358. AAV-MitoTimer to Asses Mitochondrial Turnover
Authors: Bennett Gosiker, Skylar Rizzo, Amy Donate, Christina Pacak
Journal: Molecular Therapy (2016)

Mitochondrial turnover is a key marker in the process of cellular senescence. In order to track mitochondrial turnover rates in various cell lines, an indicator is necessary to identify the formation of new mitochondria along with the degradation of old mitochondria. This measure is critical for the study of disease processes in which there is an up or down regulation of mitochondrial turnover rates. One dynamic tool to measure this balance is the pTRE-Tight-MitoTimer plasmid. The MitoTimer transgene combines the red fluorescent protein, dsRed, and a human cytochrome c oxidase subunit VIII (Cox 8) mitochondrial localization sequence to traffic the protein to the mitochondria where the color shifts from green to red over time (approximately 24 hours). Our goal is to use this as a tool to assess the increase or decrease in mitochondrial turnover as a result of mutations that cause mitochondrial dysfunction. We successfully developed two plasmids through the ligation of the MitoTimer transgene into a double stranded AAV-ITR containing plasmid with a 1) Desmin promoter pds-AAV-Des-MitoTimer to restrict expression to striated muscle and 2) CMV promoter pds-AAV-CMV-MitoTimer to enable ubiquitous expression among many cell types. Once cloned, these novel plasmids were then transfected into an immortalized C2C12 mouse muscle cell line to confirm successful generation of functional plasmids. An immediate application for these novel plasmids in our laboratory is the characterization of mitochondrial turnover rates in Barth Syndrome (BTHS) patient derived cells. BTHS is an X-linked mitochondrial disease that results from mutations of the gene TAZ, which encodes tafazzin. This gene serves to orchestrate the production of mature cardiolipin (CL) within the mitochondrial membrane. Mutations in the TAZ gene result in altered CL maturation, with implications on mitochondrial function. Thus far, mitochondrial turnover rates have not been carefully assessed in this disorder. As a preliminary experiment using human patient derived BTHS fibroblasts (harboring an exon 10: 748G &gt T nonsense mutation), pds-AAV-CMV-MitoTimer was successfully transfected and displayed variation in green and red fluorescence ratios as compared to a healthy control cell line. This demonstration of the impact of BTHS on mitochondrial turnover dynamics supports future use of these plasmids in BTHS induced pluripotent stem cells (iPSCs) that have been differentiated into cardiomyocytes or skeletal myotubes - the two cell-types most affected in BTHS. This will enable future comparisons of mitochondrial turnover in clinically relevant BTHS cells harboring a variety of different mutations and provide another metric by which to measure the success of pre-clinical therapeutics for BTHS. We are preparing an AAV Quad-mutant serotype for in vitro testing, and other serotypes will be used for in vivo models of mitochondrial dysfunction. AAV-MitoTimer will continue to be used as a measure of mitochondrial turnover, and provide an effective characterization method for metabolic diseases.