Rescue of mt DNA-derived defects by mitochondria-tareted mRNA import and translation

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PROJECT ABSTRACT Mitochondrial dysfunction is known to be involved in a number of human diseases. Many mitochondrial proteins are encoded by genes located in nuclear DNA (nDNA), however mitochondria possess their own genome (mtDNA) that encodes 37 genes in humans. Among them, 13 encode polypeptides that are critical subunits for electron transport chain (ETC) complex holoenzyme activity, making them essential components for mitochondrial function. Investigation into nuclear encoded mitochondrial proteins has been simplified due to an abundance of methods available for manipulating the nuclear genome. However, methods for studying the effects of mutations found in Mt-DNA are limited and indirect. Specifically, our ability to generate mtDNA “transgenic” cell lines and animals remains quite limited. This leaves a significant gap in our ability to address fundamental questions about mitochondrial contribution to disease. In an attempt to bridge this gap, our lab recently used an RNA localization sequence previously discovered by the Koehler and Teitell group to deliver mRNA of both native (mtCO2 and mtCO3) and non- native, engineered (eGFP and DNase1) mRNA to mitochondria. Our results indicate successful import and translation of these mRNA's within the mitochondrial matrix of SH-SY5Y human neuroblastoma cells. To our knowledge this is the first successful demonstration of translation of a non-native protein within mitochondria. The objective of this study is to further develop this method as a means of studying mitochondrial dysfunction by targeting nucleus-transcribed mitochondrial mRNA (mt-mRNA) transcripts to mitochondria for translation and incorporation into ETC holoenzymes. Our central hypothesis is that nucleus-transcribed, mitochondria-translated mRNA can produce functional respiratory chain proteins and will facilitate modeling and rescue of mtDNA-derived mitochondrial dysfunction. Our long-term goals are to study the functional consequences of disease-associated single nucleotide polymorphisms on mitochondrial function and brain aging in mice. (Aim 1) We will determine the efficiency and functional effects of mitochondrial-targeted delivery and translation of exogenous transcripts for incorporation into ETC holoenzymes in neuronal cells. (Aim 2) We will investigate whether mitochondrial-targeted delivery and translation of exogenous transcripts rescues mtDNA-derived mitochondrial dysfunction in mouse neurons of a mitochondrial disease model. Our proposed studies are expected to generate a novel approach, both in vitro and in vivo, to analyzing the role of mitochondrial dysfunction in brain aging that can be further utilized for basic and translational research. We believe that our approach will provide insight into mtDNA mutation pathology and offer a pathway-specific method for potential intervention.