"We have identified two markers of small intestine aging: low-frequency point mutations in mitochondrial DNA and the mitochondrial LONP1 protein, and have developed multiple strategies to reverse the aging of the small intestine. These could serve as targets or candidate drugs, ultimately generating significant economic value," said Liu Xingguo, a researcher at the Guangzhou Institutes of Biomedicine and Health.

In a recent study, he and his team used a premature aging mouse model, stem cell labeling technology, and organoid technology to demonstrate that the accumulation of mitochondrial DNA mutations leads to the consumption of nicotinamide adenine dinucleotide (NAD+) during the aging process of the intestine.

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By activating the mitochondrial unfolded protein response (UPRmt), they depleted intestinal stem cells, thereby inducing intestinal aging. They found that supplementing with the NAD+ precursor nicotinamide mononucleotide (NMN, β-Nicotinamide Mononucleotide) can delay the aging of the small intestine.

Overall, this study provides a mechanism linking NAD+ deficiency to aging and supports its practicality as a biomarker for monitoring the aging of different tissues.

At the same time, this work for the first time addresses the fundamental scientific question of the causal relationship between the accumulation of aging mitochondrial DNA mutations and the aging of mammalian intestines.Based on this, the research team has developed several anti-aging schemes for the small intestine targeting this pathway, and then discovered a multi-level spatiotemporal mechanism from mitochondrial DNA mutations, to mitochondrial reverse signaling, to stem cell exhaustion, to intercellular signaling, and then to organ aging, providing a brand new idea, target, and strategy for delaying intestinal aging.

Recently, the related paper was published in Nature Communications[1] with the title "NAD+ dependent UPRmt activation underlies intestinal aging caused by mitochondrial DNA mutations."

Yang Liang is the first author, and Liu Xingguo serves as the corresponding author.

Exploring the mechanism of intestinal aging to better cope with the aging population

It is reported that the intestine is the main digestive organ and the largest immune detoxification organ in the human body. Once the intestine has a dysfunction, it will affect the physical and mental health through metabolism, nerves, endocrine, and immune aspects.The basic unit of the small intestine consists of two parts: villi and crypt structures, which have a relatively fast rate of self-renewal. Intestinal stem cells can differentiate into different epithelial cells, thereby forming an orderly intestinal structure to maintain the intestinal homeostasis and basic functions such as absorption, secretion, barrier, and antimicrobial functions.

Imbalance of intestinal homeostasis is considered to be closely related to aging, but the markers and response signal pathways of intestinal aging are still unclear.

The theory of oxidative damage is one of the main theories of aging mechanisms. In detail: mitochondria, as the central control of cellular metabolism, oxygen consumption, reactive oxygen species (ROS) production, and apoptosis, play a key role in cellular aging.

The accumulation of mutations in mitochondrial DNA, the genetic material of mitochondria, is an important cause of aging and the occurrence of age-related diseases.

Regarding mitochondria, Liu Xingguo also wrote a poem titled "Mitochondria Heartbreak": "The red shadow fades in the late spring light, while the white hair grows in the slanting sun. Asking what the three tears resemble, a thread and a grain break the heart."During the aging process of the body, organs such as the small intestine are affected first and accumulate mitochondrial DNA mutations. However, whether there is any correlation between the accumulation of mitochondrial DNA mutations and the aging of the small intestine is currently unclear.

Currently, countries around the world are facing a serious problem of an aging population. Data shows that by 2050, the elderly population in China will account for 1/3.

By 2025, the market for treating age-related diseases and extending healthy life expectancy is expected to exceed 600 billion US dollars.

Despite the huge market demand and scale for anti-aging drugs, there are currently no anti-aging drugs on the market internationally. In summary, the study of small intestine aging is a field full of challenges and opportunities.

By deeply understanding the mechanisms and influencing factors of small intestine aging, new ideas and methods can be provided for the prevention and treatment of age-related diseases.Eleven years ago, a mouse model was introduced from the United States.

As previously mentioned, the oxidative damage theory is one of the main theories of aging mechanisms. Mitochondria, as the center of reactive oxygen species production, play a key role in cellular aging.

In order to study whether and how mitochondria participate in the regulation of aging in various organs and tissues, Liu Xingguo's team introduced the world's only recognized mitochondrial premature aging mouse model - POLGAD257A mutant mice - from the Jackson Laboratory in the United States at the end of 2013.

Later, they first established a POLG mouse population through breeding and continuously observed the homozygous POLG mutant mice.It was found that after the mice reached 9 months of age, they began to exhibit obvious signs of premature aging, including whitening and loss of hair, as well as hunching over.

In order to study whether mitochondrial dysfunction is the cause of mitochondrial aging, they started to focus on the phenotypes of various organs in POLG premature aging mice before they reached 9 months of age.

Through this, the research team found that the mice's small intestine showed signs of premature aging, mainly characterized by the elongation of the villi in the small intestine, a reduction in the number of crypts, and the fertility of female mice was affected.

In fact, during the process of individual aging, the human small intestine and female reproductive system are the first to be "affected". Therefore, they set such a topic: to study the impact of mitochondrial dysfunction on the aging of the small intestine and female reproduction.

Due to the particularly obvious fertility phenotype, coupled with the fact that they usually test the fertility of female mice at 2 months of age, the detection cycle for the fertility of female mice is relatively short.In 2020, the team completed a paper on fertility-related topics and published it in the journal Aging Cell. Since significant aging phenotypes in the small intestine only appear at 8 months of age, this topic requires a longer period of time.

Therefore, after the fertility topic was concluded, they shifted their focus to the topic of small intestine aging. In their research, they used small intestine organoid technology and endogenous tracing technology of small intestine stem cells to study how mitochondrial dysfunction regulates small intestine aging.

Through this, the research group discovered the role of NAD+ in small intestine aging and explained how NAD+ regulates the Wnt signaling in the small intestine through the mitochondrial retrograde signaling UPRmt to trigger small intestine aging.

In the paper submission, the reviewers provided many valuable suggestions, including whether the small intestine undergoes similar aging phenotypes during the natural aging process, and whether mitochondrial DNA mutations accumulate.

Following the reviewers' suggestions, they supplemented some experiments and found that the aged small intestine, like the POLG mice, mainly accumulates low-frequency mitochondrial DNA point mutations, indicating that the POLG mice are a good model for studying the physiological aging of the small intestine.In fact, the many suggestions and questions raised by the reviewers, although it seemed quite challenging at the time, were all very valuable," said Liu Xingguo.

It is also reported that a large number of studies have shown that after the small intestine ages, the size of the crypts, villus length, Paneth cell count, and goblet cell count all increase. However, the number of stem cells in the small intestine and their regenerative potential decrease.

Similarly, they also found this phenomenon in prematurely aging mice. However, during the aging process, the number of stem cells in the small intestine decreases, while the length of the villi increases. Why is this the case? Further answers are needed on this issue.

In addition, unlike nuclear gene mutations, mitochondrial gene mutations are often heterogeneous. Under normal circumstances, most of the mitochondrial DNA molecules within human cells are identical, that is, mitochondrial DNA is homogeneous.

Mitochondrial DNA is in a highly reactive oxygen environment, lacks protective and repair mechanisms, and is prone to mutations that accumulate over time.In the human body, wild-type mitochondrial DNA (mtDNA) and mutated mitochondrial DNA coexist, with the proportion of mutated mtDNA varying by tissue and age. The proportion of mtDNA mutations determines the clinical symptoms of mitochondrial diseases.

Clinically, mtDNA mutations exhibit a threshold effect, where only when the proportion of mutated mtDNA reaches a high level does it affect respiratory function and lead to the onset of disease.

This research team found that the small intestine of the POLG premature aging mice accumulated a large number of low-frequency point mutations in mtDNA. However, whether and how these low-frequency mtDNA point mutations play a role in the aging of the small intestine requires further exploration.

In addition, Liu Xingguo and others revealed the pattern of changes in intestinal mtDNA mutations with age in naturally aging animals and identified the type as low-frequency point mutations in mtDNA.

To address the causal role of mtDNA mutations in intestinal aging, the research team used mice with mtDNA mutations, which are an experimental model widely used to study the aging caused by mtDNA mutations.At the same time, they used POLG mutant mice and wild-type mice to generate four types of mice with different mitochondrial DNA mutation rates.

Among them, the POLG mutant homozygous mice had the highest mitochondrial DNA mutation rate and, like naturally aging mice, mainly accumulated low-frequency point mutations.

Combining intestinal organoid technology and endogenous labeling technology of intestinal stem cells, they found that under the same age, only the POLG mutant homozygous mice exhibited intestinal aging phenotypes earlier, showing a low degree of intestinal organoid differentiation and a sharp decrease in the number of intestinal stem cells.

This indicates that a high burden of mitochondrial DNA mutations can promote the occurrence of intestinal aging, thus providing a new biological marker of low-frequency point mutations in mitochondrial DNA for intestinal aging.

However, how mitochondrial DNA mutations regulate the expression of nuclear genes through the UPRmt signal still requires further research. And these will be the directions of their future efforts.