Investigation of the effects of ATP13A2 mutations on intracellular iron accumulation

Abstract

Kufor-Rakeb Syndrome (KRS), characterised by spasticity, dementia and supranuclear gaze paresis, is a form of early-onset atypical Parkinson's disease. Autosomal recessive and loss-of-function mutations in the ATP13A2 gene have been shown to cause KRS. The ATP13A2 gene is located on chromosome 1 and encodes a large transmembrane protein. ATP13A2 belongs to the P5 type ATPase protein family and is mainly located in the membranes of lysosomes and late endosomes. By transporting polyamines that can chelate iron, ATP13A2 is involved in maintaining iron homeostasis in cells. Because of this function, ATP13A2 mutations can lead to iron accumulation in cells. In addition, this accumulated iron can lead to cell death because iron deposition can induce endoplasmic reticulum (ER) stress in cells. However, it is controversial whether all ATP13A2 mutations cause iron deposition in patients based on their magnetic resonance images. In this study, ATP13A2-related iron deposition was analysed at the cellular level. Iron accumulation and its effects on cell viability were studied for three different ATP13A2 mutations, namely frameshift (FS), deletion (del) and missense (MS). These mutations were identified in three different KRS patients. To analyze the effects of these mutations, MCF7 cells overexpressing either wt or mutant ATP13A2 proteins (FS-ATP13A2, del-ATP13A2 and MS-ATP13A2) and primary fibroblasts from patients subjected to FeCl₃-6H₂O treatment were used. Iron ions were detected in the cells by Prussian blue staining and it was observed that frameshift and deletion mutations of ATP13A2 caused the most iron accumulation in the cells. However, missense ATP13A2 also contributed to iron deposition compared to control cells expressing wt ATP13A2 protein. On the other hand, iron accumulation is known to lead to cell death via the ER stress pathway. Therefore, cell viability was analysed for these three ATP13A2 mutations. Consistently, most cell death was detected in cells expressing FS-ATP13A2 and del-ATP13A2 in both MCF7 cells and fibroblasts. In addition, the viability of cells overexpressing MS-ATP13A2 was also decreased in association with the iron accumulation. This suggests that mutations in ATP13A2 contribute to iron accumulation in cells and that this iron accumulation can lead to cell death.