臨床上抗愛滋病毒的藥物治療是利用合併治療的策略(俗稱雞尾酒療法)，最重要的基礎藥物是核甘酸反轉錄酶抑制劑，此基礎藥物會與蛋白質酵素抑制劑或是非核甘酸酵素抑制劑共同合併治療。但是長時間服用核甘酸反轉錄酶抑制劑具有許多短期和長期使用相關的副作用，其中一種副作用是神經病變，也是促使本研究計畫展開的動機。核甘酸反轉錄酶抑制劑的毒理機轉被認為是抑制粒線體DNA聚合酶γ，影響粒線體DNA的生成，造成粒線體DNA含量減少，這個過程被認為會導致粒線體受損，又稱為DNA聚合酶γ假說。但近期越來越多在其他細胞中的研究質疑核甘酸反轉錄酶抑制劑導致的粒線體受損並不需要粒線體DNA含量下降。但在神經細胞中還未被驗證。因此我們利用小鼠初代皮質神經細胞建立一套系統去檢測毒物對粒線體不同功能的影響，包含粒線體生成、粒線體DNA含量、氧化磷酸化、型態、移動和粒線體未折疊蛋白反應。首先我們使用已知道會抑制粒線體DNA聚合酶γ的溴化乙錠來建立系統。結果顯示溴化乙錠會損害粒線體的生成、移動、氧化磷酸化和粒線體未折疊反應並增加粒線體的分裂，但並不影響粒線體DNA含量。接著，我們使用6種不同的核甘酸反轉錄酶抑制劑，這些抑制劑對於粒線體DNA聚合酶γ的抑制和造成神經毒性的能力皆有不同。結果發現只有具有抑制粒線體DNA聚合酶γ 能力強的ddC 和ddI此兩種藥物，會損壞粒線體DNA生成，造成粒線體DNA含量下降。更進一步，只有ddC會些微的影響氧化磷酸化。另外，觀察到AZT和d4T雖然沒有抑制粒線體DNA的合成或減少粒線體DNA含量，但卻輕微影響粒線體的型態和減少粒線體的移動。綜合以上結果，我們知道不同的核甘酸反轉錄酶抑制劑會造成不同層面的粒線體受損並在初代皮質神經細胞所造成的粒線體毒性是不需要粒線體DNA含量下降。

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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