吳建國教授團(tuán)隊(duì)發(fā)現(xiàn)抑制衰老相關(guān)疾病和延長壽命的關(guān)鍵因子
衰老是生物隨著時(shí)間推移的必然過程，是復(fù)雜的和自然的生命現(xiàn)象。人體衰老過程中的生理變化主要體現(xiàn)在細(xì)胞功能喪失，機(jī)體代謝減慢，組織器官衰竭，最終導(dǎo)致死亡。衰老是生命的一個(gè)普遍特征，是很多疾病的危險(xiǎn)因素，是不可避免的，但延緩衰老卻是可能的。闡述導(dǎo)致衰老的機(jī)制和發(fā)展延緩衰老的技術(shù)，對于防治衰老相關(guān)疾病和延長健康壽命至關(guān)重要。
機(jī)體基因組DNA組裝形成染色質(zhì)并存儲在細(xì)胞核中，核小體是染色質(zhì)的基本結(jié)構(gòu)單元，主要由組蛋白和基因組DNA組成，在DNA復(fù)制與基因轉(zhuǎn)錄過程中發(fā)揮著高度動(dòng)態(tài)調(diào)節(jié)作用。組蛋白H2A泛素化修飾是一類重要的修飾方式，在調(diào)控染色質(zhì)空間構(gòu)象、核小體動(dòng)態(tài)結(jié)構(gòu)、基因轉(zhuǎn)錄、細(xì)胞周期及DNA損傷修復(fù)等過程中起著重要的作用。
MYSM1是一種去泛素化酶，其主要功能是去除組蛋白H2A的泛素化修飾。MYSM1在造血功能，免疫系統(tǒng)，皮膚、組織、視力、骨骼發(fā)育，漿細(xì)胞分化，干細(xì)胞免疫調(diào)節(jié)，樹突狀細(xì)胞抗原提呈，以及炎性因子分泌等功能中起著非常重要作用。因此，MYSM1是一種不可替代的表觀遺傳學(xué)修飾的關(guān)鍵因子。
2020年9月30日，國際權(quán)威性雜志《Advanced Science》《尖端科學(xué)》在線公開發(fā)表了吳建國團(tuán)隊(duì)的研究成果（https://doi.org/10.1002/advs.202001950），文章題目是“MYSM1 suppresses cellular senescence and the aging process to prolong lifespan”，第一作者為田明富博士生，通訊作者為吳建國教授，該研究得到了國家自然科學(xué)基金重點(diǎn)項(xiàng)目（項(xiàng)目編號81730061） 和面上項(xiàng)目（項(xiàng)目編號81471942）的資助。
該團(tuán)隊(duì)發(fā)現(xiàn)MYSM1能顯著抑制細(xì)胞衰老、阻礙衰老相關(guān)疾病發(fā)生發(fā)展、延緩機(jī)體衰老進(jìn)程、從而延長壽命。通過詳細(xì)和深入研究，證明了MYSM1具有能抑制人和小鼠的原代細(xì)胞的細(xì)胞衰老現(xiàn)象以及延緩小鼠組織器官病理發(fā)生發(fā)展的功能；揭示了MYSM1通過促進(jìn)機(jī)體DNA損傷修復(fù)，從而阻礙細(xì)胞衰老、延緩衰老進(jìn)程、降低病理發(fā)生發(fā)展的機(jī)制。通過動(dòng)物試驗(yàn)研究發(fā)現(xiàn)，MYSM1敲除的小鼠，衰老進(jìn)程明顯加快、衰老相關(guān)疾病顯著加重；與其相反，MYSM1治療的小鼠，衰老進(jìn)程明顯延緩、衰老相關(guān)疾病顯著減輕。更重要的是，與未處理的正常小鼠的平均壽命（20–26個(gè)月）相比，MYSM1敲除的小鼠的平均壽命（13–16個(gè)月）明顯縮短，MYSM1治療的小鼠的平均壽命（28–30個(gè)月）顯著延長。可見，MYSM1是一種阻礙細(xì)胞衰老進(jìn)程、防止衰老相關(guān)疾病發(fā)生、延緩生命周期的關(guān)鍵細(xì)胞因子。因此，MYSM1很有希望成為預(yù)防衰老相關(guān)疾病以及延長健康壽命的有效和創(chuàng)新藥物。
Figure 1. MYSM1 promotes HR mediated DNA repair.(A?and B) of WT MEFs or Mysm1-/-?MEFs were treated with DOX. Cell images showing the cell cytotoxicity (A). Cell viability was measured using the CELL COUNTING kit-8 (B). (C–E) WT MEFs and Mysm1-/-?MEFs (C, D) or WT MEFs, Mysm1-/-?MEFs, and Mysm1-/-?MEFs transfected with Flag-Mysm1 (E) were treated with DOX to induce DNA damage. g-H2AX and GAPDH protein levels were detected by Western blotting (WB) (C). g-H2AX protein was detected by anti-g-H2AX antibody and visualized under a confocal microscope (D, E). (F) MEFs were treated with DOX for 2 days to induce DNA damage. g-H2AX protein was detected by anti-g-H2AX antibody and visualized under confocal microscope. (G) 2-month old WT and Mysm1-/-?mice (n=3 per group) were subjected to IR. The levels of g-H2AX in the lung were determined by IHC. Scale bars,?50μm. (H) MEFs were grown continuously for different passages to induce DNA damage. g-H2AX levels were detected and visualized under a confocal microscope. (I) MEFs were grown continuously for 10 passages to induce DNA damage. g-H2AX levels were detected and visualized under a confocal microscope. (J–K) Liver, spleen, and lung were excised from 10-month-old WT and Mysm1-/-?mice (n=3 per group). The levels of g-H2AX were determined by IHC. Scale bars,?100× =100μm, 200× =50μm. (M–P) Human HEK293T cells (M, N) and mice MEFs (O, P) were co-transfected with a HR reporter system (M, O) or a NHEJ reporter system (N, P), along with pCMV-DsRed and HA-Ctrl or HA-Mysm1. The efficiencies of HR (M) and NHEJ (N) were determined. (Q) MEFs were co-transfected with the HR reporter along with pCMV-DsRed and Flag-Ctrl or Flag-Mysm1. The efficiencies of HR were determined. Data are presented as the means ± SD. Statistical analyses were performed using Prism software by Unpaired t-tests. *p<0.05, **p<0.01, ***p<0.001, n.s.= no signi?cance.
Figure 2. MYSM1 correlates with senescence. (A–C)?C57BL/6 mice MEFs were treated with ETO or DOX (A). Kidney (B) and lung (C) tissues were excised from 2- and 22-month old WT mice (n=4 per group). MYSM1 and GAPDH protein levels were assessed by WB. (D–F) WT or Mysm1-/-?mice MEFs were pre-treated with ETO or DOX for 48 h to induce senescence. Cell proliferation was determined (D). Colony formation was analyzed by crystal violet staining (E).?SA-b-Gal activity was?determined by X-gal staining (F). (G) 2-months old WT and Mysm1-/-?mice were exposed to ionizing radiation (n=3 per group). SA-b-Gal activities in the lung and liver were determined by IHC. (H–K)?WT or Mysm1-/-?MEFs were grown continuously for different passages (P) or on different days (D). Population doubling levels were determined (H). Cell proliferation was measured (I).Colony formation was analyzed (J). SA-b-Gal activities were determined by X-gal staining (K). (L–N) SA-b-Gal activities in the lung (L), liver (M), and spleen (N) of 2- and 6-month-old WT and Mysm1-/-?mice were determined by IHC analysis(n=3 per group). Scale bars =?50μm. Data presented as the mean ± SD. Statistical analyses were performed using an Unpaired t-test in Prism software. *p<0.05, **p<0.01, ***p<0.001. n.s. = no signi?cance.
Figure 3. MYSM1 suppresses DDR-associated SASP. (A–D)IL-6, IL-1?, and MMP3 mRNA levels in the kidney (A), lung (B), liver (C), and spleen (D) excised from 6-month-old WT and Mysm1-/-?mice (n=3 per group) determined by RT-PCR. (E–H) IL-6 protein in the kidney (E), lung (F), liver (G), and spleen (H) excised from 6-month-old WT and Mysm1-/-?mice (n=3 per group) analyzed by IHC. Scale bars = 50?μm. (I) IL-6 protein in the sera from 6-month-old WT and Mysm1-/-?mice (n=3 per group) measured by ELISA. (J and K) 2-month-old WT and Mysm1-/-?mice were exposed to IR (n=3 per group). IL-6 protein in the sera was measured by ELISA (J). IL-6 protein in the lung and liver was analyzed by IHC. Scale bars = 50?μm (K). Data are means ± SD. Statistical analyses were performed using an Unpaired t-test in Prism software. *p<0.05, **p<0.01, ***p<0.001. n.s. = no signi?cance.
Figure 4. MYSM1 represses senescence in aged mice. (A–D) MEFs were treated with ETO (A) or H2O2 (B), infected with lentivirus expressing Mysm1 (C), or grown for different passages (D). p16, MYSM1, and GAPDH proteins were detected by WB. (E–H) p16 and p21 mRNA in the kidney (E), lung (F), liver (G), and spleen (H) of 6-month old WT and Mysm1-/-?mice (n=3 per group) were determined by RT-PCR. (I and J) p16, MYSM1, and GAPDH proteins in the lung (I) and liver (J) of 2 and 6-month old WT and Mysm1-/-?mice (n=3 per group) were detected by WB. (K?and L) p16 protein (K) and p21 protein (L) in the lung, liver, and spleen excised from 6-month old WT and Mysm1-/-?mice (n=3 per group) were measured by IHC. Scale bars, 50?μm. (M?and N) 2-month-old WT and Mysm1-/-?mice (n=3 per group) were exposed to IR. p16, MYSM1, and GAPDH protein levels in the lung and liver were determined by WB (M). p16 and p21 protein levels in the lung, liver, and spleen were analyzed by IHC. Scale bars =?50?μm (N). Data are means ± SD. Statistical analyses were performed using an Unpaired t-test in Prism software. n = 3 for each sample group. n.s. = no signi?cance.
Figure 5. MYSM1 deficiency reduces the lifespan of mice. (A–D)Images of the body size (A), body form (B), chest (C), and head (D) of 10-month old WT and Mysm1-/-?mice. (E) Analysis of body lengths (cm) of 2-month old?WT and Mysm1-/-?mice (n=7 per group). (F) Analysis of body weights of WT and Mysm1-/-?mice (n=3 for each time-point). (G) Images of the eyes of 6-month old WT and Mysm1-/-?mice. (H) Frequencies of cataracts and eye diseases in WT mice (n=16) and Mysm1-/-?mice (n=17). (I) Images of spleens?of 6-month old WT and Mysm1-/-?mice (n=5 per group). (J) Images of livers of 2-month oldWT and Mysm1-/-?mice (n=5 per group). (K) Images of liver of 6-month old WT and Mysm1-/-?mice (n=5 per group). (L) Survival rates of WT mice (n=6) and Mysm1-/-?mice (n=7). (M–Q) Masson’s trichrome staining?images of kidney (M), heart (N), muscle (O), lung (P), and liver (Q) of 6-month-old WT and Mysm1-/-?mice (n=3 per group). Scale bars,?100×=100μm, 200×=50μm. (R) Periodic acid Schiff (PAS) histochemical staining?analyses of 6-month old WT and Mysm1-/-?mice kidney (n=3 per group). Scale bars,?100×=100μm, 200×=50μm. Data are means ± SD. Statistical analyses were done by using Prism software by Unpaired t-tests. ***p<0.001.
Figure 6. MYSM1 overexpression enhances mice lifespan. (A–D) 14-month-old WT mice were treated with AAV9-Ctrl (n=4) or AAV9-Mysm1 (n=5) for 2 months. Images of 16-month old mice after the treatments (A, top). p16 and p21 proteins in the kidney and heart were measured by WB (a, middle and bottom). IL-6 (left), p16 (middle), and p21 (right) in the liver (B), kidney (C), and heart (D) of the treated mice were measured by IHC. Scale bars,?100×=100μm, 200×=50μm. (E–Q) 16-month old WT mice were treated with AAV9-Ctrl or AAV9-Mysml for 6 months (n=5 per group). Images of mice before and after the treatments (E, top), images of mice furs after the treatments (E, middle), and analysis of fur changes intreated mice (E, bottom). Survival rates of treated mice treated with AAV9-Ctrl or AAV9-Mysml for 14 months (n=5 per group)?(F). Images of the eyes of the treated mice and analysis of frequencies of cataracts and eye diseases (right) (G). g-H2AX produced in the liver, kidney, and heart of the treated mice were detected by IHC (H–J). H&E stained images of the liver, kidney, and heart of the treated mice (K–M). Masson trichrome staining analyses of the liver, kidney, and heart of the treated mice (N–P). PAS analyses of the treated mice kidney (Q). Scale bars,?10× = 100μm, 20× = 50μm. Data are presented as the means ± SD. Statistical analyses were performed using Unpaired t-tests in Prism software.
Figure 7. MYSM1 is a key suppressor of aging and aging-related pathology. (A) The role of MYSM1 in promoting DNA repair. (B) The function of MYSM1 in repressing cellular senescence and the aging process. (C) MYSM1 deficiency facilitates aging, promotes aging-related pathology, and reduces the lifespan of mice. (D) MYSM1 over-expression attenuates the aging process, reduces aging-related pathology, and extends the lifespan of mice.
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