酪蛋白激酶1α藉由數種訊息路徑如雷帕黴素標靶路徑及細胞自噬等參與許多生理機制，包含發育、生理時鐘等，然而，酪蛋白激酶1α是否可調控壽命以及其分子機制皆仍尚未知。在此，我們報導酪蛋白激酶1α突變果蠅株可延長壽命，並有數種長壽果蠅常見的表徵型，例如較短小的果蠅一齡幼蟲、生殖力的下降、抗飢餓能力的提升等。為了找出酪蛋白激酶1α調控壽命的標的組織，我們發現在全身、神經、以及脂肪體中降低酪蛋白激酶1α的表現量，可以延長壽命。另一方面，全身性地過度表現酪蛋白激酶1α會使壽命縮短。此外，酪蛋白激酶1α長壽突變株有較低的雷帕黴素標靶及訊息，伴隨著降低的磷酸化S6激酶與磷酸化蛋白激酶B的表現量，以及被提高的細胞自噬能力，並伴隨著提升的微管相關蛋白輕鏈3II的表現量。另外，藉由處以雷帕黴素的藥理抑制雷帕黴素訊息，發現突變果蠅株並沒有進一步的延長壽命，意味著酪蛋白激酶1α可能藉由雷帕黴素標靶訊息調控壽命。統整以上所述，我們的研究提供了酪蛋白激酶1α如何調控壽命的新見解。

Casein kinase I alpha (CKIα) is involved in multiple biological processes including development and circadian rhythm via different pathways such as TOR and autophagy. However, whether CKIα can regulate lifespan and its molecular mechanism are unknown. Here, we reported that CKIα mutant flies display extended lifespan and longevity related phenotypes like smaller larval length, reduced fecundity, and starvation resistance. To identify the target tissue that CKIα regulates lifespan, we found that the RNAi knockdown of CKIα in whole body, neuron, and fat body can extend lifespan. On the other hand, ubiquitous overexpression of CKIα shortens lifespan. Furthermore, CKIα longevity mutants exhibit lowered TOR signaling with reduced levels of p-S6K and p-AKT, and elevated autophagy activity with increased LC3-II levels. Moreover, pharmacological inhibiting TOR by treating rapamycin indicates that CKIα longevity mutants do not show further lifespan enhancement, suggesting that CKIα regulates lifespan possibly via TOR signaling. Together, our finding provides new insights into how CKIα regulates lifespan in Drosophila.

Abstract .......................................................................................................................... i
中文摘要........................................................................................................................ ii
致謝............................................................................................................................... iii
Table of Content ........................................................................................................... 1
Introduction .................................................................................................................. 3
Materials and methods ................................................................................................ 6
Fly culture and fly strains ................................................................................... 6
Lifespan assay....................................................................................................... 6
Lifespan assay under geneswitch system ........................................................... 6
Lifespan assay under rapamycin treatment ...................................................... 7
Starvation assay ................................................................................................... 7
Oxidative stress assay .......................................................................................... 7
Pupation assay ...................................................................................................... 8
Egg laying assay ................................................................................................... 8
Larval collection and larval length measuring assay........................................ 8
Climbing assay ..................................................................................................... 8
Immunoblotting.................................................................................................... 9
RNA extraction and Reverse transcription ..................................................... 10
Results ......................................................................................................................... 11
The CKIα Drosophila mutants exhibited extended lifespan........................... 11
CKIα mutant lines displayed several longevity related phenotypes .............. 12
CKIα mutant lines showed enhanced anti-starvation ability but only one allele exhibited anti-oxidative stress ability ..................................................... 12
CKIα mutant lines did not show improved climbing decline upon aging ..... 13
Reduced TOR signaling was detected in the CKIα longevity mutants ......... 13
Elevated autophagy activity was detected in CKIα longevity mutants ......... 13
Rapamycin treatment does not further enhance the extended lifespan in CKIα heterozygous mutants .............................................................................. 14
Ubiquitous knockdown of CKIα can prolong lifespan in Drosophila ............ 14
Ubiquitous knockdown of CKIα in adulthood shortens lifespan in Drosophila ........................................................................................................... 15
Knockdown of CKIα in neuron by elav-Gal4 and Tdc-Gal4 can prolong lifespan in Drosophila ........................................................................................ 15
Knockdown of CKIα in fat body can prolong lifespan in Drosophila ........... 16
Knockdown of CKIα in germline cannot prolong lifespan in Drosophila ..... 16
Reduced TOR and elevated autophagy activity were detected in the CKIα
2
RNAi lines by da-Gal4, elav-Gal4, and Tdc-Gal4 ............................................ 17
Anti-starvation ability was not detected in CKIα RNAi lines ........................ 17
CKIα knockdown induces autophagy activity which might involve in lifespan regulation .............................................................................................. 17
Ubiquitous overexpression of CKIα shortens lifespan in Drosophila ............ 18
Discussion.................................................................................................................... 19
Figures ......................................................................................................................... 22
Tables ........................................................................................................................... 56
Supplementary ........................................................................................................... 68
References ................................................................................................................... 69

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