| dc.description.abstract | Mitochondrial proteins are encoded by both the nuclear and mitochondrial genome (mtDNA). Thus, alterations in mtDNA affect mitochondrial function and are involved in a plethora of diseases associated with mitochondrial dysfunction. Although role of nuclear DNA methylation in regulating gene expression is fairly well understood, the very existence of mtDNA methylation has been debated for the past four decades. In spite of the controversies revolving mtDNA methylation, differences in mtDNA methylation have been reported in a number of pathological conditions. However, role of mtDNA methylation in regulating transcription of mtDNA and consequently, mitochondrial function remains unexplored. Besides, whether mtDNA methylation can be altered experimentally has not been investigated. The goal of this dissertation is to establish a method to quantitate mtDNA methylation and evaluate its presence in SH-SY5Y neuroblastoma cells; examine if mtDNA methylation can be altered and if there are any associated changes in mitochondrial transcription and function; and further, determine if mtDNA methylation is altered in a model associated with mitochondrial dysfunction. In Chapter II, we aimed to investigate the presence of mtDNA methylation in SH-SY5Y neuroblastoma cells at global and site-specific levels. In order to investigate the presence of methylation at global levels in SH-SY5Y mtDNA, we established a direct, sensitive and highly specific method using HPLC-ESI-MS/MS by addressing limitations such as differential or incomplete enzymatic digestion of DNA and also, potential nuclear DNA contamination. Differential enzymatic digestion of DNA was addressed by incorporation of one-step digestion for DNA and using oligonucleotides harboring known amounts of dC and 5m-dC as calibrators. 5m-dC was detected in a linear and proportional manner in SH-SY5Y mtDNA. Around 3% methylation was found in mtDNA obtained from crude mitochondrial isolates, which was comparable to whole-cell DNA methylation levels. Interestingly, similar percentages of methylation were observed even in mtDNA obtained from gradient-purified mitochondria. Further, purity of mtDNA was characterized, indicating elimination of 99.95 % of nuclear DNA contamination with 115 fold-enrichment of mtDNA in gradient-purified mitochondria. Presence of methylation in SH-SY5Y mtDNA was further substantiated using methylation-specific PCR for a specific site in 12S rRNA. Thus, we established a direct method to quantify global methylation in mtDNA using HPLC-MS/MS and also, reported presence of methylated DNA in purified SH-SY5Y mitochondria. In Chapter III, we investigated if mtDNA methylation can be altered by experimental interventions and whether altered mtDNA methylation has any concomitant effects on mitochondrial transcription and mitochondrial function. Global mtDNA methylation was assessed by HPLC-ESI-MS/MS and site-specific methylation of 12S rRNA was studied by methylation-specific PCR assay. DNMT1 is known to localize in mitochondria but its role in mtDNA methylation is unknown. We reported that transient over-expression of DNMT1 in SH-SY5Y increased mtDNA methylation, decreased transcription of mtDNA-encoded genes and reduced total and basal-mitochondrial oxygen consumption rates. Likewise, treatment of SH-SY5Y by DNA methylation inhibitor (5-aza-dC) reduced levels of mtDNA methylation, increased transcription of mtDNA-encoded genes and altered mitochondrial oxygen consumption rate. Thus, we found that that mtDNA methylation is an alterable phenomenon, which is affected by DNMT1 levels and 5-aza-dC treatment and is associated with changes in mtDNA transcription and mitochondrial function. In chapter IV, we investigated the direct role of mtDNA in aging-associated mitochondrial dysfunction using cytoplasmic hybrid cell lines that were created by transferring mitochondria from platelets of healthy young and aged human subjects to mtDNA-depleted SH-SY5Y ρ0cells. Bioenergetic fluxes, their regulators and DNA methylation-associated parameters were characterized in these resulting cytoplasmic hybrids. Relative to the cybrids generated from young donors, mitochondrial oxygen consumption parameters and COX activity were reduced in the cybrids made from aged subjects. Interestingly, there was a compensatory increase of Complex I activity in the cybrids generated from aged subjects. However, no alterations were found in ATP levels, redox states and glycolysis fluxes. Although the mitochondrial mass was not different, mRNA levels of some of the mtDNA-encoded OXPHOS genes and nuclear-encoded genes that promote mitochondrial biogenesis were induced in the cybrids created from aged subjects. Although total DNA methylation remained unaltered, levels of DNMT1 protein, mtDNMT1 mRNA and methylation in the mtDNA were higher in the cybrids generated from aged subjects. Thus, we found that mtDNA have a direct role in age-associated mitochondrial dysfunction and mtDNA methylation is altered in age-dependent manner in cybrid model. In summary, this dissertation work established a method to detect global mtDNA methylation and quantified methylation levels in isolated mitochondria of characterized purity. Also, it established that mtDNA methylation is an alterable phenomenon, which is affected by DNMT1 levels and 5-aza-dC treatment and is associated with mitochondrial transcription and mitochondrial function. Further, it established a direct role of mtDNA in age-associated mitochondrial dysfunction and alteration in mtDNA methylation as one of the age-dependent changes in mtDNA. | |
