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Can we exploit the Klotho gene for Ageing therapy?

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Antonio Carusillo
Antonio Carusillo Dec 06, 2020
Can we develop gene therapy approaches based on the Klotho gene?

In our journey to collect and investigate about possible strategies to slow down, ameliorate or even reverse the ageing process we already discussed the possible use of genetic engineering to either discover new possible “druggable” targets for ageing therapy or also as a tool itself, likeactivating the human telomerase reverse transcriptase (hTERT) gene using CRISPR/Cas9, using senolytics based drugs to target specific signalling pathways or even equipping Chimeric Antigene Receptor (CAR) T cells to specifically targeting the senescent cells.

In this session, I would like to talk about other possible targets we didn’t ever mention so far, the Klotho gene .

What is the Klotho gene and why it may be important?

Klotho gene was discovered in 1997 when a mouse characterized by a phenotype resembling premature ageing similar to what happens in humans was described.

The Klotho gene is indeed poetically named after the homonymous Greek goddess of the fate in charge of spinning the thread of life.

Further studies of such gene revealed that it belongs to a larger family of single-pass transmembrane proteins which include different isoforms of the gene: alpha, beta and gamma . The gamma isoform remains uncharacterized to date. The beta is particularly expressed in liver, kidney gut and spleen and its function is connected to the activity of the fibroblast growth factor (FGF) . The alpha instead is composed of a large extracellular domain, a transmembrane domain and a small intracellular domain. The extracellular domain features two repeat sequences KL1 and KL2 which can be cleaved by ADAM-10 and ADAM-17, two metalloproteases. This results in a form of soluble Klotho which can be detected in blood, urine and cerebrospinal fluid . This isoform was later on described to be important for the binding of the Fibroblast Growth Factor-23 (FGF23) to its receptor FGFR1 .

Mice deficient in Klotho develop a complex progeric phenotype including poor growth, atrophy of multiple organs, sarcopenia, cardiac hypertrophy and fibrosis, cognitive impairment, and shortened lifespan . Klotho regulates many pathways involved in ageing processes, such as the regulation of phosphate homeostasis, insulin signaling and Wnt signaling. Additionally, Klotho also affects intracellular signaling pathways including p53/p21, cAMP, protein kinase C (PKC) and TGF-β .

Klotho expression levels and its circulating level decline during ageing. In humans, Klotho deficiency features medial calcification, intima hyperplasia, endothelial dysfunction, arterial stiffening, hypertension, impaired angiogenesis, and vasculogenesis (i.e., characteristics of early vascular ageing) .

Possible Therapies targeting Klotho or its axis

For these reasons Klotho seems to be an attractive therapeutic target when it comes to anti-ageing treatments. For example:
  1. AAV virus-associated anti-ageing cocktail delivering 3 longevity associated genes Fibroblast Growth Factor 21 FGF21, alpha Klotho and the soluble from of transforming growth factor-beta receptor 2 (sTGFbR2) ave been delivered using AAV viral vector and proved to ameliorate 3 age-related diseases: obesity, type II diabetes and heart failure and renal failure. These amazing results in the mouse model suggest the feasibility of the approach
  2. Systemic gamma-aminobutyric acid (GABA) can stimulate the levels of circulating Klotho, thus providing a possible approach to increase Klotho levels
  • Which other strategies can we think about developing to increase the level of Klotho?
  • Can we target the Klotho pathway instead of Klotho itself? Which would be more effective and safe?
  • Are there known side effects correlated to Klotho supraphysiological levels?
What is intriguing is that Klotho as been also been defined as a possible tumor suppressor gene in cancer . We may start a follow-up session about Klotho involvement in other human conditions besides ageing and whether or not there are some “cross-talks” between its role as an anti-ageing agent and as tumor-suppressor.

[1]Buchanan Sarah, Combet Emilie, Stenvinkel Peter, Shiels Paul G. Klotho, Aging, and the Failing Kidney, Frontiers in Endocrinology vol. 11, 2020 560 DOI=10.3389/fendo.2020.00560

[2]Kuro-o, M., Matsumura, Y., Aizawa, H. et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 390, 45–51 (1997). https://doi.org/10.1038/36285

[3]Ryozo Nagai, Makoto Kuro-o, Yo-ichi Nabeshima, Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein1The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with the accession numbers AB010088, AB010089, AB010090 and AB010091.1, FEBS Letters, Volume 424, Issues 1–2, 1998, Pages 6-10, ISSN 0014-5793, https://doi.org/10.1016/S0014-5793(98)00127-6.

[4]Yuechi Xu, Zhongjie Sun, Molecular Basis of Klotho: From Gene to Function in Aging, Endocrine Reviews, Volume 36, Issue 2, 1 April 2015, Pages 174–193, https://doi.org/10.1210/er.2013-1079

[5]van Loon EP, Pulskens WP, van der Hagen EA, Lavrijsen M, Vervloet MG, van Goor H, Bindels RJ, Hoenderop JG. Shedding of klotho by ADAMs in the kidney. Am J Physiol Renal Physiol. 2015 Aug 15;309(4):F359-68. doi: 10.1152/ajprenal.00240.2014. Epub 2015 Jul 8. PMID: 26155844.

[6]Lim K, Groen A, Molostvov G, et al. α-Klotho Expression in Human Tissues. J Clin Endocrinol Metab. 2015;100(10):E1308-E1318. doi:10.1210/jc.2015-1800

[7]Chen G, Liu Y, Goetz R, Fu L, Jayaraman S, Hu MC, Moe OW, Liang G, Li X, Mohammadi M. α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling. Nature. 2018 Jan 25;553(7689):461-466. doi: 10.1038/nature25451. Epub 2018 Jan 17. PMID: 29342138; PMCID: PMC6007875.

[8]o-ichi Nabeshima, Klotho deficient mouse: an in vivo model for human aging, Drug Discovery Today: Disease Models, Volume 1, Issue 3, 2004, Pages 223-227, ISSN 1740-6757, https://doi.org/10.1016/j.ddmod.2004.11.023.

[9]Abolghasemi M, Yousefi T, Maniati M, Qujeq D. The interplay of Klotho with signaling pathway and microRNAs in cancers. J Cell Biochem. 2019 Sep;120(9):14306-14317. doi: 10.1002/jcb.29022. Epub 2019 May 24. PMID: 31127658.

[10]Shimamura Y, Hamada K, Inoue K, Ogata K, Ishihara M, Kagawa T, Inoue M, Fujimoto S, Ikebe M, Yuasa K, Yamanaka S, Sugiura T, Terada Y. Serum levels of soluble secreted α-Klotho are decreased in the early stages of chronic kidney disease, making it a probable novel biomarker for early diagnosis. Clin Exp Nephrol. 2012 Oct;16(5):722-9. doi: 10.1007/s10157-012-0621-7. Epub 2012 Mar 29. PMID: 22457086.

[11]Mazzotta C, Manetti M, Rosa I, et al. Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells. Arthritis Res Ther. 2017;19(1):27. Published 2017 Feb 10. doi:10.1186/s13075-017-1233-0

[12]Hu MC, Shi M, Zhang J, et al. Klotho deficiency causes vascular calcification in chronic kidney disease. J Am Soc Nephrol. 2011;22(1):124-136. doi:10.1681/ASN.2009121311

[13]A single combination gene therapy treats multiple age-related diseases Noah Davidsohn, Matthew Pezone, Andyna Vernet, Amanda Graveline, Daniel Oliver, Shimyn Slomovic, Sukanya Punthambaker, Xiaoming Sun, Ronglih Liao, Joseph V. Bonventre, George M. Church

[14]Prud'homme GJ, Glinka Y, Kurt M, Liu W, Wang Q. The anti-aging protein Klotho is induced by GABA therapy and exerts protective and stimulatory effects on pancreatic beta cells. Biochem Biophys Res Commun. 2017 Dec 2;493(4):1542-1547. doi: 10.1016/j.bbrc.2017.10.029. Epub 2017 Oct 6. PMID: 28993191.

[15]Zhou, X., Fang, X., Jiang, Y. et al. Klotho, an anti-aging gene, acts as a tumor suppressor and inhibitor of IGF-1R signaling in diffuse large B cell lymphoma. J Hematol Oncol 10, 37 (2017). https://doi.org/10.1186/s13045-017-0391-5

[16]Tang, X., Wang, Y., Fan, Z. et al. Klotho: a tumor suppressor and modulator of the Wnt/β-catenin pathway in human hepatocellular carcinoma. Lab Invest 96, 197–205 (2016). https://doi.org/10.1038/labinvest.2015.86

Creative contributions

Use tools to find compounds that increase the expression of the Klotho gene.

Jamila Dec 11, 2020
So, I ran a search on a tool called Drug Gene Budger (https://maayanlab.cloud/DGB/). Using this tool, you can find potential drugs that change your target gene's expression levels. Apparently, the following small compounds/drugs have been predicted to upregulate the Klotho gene (KL).
  • Avicin D
  • Trichostatin A
  • COL-3
  • Doxorubicin
  • Cytarabine
A summary of each drug that I've mentioned:

Avicin D is a plant triterpenoid that has profound effects on cellular metabolism. Avicin D can induce AMPK, suppress mTOR, and activate autophagy. The mechanisms that avicin D impacts are known to promote longevity.

Trichostatin A is a natural compound with antifungal properties, and it can suppress class I and class II histone deacetylases - this drug can inhibit the cell cycle.

COL-3 (incyclinide) is a modified version of tetracycline that was being trialed as a treatment for rosacea, allergies, etc. COL-3 works by inhibiting matrix metalloproteinases.

Doxorubicin is a chemotherapy medicine used for various types of cancer. This drug works by preventing the DNA replication process as it directly interacts with DNA.

Cytarabine is another chemotherapy medicine, but this one is mainly used for the various types of leukemia. This drug inhibits the DNA replication process, too, as it can damage DNA and thus stop the cell cycle.

So, perhaps researchers could use these compounds to see if they can actually increase Klotho levels in the body, and then they can take it further from there.

[1]Haridas, Valsala, et al. "The anticancer plant triterpenoid, avicin D, regulates glucocorticoid receptor signaling: implications for cellular metabolism." PLoS One 6.11 (2011): e28037.

[2]Stancu, Andreea L. "AMPK activation can delay aging." Discoveries 3.4 (2015).

[3]Johnson, Simon C., Peter S. Rabinovitch, and Matt Kaeberlein. "mTOR is a key modulator of ageing and age-related disease." Nature 493.7432 (2013): 338-345.

[4]Barbosa, María Carolina, Rubén Adrián Grosso, and Claudio Marcelo Fader. "Hallmarks of aging: an autophagic perspective." Frontiers in endocrinology 9 (2019): 790.

[5]Kozeretska, Irina A., et al. "Epigenetic regulation of longevity in insects." Advances in Insect Physiology. Vol. 53. Academic Press, 2017. 87-114.

[6]Fields, Gregg B. "The rebirth of matrix metalloproteinase inhibitors: moving beyond the dogma." Cells 8.9 (2019): 984.

[7]Thorn, Caroline F., et al. "Doxorubicin pathways: pharmacodynamics and adverse effects." Pharmacogenetics and genomics 21.7 (2011): 440.

[8]Li, Zheng, et al. "Exploring the antitumor mechanism of high-dose cytarabine through the metabolic perturbations of ribonucleotide and deoxyribonucleotide in human promyelocytic leukemia HL-60 cells." Molecules 22.3 (2017): 499.

Considerations for therapeutic use of klotho

Shubhankar Kulkarni
Shubhankar Kulkarni Dec 15, 2020
  1. Transgenic mice that overexpress Kl gene (EFmKL46 and EFmKL48) have biochemical features of insulin resistance, suggesting that klotho doses as therapy might be different for different individuals. The hepatic expression of phosphoenol pyruvate carboxykinase, an enzyme that increases gluconeogenesis, was raised in the kl/kl mice.
  2. Human studies warn that Klotho may have a narrow time and the concentration window of therapeutic efficacy before it causes disease on its own.

[1]Kurosu H, Yamamoto M, Clark JD, Pastor J V, Nandi A, Gurnani P, et al. Suppression of Aging in Mice by the Hormone Klotho. Science (80- ) [Internet]. 2005 Sep 16;309(5742):1829–33. Available from: https://www.sciencemag.org/lookup/doi/10.1126/science.1112766

[2]Mori K, Yahata K, Mukoyama M, Suganami T, Makino H, Nagae T, et al. Disruption of klotho Gene Causes an Abnormal Energy Homeostasis in Mice. Biochem Biophys Res Commun [Internet]. 2000 Nov;278(3):665–70. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0006291X00938646

[3]Mengel-From J, Soerensen M, Nygaard M, McGue M, Christensen K, Christiansen L. Genetic Variants in KLOTHO Associate With Cognitive Function in the Oldest Old Group. Journals Gerontol Ser A Biol Sci Med Sci [Internet]. 2016 Sep;71(9):1151–9. Available from: https://academic.oup.com/biomedgerontology/article-lookup/doi/10.1093/gerona/glv163

[4]Brownstein CA, Adler F, Nelson-Williams C, Iijima J, Li P, Imura A, et al. A translocation causing increased -Klotho level results in hypophosphatemic rickets and hyperparathyroidism. Proc Natl Acad Sci [Internet]. 2008 Mar 4;105(9):3455–60. Available from: http://www.pnas.org/cgi/doi/10.1073/pnas.0712361105

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