Since ancient times, there has been a historical tradition of emperors and saints refining elixirs and medicines in order to seek the way to immortality. With the development of economic level, average life expectancy continues to increase, and the problem of population aging and aging is becoming more and more prominent. Therefore, both scientists and the general public hope to find a good medicine to slow down aging and even extend life span.
Recently, Elsa Logarinho's team from University of Porto, Portugal, found a good medicine - transcription factor FOXM1, and confirmed through in vivo experiments in mice that it has the effect of slowing down aging and extending lifespan. In naturally aging mice, increasing FOXM1 expression can significantly extend the median lifespan by 28% and the maximum lifespan by 29%, and also slow down aging-related weight loss!
This research result was recently published in the top aging research journal Nature Aging[1], let’s have a good time together!
Screenshot of the paper's homepage
Previous research by this research group found that about 60% of the transcription changes related to aging in human skin fibroblasts depend on the transcription factor FOXM1 [2], and FOXM1 is a key proliferation-related The transcription factor regulates the expression of G2/M phase genes. In the field of aging research, there is a view that the loss of cell proliferation ability is a significant feature of cellular aging. These evidences suggest that FOXM1 is closely related to aging, so can it be selected as a therapeutic target?
In the previous research of this research group, they overexpressed the N-terminal truncated form of FOXM1 (FOXM1-dNdK) in fibroblasts derived from HGPS (Hutchinson–Gilford progeria syndrome, progeria syndrome ) patients, which enhanced It improves the proliferation ability of cells and delays the occurrence of premature cell aging. This part of the in vitro results was published on Nature Communications in 2018 [2].
Note: HGPS, progeria, is an early-onset and severe premature aging disease. It is caused by point mutations in the LMNA gene encoding A/C type lamin. Because HGPS is caused by known genetic mutations, researchers can easily create corresponding mouse models. And because the normal aging process is a very slow process (even in mice, it usually takes 2-3 years), and HGPS greatly accelerates the aging process, so cell samples from HGPS mice or humans are often used in aging research. Models for aging research.
Since it is effective in vitro, what is the effect in vivo? In order to answer this question, the researchers used naturally aging mice and HGPS mice [3] (LmnaG609G/G609G, referred to as LAKI) to conduct research. They overexpressed FOXM1 in these two mice to observe the effects of physiological aging and aging respectively. Whether anti-aging effects can be produced in pathological aging.
First, the researchers constructed a transgenic mouse , referred to as LAKI-Foxm1. This mouse has an HGPS phenotype and can overexpress truncated FOXM1 through doxycycline (dox) induction. The researchers used ear dermal fibroblasts from this mouse to test the efficiency of short-term overexpression of FOXM1-dNdK and the changes in cellular senescence phenotypes in vitro. (This part of the experiment is the same as their experiment in Nature Communications, except that the fibroblasts from HGPS patients were replaced by fibroblasts from LAKI-Foxm1 mice.) The
results showed that this system can overexpress FOXM15- 10 times, including cell proliferation (Ki67), DNA damage (γH2AX), classic markers of cell senescence (SA-β-gal), heterochromatin-related epigenetic markers (H3K9me3/H4K20me3), nuclear membrane integrity and nuclear Area and other indicators show that overexpression of FOXM1 in can alleviate the cellular senescence phenotype of .
Overexpression of FOXM1 in mouse fibroblasts can alleviate the cell senescence phenotype
The above are only the results detected immediately after short-term overexpression. Can the improved cell senescence benefit be maintained after the removal of FOXM1 overexpression? To this end, we tested a cyclic induction protocol in which fibroblasts from LAKI-Foxm1 mice were treated with dox for 4 days, followed by withdrawal of dox, and the cellular phenotypes were assessed 2 and 5 days after withdrawal.The results of
show that even if removes FOXM1 overexpression for 5 days, although there is a certain accumulation of senescence phenotype, it can still better maintain the improvement of cell senescence phenotype caused by overexpression of FOXM1 for the previous 4 days, which is different from untreated fibroblasts. Significantly . What is even more exciting is that administration of dox again to overexpress FOXM1 5 days after withdrawing dox can further prevent the reaccumulation of the aging phenotype.
The anti-aging benefits of overexpressing FOXM1 have sustained effects
The above proves that the mouse model system can operate effectively, and a cyclic overexpression scheme has been explored. Next, the authors wanted to see whether FOXM1 has a therapeutic effect on pathological aging mice. They set up three groups of mice, LAKI-Foxm1, LAKI, and WT. Based on the in vitro results, they used the strategy of "give dox for 3 days and stop dox for 4 days" to induce FOXM1 is overexpressed.
The researchers first wanted to see whether this potential treatment had a preventive effect on pathological aging, rather than a reversal effect (early intervention may be more likely to achieve positive results than late reversal), so the researchers chose 4-week-old mice. Start to continue the above dox administration strategy for 12 weeks until the mice are 16 weeks old.
results showed that the median and maximum lifespan of LAKI-Foxm1 mice on the dox diet were extended by approximately 25% compared with the LAKI control group (median, 18.1 weeks vs 14.4 weeks; maximum, 21.9 weeks vs 17.7 weeks), and the weight loss and growth retardation of in HGPS mice were also improved after treatment of . It is worth noting that postmortem analysis of dox-treated LAKI-Foxm1 mice did not show any obvious signs of hyperplastic tissue growth, indicating a certain degree of safety (at least no tumors visible to the naked eye).
Overexpression of FOXM1 in LAKI (HGPS) mice can extend the lifespan of mice.
In addition, researchers also used various phenotypic detection methods, such as 3D-micro-CT and small animal ultrasound, to detect progeria expression in HGPS mice. Types such as bone defects, lipodystrophy (shown as a reduction in fat accumulation), and whether cardiac dysfunction can be treated and recovered. The results showed that craniofacial abnormalities and kyphosis ( hunchback ) were significantly improved, subcutaneous fat accumulation was increased, and cardiac systolic function and rhythm were improved.
Overexpression of FOXM1 improves kyphosis symptoms in pathologically aged mice
Taken together, these results indicate that overexpression of FOXM1 in vivo significantly improves various features of progeria, including lethal cardiac dysfunction and prolongs progeria. The lifespan of mice with schizophrenia .
Next, the researchers further verified the therapeutic improvement effect of FOXM1 on pathological aging phenotypes at the tissue level (including skin, fat, aorta, bone) and molecular level. The aging changes of various tissues at the pathological level were improved.
Importantly, several aging markers upregulated in the skin, gonadal fat, and aorta of LAKI mice were restored to WT levels in LAKI-foxm1 mice .
The above are the results obtained in pathological aging, that is, HGPS model mice. However, pathological aging does not completely represent physiological aging. From its pathogenesis, it can be seen that pathological aging is mainly caused by a certain gene mutation. Natural aging is a global and extensive change, and there is an essential difference between the two.
Therefore, the researchers wanted to further explore whether FOXM1 overexpression can improve the aging phenotype of naturally aging mice? As an important cell cycle transcription factor,
FOXM1's overexpression will indeed promote cell proliferation and division. However, its carcinogenicity must be considered when it is overexpressed for a long time in "circulation". And studies have reported that there is an association between high FOXM1 levels in cancer and adverse clinical outcomes. Therefore, researchers have questioned the oncogenic risk of this approach. First, major risks must be eliminated before we can talk about the therapeutic effect~
By coincidence, recent evidence has revealed that the non-transcribed end (N-terminus) of FOXM1 has a tumor suppressor function [4]. And the researchers conducted in vitro experiments using 88-week-old Foxm1-dNdk old mouse fibroblasts (MAF) and 4-week-old MAF, confirming that induction of FOXM1-dNdK can indeed reverse the tumorigenic phenotype , but specifically through up-regulation of Endogenous Foxm1 plays a role, which is confirmed by the restoration of the tumorigenic phenotype after siRNA interference with endogenous Foxm1.
FOXM1 overexpression has been confirmed to have a tumor suppressive effect in naturally aging mouse fibroblasts
(F-actin: cortical actin, positively related to tumorigenesis and metastasis formation)
Interestingly, even using siFoxm1 to interfere with endogenous Expression of Foxm1, the aging phenotype of naturally aging mouse fibroblasts is still improved by the transcriptional function of transgenic FOXM1, indicating that its anti-aging effect is independent of the expression of endogenous Foxm1.
The senescent phenotype of naturally aging mouse fibroblasts is ameliorated by the transcriptional function of transgenic FOXM1
Therefore, this result demonstrates that FOXM1 transgenic expression can correct the tumorigenic phenotype by upregulating endogenous Foxm1 and its N-terminal function, providing It lays the foundation for assessing the longevity and aging phenotypes of animals under long-term induction.
Now that the current evidence shows that this treatment is safe in terms of tumorigenicity, the next step is to look at the anti-aging effects in naturally aging mice.
researchers used 8-week-old Foxm1-dNdK mice to overexpress FOXM1 according to the strategy of "give dox for 3 days and stop dox for 4 days" for a total of 80 weeks. By 88 weeks, the aging tissue phenotypes (skin, muscle, fat) were detected. , aorta) and changes at the molecular level (RNA-seq). Survival curves and body weight changes were continuously recorded throughout the process.
Overexpression of FOXM1 slows down the aging process and extends the lifespan of naturally aging mice
Surprisingly, the treatment group significantly extended the median lifespan (increased by 28%) and maximum lifespan (increased by 29%) compared with the control group, and also slowed down the aging process. aging-related weight loss. The results of tissue phenotype testing by
showed that compared with the control group, the dorsal skin tissue homeostasis of the treatment group was improved, and subcutaneous fat increased significantly; the size of adipocytes in gonadal fat deposition was slightly improved; age-related muscle fiber atrophy was slightly reversed; Aortic aging phenotypes, namely adventitial fibrosis, aortic wall thickening, and vascular smooth muscle cell loss, were significantly reversed.
It is worth noting that to demonstrate that the senescence phenotype is indeed attenuated and not just due to the broad-spectrum gain-of-function of FOXM1 overexpression, the researchers conducted a phase in which senescence-related phenotypes were absent (14- 16 weeks of age), evaluated the effects of transgenic overexpression of FOXM1 on tissues of young animals. The results showed that there were no significant changes in the above phenotypic indicators detected at the tissue level . It is suggested that this is not a simple gain-of-function, but indeed a therapeutic effect on the aging phenotype.
Next, the researchers conducted bulk RNA-seq on the aorta and gastrocnemius tissues of two groups of mice to observe changes at the molecular level and try to analyze the potential anti-aging molecular mechanisms. GSEA analysis showed that the pathways altered in both tissues were mainly inflammatory and metabolic pathways. The changes in the molecular profile are similar to those of other known anti-aging treatments.
Circulating expression of N-terminally truncated FOXM1 improves health and lifespan
Taken together, these data support that FOXM1 transgenic overexpression restores the loss of FOXM1 transcriptional and non-transcriptional functions during aging, mitigates aging-associated functional decline, and regulates pro-inflammation and tissue-specific molecular signatures of metabolic pathways to counteract the accumulation of physiological aging phenotypes, and are positively correlated with molecular signatures of some well-established anti-aging regimens .
It should be noted that the intervention measures used in the above-mentioned pathological aging and physiological aging mouse experiments are preventive long-term interventions, rather than real treatments in the middle and late stages of aging. The preventive treatment effect of
in cells and mice is so good. We look forward to researchers further promoting the treatment of middle and late stages of aging and related large animal experiments and elucidating the specific molecular mechanisms. We hope that it can become a therapeutic target for progeria and natural aging.
Reference:
. Ribeiro, R., et al., In vivo cyclic induction of the FOXM1 transcription factor delays natural and progeroid aging phenotypes and extends healthspan. Nature Aging, 2022. 2(5): p. 397-411. . Macedo, J.C., et al., FoxM1 repression during human aging leads to mitotic decline and aneuploidy-driven full senescence. Nat Commun, 2018. 9(1): p. 2834.. Osorio, F.G., et al., Splicing-directed therapy in a new mouse model of human accelerated aging. Sci Transl Med, 2011. 3(106): p. 106ra107.. Limzerwala, J.F., et al., FoxM1 insufficiency hyperactivates Ect2-RhoA-mDia1 signaling to drive cancer. Nat Cancer, 2020. 1(10): p. 1010-1024.Author of this article丨Li Haotong
Editor in charge丨Dai Siyu
