using a rabbit polyclonal anti-ALADIN antibody (Proteintech Europe). MitoTracker identified the mitochondria. Confocal microscopy identified the subcellular localisation of ALADIN relative to mitochondria. ALADIN deplete AAAS-KD SHSY5Y cells established any non-specific staining. Mitochondrial volume was measured by 3D reconstruction of the fluorescent images, using Imaris.
Results: Microarray revealed that reduced levels of ALADIN altered the expression of 8 genes in oxidative stress pathways (ACTB, SOD1, PXDN, PRDX6, HSPA1A, GPX3, CCS, and ALB). Confocal imaging demonstrated ALADIN co-localisation with the mitochondria in WT SHSY5Y cells. Additionally, mitochondrial volume was increased in AAAS-KD (n=5) compared to WT cells (n=6): mean 73.29 um3, SEM ±23.14 vs mean 34.82 um3, SEM ±7.681 (P=0.122), respectively.
Conclusion: In this model, ALADIN deficiency impacts the transcription of genes involved in oxidative stress pathways. ALADIN appears to co-localise with mitochondrial marker MitoTracker. ALADIN deficiency is associated with an increase in mitochondrial volume. This supports the further exploration of ALADINs role in mitochondrial physiology.
22 - 24 Nov 2017
British Society for Paediatric Endocrinology and Diabetes