In Molecular Therapy, a team of researchers has described how increasing the expression of a form of Klotho, a protein that has been frequently found to have rejuvenative effects, leads to longer lifespans in male mice.
Klotho has various forms
In their introduction, the researchers distinguish between the various forms of Klotho. The full mRNA sequence that generates Klotho creates two homologous effective sections (KL1 and KL2) and a membrane that is meant for transportation between cells: this full version is m-KL [1]. Once enzymes take away this membrane, however, it becomes p-KL, with each section being p-KL1 and p-KL2.
However, this full version interferes with the function of FGF23, a protein that manages the metabolism of minerals [2]. Artificially upregulating this protein, therefore, is not safe [3].
Another form of Klotho, secreted Klotho (s-KL), does not have this problem. s-KL has been found to have multiple anti-aging effects according to a review of 65 studies [4]. Of course, without the transmembrane protein, it is not fit for intercellular transportation. Therefore, the researchers used an adeno-associated virus (AAV) to deliver a gene therapy that upregulates Klotho in the mice in this experiment.
Improvements in lifespan
The researchers used a total of 96 mice of the Black 6 strain: a group that received the AAV at 6 months, a group that received the AAV at 12 months, and a control group that received an ineffective AAV at 6 months. Both male and female mice were included in this experiment.
The treatment had far different effects in males and females. In female mice, the treatment had similar effects at 6 months and 12 months, but the increase in s-KL was accompanied by serious health problems during the course of the experiment, including skin ulcers and bleeding from the anus. In male mice, the AAV upregulated s-KL much more than in female mice, and it was much more effective at 12 months than at 6 months. Despite having far more s-KL, the males did not experience any of the health problems that the females did; instead, they received significant improvements to their lifespan.
Improvements to tissues and performance
Interestingly, at the age of 24 months, females showed improvements on the rotarod balance test that males did not. Both sexes given the s-KL AAV at 12 months were able to hold onto a horizontal bar longer than their control groups. In a three-trial grip strength test, the AAV-treated males performed far better, while the females performed better during only the first trial. The males also had significant reductions in fibrosis.
Regeneration capability was tested by transplanting muscles from old mice into younger mice. The muscle fibers in the AAV-treated animals became much larger than those from the control group. Muscles derived from the animals treated at 6 months old grew a wider variety of fiber sizes than those derived from animals treated at 12 months. Proliferation markers, and markers relating to a muscle-related fate of stem cells, were increased in the muscles derived from the mice that had received the s-KL AAV.
The researchers also tested bone tissue, seeing significant improvements to bone structure in females treated at 6 months and non-significant improvements in males treated at 12 months. Curiously, while FGF23 was upregulated along with many other bone-related factors in male mice, it was downregulated in female mice. This may be beneficial for females, as age-related increases in FGF23 have been linked to osteoporosis [5].
While no behavior testing was done in this study, the researchers did examine the mice’s brains. They found that, in the treated animals, there were more functional neurons and a thicker cellular layer, and markers of cellular proliferation were increased in the hippocampus. An examination of differently expressed genes revealed that the treated animals had fewer age-related changes than the control group.
The researchers note that this is the first time an AAV for s-KL has demonstrated lifespan increases in wild-type mice; previous experiments used transgenic mice. They believe that further experiments should test mice with different genetic backgrounds, because the side effects they saw in this experiment may or may not be limited to the AAV’s effects on Black 6 mice. Further work may elucidate exactly why klotho treatment has such different effects on males and females.
Literature
[1] Chen, C. D., Tung, T. Y., Liang, J., Zeldich, E., Tucker Zhou, T. B., Turk, B. E., & Abraham, C. R. (2014). Identification of cleavage sites leading to the shed form of the anti-aging protein klotho. Biochemistry, 53(34), 5579-5587.
[2] Kurosu, H., Ogawa, Y., Miyoshi, M., Yamamoto, M., Nandi, A., Rosenblatt, K. P., … & Kuro-o, M. (2006). Regulation of fibroblast growth factor-23 signaling by klotho. Journal of Biological Chemistry, 281(10), 6120-6123.
[3] Roig-Soriano, J., Sánchez-de-Diego, C., Esandi-Jauregui, J., Verdés, S., Abraham, C. R., Bosch, A., … & Chillón, M. (2023). Differential toxicity profile of secreted and processed α-Klotho expression over mineral metabolism and bone microstructure. Scientific reports, 13(1), 4211.
[4] Abraham, C. R., & Li, A. (2022). Aging-suppressor Klotho: Prospects in diagnostics and therapeutics. Ageing Research Reviews, 82, 101766.
[5] Sirikul, W., Siri-Angkul, N., Chattipakorn, N., & Chattipakorn, S. C. (2022). Fibroblast growth factor 23 and osteoporosis: evidence from bench to bedside. International Journal of Molecular Sciences, 23(5), 2500.
