跳耀子調控的嚴謹程度與減緩老化和老化相關疾病進程的關聯

Abstract

老化是一個複雜及由多因子造成的生物性過程。在這過程中，表觀遺傳的改變是一個重要因素。隨著老化，染色質的鬆散及異染色質的下降，可能會使原本受抑制的基因及跳耀子表現。跳耀子的移位作用對基因體所造成的威脅是廣為熟知。然而在跳耀子被轉譯成蛋白前，部分跳耀子 RNA 序列也被證明具有一些生物性功能，包括生理性或是病理性的重要功能。例如：特定亞型的跳耀子轉錄體對於著床前胚胎及胚胎幹細胞的建立或維持是必要的。跳耀子的表現會受到表觀遺傳修飾調控，包括 DNA 甲基化、組蛋白修飾及以 piRNA 媒介的轉錄後及轉錄抑制。此外，跳耀子對於環境刺激相當敏感。在這篇研究，我們利用了兩種老鼠模型，包括了飲食節制老鼠模型及阿茲海默症模式鼠，研究環境刺激與遺傳變異對於老化相關跳耀子序列活化之影響。飲食限制已知可以延緩老化並延長生命週期，因此相較於任飼，被視為一種健康老化模式。阿茲海默症模式在此則是作為一個老化相關性疾病的例子。我們假設了跳耀子在這兩種老鼠模型中與其分別的對照組有表現上的差異。我們試圖想探討是否健康老化及不健康老化狀態能影響到跳耀子的表現，再進而探討可能造成的原因及未來的應用。我們利用 RT-qPCR 來篩選飲食節制及任飼老鼠不同腦區及肝臟的跳耀子表現量，並發現肝臟的 L1s 跳耀子表現量在飲食節制的老鼠中相較任飼的對照組是顯著降低的。令人振奮的是，飲食節制所造成跳耀子抑制現象也在血液的樣本中被觀察到。這顯示跳耀子表現具有作為診斷工具的潛力。第二部分，我們探討跳耀子在老化相關神經退化性的阿茲海默症模式鼠中是否有失調的狀況。我們發現跳耀子在阿茲海默症的模式鼠的小腦中相較對照組有異常表現量上升的現象。其中，抑制型的組蛋白修飾在阿茲海默症模式鼠的小腦蛋白中是下降的，這意味著這可能是造成跳耀子異常表現的原因之一，並開啟小腦中表觀基因體變異對阿茲海默症病程影響之研究方向。第三部分，我們試圖找到可以調控跳耀子的治療策略。我們從運用一個表觀遺傳調控的共同調控者 DNA 甲基轉移酶類三號蛋白 (Dnmt3L) 來著手，因為 Dnmt3L 已知在生殖細胞中對於跳耀子的抑制扮演重要的角色。作為一個原理驗證的實驗，我們測試倘若外加 Dnmt3L 到老化的人類纖維母細胞中是否能延緩老化及再次抑制老化所造成跳耀子表現的現象。我們發現當我們外加 Dnmt3L 可以恢復核蛋白的表現量，暗示染色質的結構變得更緊密。總結以上，跳耀子在任飼及阿茲海默症的老鼠的不同器官的異常表現現象可能是由於染色質結構的改變。再者，L1跳耀子家族的異常表現，也具有潛力作為診斷由代謝症候群所造成不健康老化的生物指標。最後我們也有發現外加Dnmt3L到老化的人類纖維母細胞中能使染色質的構形變得更緊，也意味其具有應用在治療上的潛力。

Aging is a complicated and multifactorial biological process. Epigenetic alteration is an important factor in the processing. During aging, chromatin relaxation and reduced heterochromatin may de-repress some originally well-repressed genes, as well as transposable elements (TEs). Transposition is a well-known threat to genome. Even before translating into protein, TEs derived RNA sequences may already have some physiologically or pathologically functions. For example, programmed activation of specific subtype of TE transcripts may be necessary for establishing or maintaining the potency of preimplantation embryos and embryonic stem cells. TEs can be modulated via epigenetic regulation, including DNA methylation, histone modifications and piRNA-mediated post-transcriptional and transcriptional repressions. TEs can also response to environmental stimuli in a relatively sensitive manner. In this study, we utilized two mouse models, including Dietary restriction (DR) model and Alzheimer’s disease (AD) models. CR/DR can retard aging and extend life span and therefore regarded as healthy aging in contrast to ad libitum (AL). AD model is used as an example for aging associated diseases. We hypothesized that there are differences of TEs in theses two models. We intended to investigate whether healthy aging and diet-induced aging can influence TEs and further seek for possible etiology of TE derepression and clinical applications. We screened TE expression levels in the different brain regions and liver of the DR model by RT-qPCR, and found that the level of L1 retro-transposons expression was significantly decreased in the liver of DR mice compared to AL controls. Surprisingly, DR associated repression of TE derepression could also be observed in blood, indicating its potential as diagnostic tools. On the other hand, we investigated whether the TE levels may be deregulated in the aging related neurodegenerative disease AD mice. We found that TEs derepressed in the cerebellum of the AD model compared to WT controls. Repressive histone marks decreased in the AD model, which may be one of the reasons for TEs derepression. At last, we investigated possible therapeutic strategies for TE modulation. We tackled the possibility of applying an epigenetic modulating co-factor, DNA methyltransferase 3-like (Dnmt3L), known to be critical for TE silencing in germ cells, used for an anti-aging targets. As a proof of principle experiment, we tested whether ectopic Dnmt3L expression in the aging human fibroblast can defer aging and re-repress TEs. We found that ectopic Dnmt3L could restore nuclear proteins and tighten up the chromatin structure in aging cells. Taken together, TE derepression is observed in the AL controls and AD models at different organs, which may both result from chromatin structure changes. Additionally, L1 retro-transposons derepression has potential to be a biomarker for diet-induced aging. Finally, we found that ectopic Dnmt3L expression in the human fibroblast can lead to tighten a chromatin conformation, the extended study from this direction may pave a way to new therapeutic strategies of aging-dependent chromatin relaxation.