Biochemical and Molecular Methods for the Study of Mitochondrial Disorders

Mitochondrial disorders are a group of genetically heterogeneous complex diseases [1–6]. Although mitochondrial structure and function involve two genomes, the biogenesis of mitochondrion and more than 99 % of its protein contents are encoded by the nuclear genome [7]. As a result, the majority of the mitochondrial disorders are caused by molecular defects in the nuclear genome [5, 6, 8–10]. Early studies of mitochondrial disorders focused on mutations in the 16.6 kb tiny mitochondrial DNA (mtDNA) because of its genomic simplicity and maternal inheritance [3, 11]. In the past decade, autosomal recessive mitochondrial disorders have been increasingly recognized [4, 5, 8, 10, 12–22]. Recent studies through integrated genomics and system biology identified approximately 1,500 nuclear-encoded proteins targeted to mitochondria [7], although mutations have only been identified in about 150 nuclear genes. The biogenesis of mtDNA requires nuclear-encoded genes, including DNA polymerase gamma (POLG) and DNA helicase (TWINKLE) for replication as well as purine/pyrimidine nucleoside kinases, DGUOK and TK2, for salvage synthesis and maintenance of deoxynucleotide (dNTP) pools. Defects in any of these genes cause mtDNA depletion [23, 24] or mtDNA multiple deletions [25, 26]. In addition, mtDNA transcription and mitochondrial protein biosynthesis require nuclear DNA (nDNA)-encoded factors [7, 27–29], including RNA polymerase, various transcription factors, small and large subunits (> 80) of mitochondrial ribosomal proteins, translation initiation and elongation factors, and all mitochondrial aminoacyl tRNA synthetases [22, 30–35]. The majority of mitochondrial respiratory chain complex subunits and all complex assembly factors are also nuclear encoded [28, 36–44]. Furthermore, the roles of mitochondria in autophagy, apoptosis, and reactive oxygen species (ROS) production have recently proven to be indispensible in the pathogenic mechanisms of neurodegenerative diseases (Parkinson, Alzheimer, Huntington, etc.)