*2.5. Aging*

Mitochondrial dysfunction has been shown to lead to oxidative stress due to the generation of excessive reactive oxygen species, triggering senescence and age-related diseases. Since cell lines with MITOL deficiency induces cellular senescence [27], we also focused on the association between reduced MITOL function and senescence and age-related diseases in vivo. In our in vivo analysis, mice lacking MITOL in a skin-specific manner showed significant signs of aging, including gray hair and hair loss. These results and MITOL downregulation during aging sugges<sup>t</sup> that MITOL may block the induction of physiological aging. We hoped that drugs that upregulate MITOL expression could inhibit or delay aging, and in a collaborative study with pharmaceutical companies, we screened drugs that upregulate MITOL mRNA from a Chinese medicine library using cultured human keratinocytes. We found that extracts of *Coptis japonica* and *Phellodendron amurense* in a Chinese medicine library upregulated MITOL mRNA approximately threefold (Patent No: P2019-52145A). When berberine, common compound of *Coptis japonica* and *Phellodendron amurense*, was mixed with drinking water, the expression of mRNA and protein of MITOL increased in various organs and tissues, including skin. To investigate the anti-aging activity of berberine, mice treated with it were irradiated with ultraviolet light to induce the formation of wrinkles in the skin, and a significant inhibitory e ffect on this was observed compared with that in control mice. In addition, with collaboration research, we found that the mice that took berberine for more than 1 year were significantly less likely to show signs of aging, such as skin hair loss and thickening of the epidermis, than control mice. Because the anti-aging e ffect of berberine on mice deficient of MITOL specifically in the skin was not confirmed, anti-aging e ffects are induced especially via the upregulation of MITOL expression levels. These results strongly indicate that MITOL has the potential to be a target for anti-aging drugs. In the future, it is anticipated that new MITOL activators other than berberine will be identified and applied to various aging-related diseases such as neurodegenerative diseases and heart failure. Interestingly, the expression of MITOL has been observed to gradually decrease with aging in many organs such as the brain and heart of mice. Moreover, Alzbase, a database for gene dysregulation in Alzheimer's disease (AD), suggested that the expression level of the mitol/march5 gene was significantly reduced in the brain of AD patients. If the worsening of AD is linked to the reduced MITOL expression, the compounds we have identified may exert a good therapeutic e ffect on AD. Therefore, we speculate that the decreased degradation of denatured proteins caused by decreased expression of MITOL may be one of the factors exacerbating the development of neurodegenerative diseases with aging (Figure 2).

**Figure 2.** Relativity between MITOL and aging. Since MITOL gradually decrease following aging, upregulation of MITOL by a Chinese herb might be able to e ffect anti-aging.

### **3. Regulation of Mitochondrial Dynamics by MITOL**
