脂質代謝在健康與老化中扮演著很重要的腳色。改變脂質的代謝在許多物種中會影響壽命的長短。然而，對於脂質代謝的改變影響壽命的下游調節機制仍然還有許多地方需要釐清。我先前於「老化細胞」期刊中已經證實了大量表現參與磷脂酰肌醇循環代謝的二醯甘油酯脂解酶或者是降低二醯甘油酯酵素的基因表現會經由降低雷帕黴素標靶 (TOR)訊號傳遞而增加了果蠅與線蟲的壽命與氧化壓力反應。磷脂酰肌醇運輸蛋白質-1(PITP-1)是位於磷酸肌醇(PPIs)代謝循環路徑中做為磷脂酰肌醇(PI)的運輸者幫助回收磷酸肌醇這一些磷脂質。然而，對於參與同一代謝路徑的磷脂酰肌醇運輸蛋白質-1是否能調控壽命依然未知。在這一份論文中，我報導了降低磷脂酰肌醇運輸蛋白質-1延長了線蟲的壽命，而大量表現磷脂酰肌醇運輸蛋白質-1則縮短線蟲的壽命。除此之外，降低脂酰肌醇運輸蛋白質-1基因表現會舒緩老化引起的癱瘓以及降低的行動能力，這建議著降低磷脂酰肌醇運輸蛋白質-1不但能夠促進長壽還能延緩老化相關的失調。我也發現在進入繁殖後期年齡之前於神經組織中降低磷脂酰肌醇運輸蛋白質-1對於延長壽命是很重要的。使壽命延長的至少其中一個機制是由於減少的TOR訊號，這使得減少的磷酸化S6蛋白質在低磷脂酰肌醇運輸蛋白質-1表現的線蟲中被偵測到，而且以遺傳地或是藥物地方式抑制TOR活性並不能進一步地增加低磷脂酰肌醇運輸蛋白質-1表現的線蟲那延長的壽命。有趣的是，降低磷脂酰肌醇運輸蛋白質-1雖然會減少磷酸化的AKT，但是這並不會促進DAF-16進入細胞核中，也不會增強DAF-16在核內的活性。而且降低磷脂酰肌醇運輸蛋白質-1所延長的壽命是和DAF-16無關的。代替DAF-16，胰島素/類胰島素生長因子(IGF-1)訊號(IIS)的下游轉錄因子SKN-1對於降低磷脂酰肌醇運輸蛋白質-1所延長的壽命是必要的。雖然降低磷脂酰肌醇運輸蛋白質-1後所延長的壽命並不需要DAF-16並參與，但是DAF-16會扮演一個上游的因子調控磷脂酰肌醇運輸蛋白質-1的轉錄。而在磷脂酰肌醇運輸蛋白質-1突變株中偵測到降低的磷脂酸可能說明了降低的TOR訊號中的磷酸化S6蛋白質以及磷酸化AKT。更進一步地，次世代定序資料指出一些被SKN-1調控的膠原蛋白基因表現都在磷脂酰肌醇運輸蛋白質-1突變株中上升，這建議著增加的膠原蛋白也對降低磷脂酰肌醇運輸蛋白質-1的長壽有著貢獻。我的論文揭發了一個磷脂酰肌醇運輸蛋白質-1的新功能，這個功能連接了脂質代謝與IIS與TOR訊號參與的壽命調控。這將帶給磷脂酰肌醇運輸蛋白質-1一個對於壽明調控的新的見解。這個訊息將會幫忙促進一個新的治療老化相關疾病的設計方向。

Lipid metabolism plays an important role in health and aging. Altering lipid metabolism affects lifespan in many species. Yet, the underlying regulatory mechanisms how altering lipid metabolism affects lifespan remain to be more elucidated. My previous study in Aging Cell has demonstrated that overexpression of diacylglycerol lipase or knockdown of DAG kinase, which are involved in phosphoinositides (PPIs) turnover, increases lifespan and oxidative stress response by reducing target of rapamycin (TOR) signaling in both Drosophila and C. elegans. Phosphatidylinositol transfer protein-1 (PITP-1) functions as phosphatidylinositol (PI) transporter in PPIs turnover to help the phospholipids recycling. However, whether PITP-1 can regulate lifespan remains unknown. In this thesis, I report that reduced pitp-1 expression extends lifespan, whereas overexpression of pitp-1 reduces lifespan in C. elegans. In addition, pitp-1 knockdown attenuates age-induced paralysis and decreased motility, suggesting reduced pitp-1 not only can promote longevity but also retard aging-related disorder. I also reveal reducing pitp-1 in neuronal tissue and before post-reproductive age both are critical for the lifespan extension. The lifespan extension at least for one mechanism is due to decreased TOR signaling which decreased p-S6K level is detected in pitp-1-knockdown worms and genetically or pharmacologically blockage of TOR activity cannot further enhance the extended lifespan in pitp-1-knockdown worms. Interestingly, knockdown of pitp-1 decreases p-AKT levels but it does not promote DAF-16 nuclear localization neither enhance DAF-16 nuclear activity. The longevity of reduced pitp-1 is also independent of DAF-16. Instead of DAF-16, the Insulin/ insulin-like growth factor (IGF-1) signaling (IIS) downstream transcription factor, SKN-1, is required for the lifespan extension by reducing pitp-1. Although DAF-16 is not required in reduced-pitp-1-mediated longevity, DAF-16 acts as a upstream factor of pitp-1 to negatively regulate the transcription of pitp-1 for lifespan regulation. The lowered PA levels are detected in pitp-1 mutants which may account for the lowered p-S6K of TOR and p-AKT levels. Furthermore, RNAseq data indicate that the upregulation of some collagen genes regulated by SKN-1 is detected in two pitp-1 mutants, suggesting the increased collagen by pitp-1 reduction may also contribute to the longevity. My thesis findings uncover a novel function of pitp-1 connecting lipid metabolism to lifespan regulation which participates in IIS and TOR signaling. It sheds new insight into pitp-1 in lifespan regulation. The information may help promoting a new therapeutic design for aging-related diseases.

Content
Abstract....................................................................................................................... I
中文摘要.................................................................................................................... III
誌謝................................................................................................................................V
Content ....................................................................................................................VII
Introduction.............................................................................................................. 1
Materials and methods ....................................................................................... 7
C. elegans strains and maintenance ............................................................. 7
RNA interference (RNAi) .................................................................................... 7
Lifespan analysis................................................................................................... 8
RNA extraction and Reverse transcription ................................................................. 8
Real-time PCR (Q-PCR) ........................................................................................ 9
Paralysis assay ..................................................................................................... 10
Motility assay ........................................................................................................ 10
Fecundity assay .................................................................................................... 10
Development assay ............................................................................................. 11
Oxidative stress test ........................................................................................... 11
Western blots ......................................................................................................... 11
Transgenic reporter assay ............................................................................... 12
Lipid analysis.......................................................................................................... 13
RNA sequencing .................................................................................................... 13
Results......................................................................................................................... 15
Reduced pitp-1 expression extends lifespan in C. eleganspitp-1 negatively
regulates lifespan ................................................................................................. 15
Neuronal pitp-1 knockdown is critical for the lifespan extension ........................... 16
Reduced pitp-1 attenuates age-induced motility degeneration in C. elegans ........ 17
Reduced pitp-1 does not affect fecundity and body size but delays development 17
VIII
pitp-1 knockdown-mediated lifespan extension occurs before post-reproductive
age ............................................................................................................................ 18
Reduced pitp-1 does not increase oxidative stress tolerance ................................. 19
Reduced pitp-1 or dgk-5 extends lifespan via common pathways .......................... 19
Decreased pitp-1 reduces TOR signaling.................................................................. 20
Reduced TOR signaling is one of the mechanisms which contributes to pitp-1-
mediated lifespan extension .................................................................................... 20
Knockdown of pitp-1 reduces p-AKT level in day-7 adult worms ............................ 21
pitp-1-mediated lifespan extension is independent of DAF-16 ............................... 22
Reduced Insulin/IGF-1 signaling and pitp-1 probably share a common pathway to
promote longevity.................................................................................................... 23
IIS signaling regulates pitp-1 transcription by DAF-16 ............................................. 23
pitp-1 mutants show a trend of decreased PA level ................................................ 24
Collagen synthesis genes are upregulated in pitp-1 mutants by RNA seq .............. 25
Discussion .................................................................................................................... 26
Figures and Tables ....................................................................................................... 31
References ................................................................................................................... 61

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