Mitochondrial Functional Study Associated With M.1555A>G Mutation Based On Deaf-Specific Induced Pluripotent Stem Cells

Hearing loss is one of the most important human health problems, affecting 360 million people in the world. m.1555A>G is a hot spot and main molecular mechanism of non-syndromic hearing loss. Due to the lack of appropriate disease models, research in the underlying mechanisms of hearing loss associated with mitochondria DNA is limited. iPSCs induced from patients’somatic cells could be differentiated into various kinds of cells like inner ear hair cells, which maintains the mutation from the patient and could be used to further illustrate the underlying mechanisms of tissue specific diseases at the same time. We established and characterized deaf patient-specific iPSCs associated with m.1555A>G, here in this article we further study the molecular mechanisms of hearing loss associated with m.1555A>G.m.1555A>G is in the 12S rRNA, which is the subunit of mitochondrial ribosome and is responsible for protein translation. In order to study the influence of cell structure and function brought by m.1555A>G, we analyzed the transcription and translation level of 12S rRNA and OXPHOS subunits, ATP and ROS production and mitochondria ultramarine structur. The transcriptional level of 12S rRNA, subunits of OXPHOS encoded by mitochondrial DNA ND5, CYTB, COX2, ATP8 and subunits of OXPHOS encoded by nuclear genes NDUFB8, SDHB, UQCRC2, COX4, ATP5A in cells associated with m.1555A>G showed a decrease at various levels, and the decrease in iPSCs was smaller than that in renal epithelial cells. Western blotting analysis showed that translational level of subunits of OXPHOS encoded by mitochondrial DNA COX2, ATP8 and subunits of OXPHOS encoded by nuclear genes NDUFB8, SDHB, UQCRC2, COX4 and ATP5A in renal epithelial cells associated with m.1555A>G showed a decrease at various levels when compared with wild type cells(P<0.01 or P<0.05). m.1555A>G had an inconspicuous influence in iPSCs. Mitochondrial functional analysis showed that total ATP production and mitochondrial ATP production decreased 168.8% and 299.3% respectively (P<0.05) in renal epithelial cells associated with m.1555A>G, the influence of the mutation in iPSCs is unapparent; ROS production caused by mitochondrial dysfunction increased about 8.9%(P<0.05) in renal epithelial cells and 13.4%(P>0.05) in iPSCs. Electron microscopic analysis showed that mitochondrial is in a long stick shape while a round shape in iPSCs. Mitochondrial DNA copy number detection showed that mtDNA copy number in renal epithelial cells associated with m.1555A>G is slightly increased (P>0.05), while increased 35.7% in iPSCs (P<0.01), in addition, mtDNA copy number in renal epithelial cells is higher than that in iPSCs.In summary, m.1555A>G resulted in decreased transcriptional and translational level of mitochondrial genes and nuclear genes, led to mitochondrial dysfunction. Insufficient energy supply excessive oxidative damage to inner ear hair cells might be the main mechanism of hearing loss associated with m.1555A>G. We established deaf patient-specific iPSCs associated with m.1555A>G for the first time. Accompanied by patient renal epithelial cells, we studied the molecular mechanism of hearing loss caused by m.1555A>G systematically. Our study will provide a new insight in the diagnosis and treatment of hearing loss.