| Summary: | 碩士 === 國立成功大學 === 生理學研究所 === 104 === The cornerstone of current HIV treatment is nucleoside reverse transcriptase inhibitors (NRTIs). Patients who receive long term treatment with NRTIs often develop severe side effects, including neuropathy. The putative toxic mechanism is the inhibition of mitochondrial DNA polymerase γ (pol γ), which impairs mitochondrial DNA (mtDNA) synthesis and leads to mtDNA depletion. This process is expected to cause mitochondrial dysfunction and is referred to as the ‘pol γ hypothesis.’ However recent studies have called into question whether NRTI damage to mitochondria may be independent of mtDNA depletion. This has not been tested in neurons. Therefore, we established a panel of endpoints in murine primary cortical neurons to systematically investigate mitochondrial regulation during toxicity including biogenesis, mtDNA synthesis, mtDNA content, oxidative phosphorylation, morphology, motility, and the mitochondrial unfolded protein response (mtUPR). First, we applied this analysis to ethidium bromide (EtBr), which is a prototypical inhibitor of pol γ. Our results showed that EtBr impaired mitochondrial biogenesis, mtUPR, motility and oxidative phosphorylation, but increased mitochondrial fission and did not affect mtDNA content. After validating the methods, we investigated the effects of six different NRTIs which have variable pol γ inhibitory activity and neurotoxicity. Among the NRTIs that we tested, only strong pol γ inhibitors, ddC and ddI, attenuated mtDNA synthesis and led to decreased mtDNA content. Further, only ddC decreased mtDNA transcription and produced a mild reduction in oxidative phosphorylation. Minor effects on motility and morphology were observed after AZT and d4T treatment without mtDNA depletion or inhibition of mtDNA synthesis. Overall, the results suggest that NRTIs induce distinct profiles of mitochondrial dysfunction in neurons which likely reflect multiple mechanisms of toxicity. Furthermore, the NRTI-induced mitochondrial toxicity in primary cortical neurons is largely independent of mtDNA depletion.
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