ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2017) 51 OC5.3 | DOI: 10.1530/endoabs.51.OC5.3

Novel evidence implies that ALADIN, the triple A syndrome gene product is involved in mitochondrial physiology

Alexandra Rodrigues Da Costa, Eirini Meimaridou, Rathi Prasad, Louise A. Metherell, J. Paul Chapple & Helen L. Storr

William Harvey Research Institute, Centre for Endocrinology, Barts and the London School of Medicine, Queen Mary University of London, London, UK.

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. ALADIN’s 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 ALADIN’s role in mitochondrial physiology.

Article tools

My recent searches

No recent searches.