Impaired ROS Scavenging System in Human Induced Pluripotent Stem Cells Generated from Patients with MERRF Syndrome.

Shih-Jie Chou,Chien-Tsun Chen,Yu-Fen Lai,Yau-Huei Wei,Yuh-Lih Chang,Shih-Hwa Chiou,Hsin-Chen Lee,Chian-Shiu Chien,Wei-Lien Tseng

doi:10.1038/srep23661

Abstract

Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a mitochondrial disorder characterized by myoclonus epilepsy, generalized seizures, ataxia and myopathy. MERRF syndrome is primarily due to an A to G mutation at mtDNA 8344 that disrupts the mitochondrial gene for tRNA(Lys). However, the detailed mechanism by which this tRNA(Lys) mutation causes mitochondrial dysfunction in cardiomyocytes or neurons remains unclear. In this study, we generated human induced pluripotent stem cells (hiPSCs) that carry the A8344G genetic mutation from patients with MERRF syndrome. Compared with mutation-free isogenic hiPSCs, MERRF-specific hiPSCs (MERRF-hiPSCs) exhibited reduced oxygen consumption, elevated reactive oxygen species (ROS) production, reduced growth, and fragmented mitochondrial morphology. We sought to investigate the induction ability and mitochondrial function of cardiomyocyte-like cells differentiated from MERRF-hiPSCs. Our data demonstrate that that cardiomyocyte-like cells (MERRF-CMs) or neural progenitor cells (MERRF-NPCs) differentiated from MERRF-iPSCs also exhibited increased ROS levels and altered antioxidant gene expression. Furthermore, MERRF-CMs or -NPCs contained fragmented mitochondria, as evidenced by MitoTracker Red staining and transmission electron microscopy. Taken together, these findings showed that MERRF-hiPSCs and MERRF-CM or –NPC harboring the A8344G genetic mutation displayed contained mitochondria with an abnormal ultrastructure, produced increased ROS levels, and expressed upregulated antioxidant genes.

Highlights

Human mitochondrial DNA is consisted of 16,569 bp encoded 37 genes, which includes 13 polypeptides of the mitochondrial respiratory chain responsible for mitochondrial oxidative phosphorylation (OXPHOS) as well as 2 rRNAs and 22 tRNAs for mitochondrial protein synthesis[1]
In over 80% of the cases, an A to G mutation at mitochondrial DNA (mtDNA) 8344 disrupts the mitochondrial gene for tRNA-Lys, which is associated with severe defects in protein synthesis, leading to impaired OXPHOS4,6. mtDNA mutation-elicited oxidative stress, oxidative damage and altered gene expression are involved in the pathogenesis and progression of Myoclonus epilepsy associated with ragged-red fibers (MERRF) syndrome[7,8]
The A8344G mutation blocks the modification of tRNALys, which impairs the synthesis of mitochondrial proteins that are essential for OXPHOS8

Summary

Introduction

Human mitochondrial DNA (mtDNA) is consisted of 16,569 bp encoded 37 genes, which includes 13 polypeptides of the mitochondrial respiratory chain responsible for mitochondrial oxidative phosphorylation (OXPHOS) as well as 2 rRNAs and 22 tRNAs for mitochondrial protein synthesis[1]. Recent progress in induced pluripotent stem cells (iPSCs) technology has facilitated the successful generation of human iPSCs via the forced expression of transcription factors such as OCT4, SOX2, KLF4, and c-MYC or OCT4, SOX2, LIN28 and NANOG in somatic cells. These cells have provided new opportunities for regenerative medicine and in vitro disease modeling[10,11,12,13]. Patient-specific iPSC models have been established to study the mechanisms of mtDNA mutation-associated diseases such as MELAS syndrome and Pearson marrow pancreas syndrome (PS)[18,19]. We generated MERRF-specific hiPSC as an in vitro models for investigating the pathophysiologic mechanism of mitochondrial diseases

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