Named after the Greek goddess who spins the thread of life, the klotho gene not only extends lifespan in mice and humans but also bestows greater intelligence, according to a paper published open-access in Cell Reports on May 8. Scientists led by Lennart Mucke, Gladstone Institute of Neurological Disease, San Francisco, report that one copy—but, intriguingly, not two—of a genetic variant of klotho boosts the concentration of the protein in blood and improves cognitive function in men and women of all ages. People carrying the variant outperformed non-carriers on a range of cognitive tests. Mice that overexpressed the normal protein also did better on tasks of learning and memory, as well as measures of neuroplasticity. Together, the findings offer new insight into age-related cognitive decline and hint at new targets for therapeutics. 

“Age is the highest risk factor for late-onset Alzheimer’s disease, so anything that can correct for this age-related cognitive vulnerability would be great,” said Hui Zheng, Baylor College of Medicine, Houston, who was not involved in the study. While she said the findings did not come as a complete surprise, given that anything that increases the lifespan can be expected to be beneficial overall, she was intrigued that klotho, best known for suppressing aging, may improve cognition in younger people. 

Klotho is a transmembrane protein produced in the kidney and also in the choroid plexus and pituitary glands in the brain. Proteases including α-secretase cleave the protein to release a fragment into the extracellular space, and from there it makes its way into blood. A variant called KL-VS appears to regulate longevity. It contains six single-nucleotide polymorphisms, two of which cause amino acid substitutions. One copy of KL-VS lengthens lifespan in people. This may be related to increased signaling through the extracellular fragment of the protein, because cells harboring the variant secrete higher amounts of that domain (see Arking et al., 2002). Strangely, people with two copies of the KL-VS allele die sooner than non-carriers, and have a higher risk for cardiovascular disease (see Arking et al., 2005). As people age, the concentration of klotho fragments in their circulation declines in parallel with cognition, and people with Alzheimer’s have even lower levels (see Semba et al., 2014). For those reasons, some scientists wonder if the protein influences cognition as well as lifespan. 

Studies in animal models support the notion. For example, klotho-deficient adult animals have trouble in learning and memory tasks (see Nagai et al., 2003). 

To see if klotho affects human cognition, first author Dena Dubal and colleagues tested language, executive function, visuospatial ability, learning, and memory in 718 cognitively healthy people aged 52 to 85 from three study cohorts. Of these volunteers, 188 carried one copy of KL-VS and about 20 had two. Because they were few, these homozygotes were excluded from analysis. The heterozygotes had 10 to 20 percent more klotho in their blood than non-carriers and performed better on all cognitive tests, the authors report. To their surprise, all age groups benefitted. “This suggested that increased levels of klotho were not simply counteracting brain aging,” Mucke told Alzforum. However, the cognitive benefit was weaker in the oldest people. The authors attributed this to a general age-related decline in circulating klotho that occurs regardless of genotype.

To figure out how boosting klotho might enhance cognition, the researchers turned to mice that systemically overexpress the wild-type, murine version (see Kuro-O et al., 1997). These animals, called KL mice, harbor 2.5 times more klotho in the brain than controls and live up to 30 percent longer (see Kurosu et al., 2005). In keeping with the results in humans, the authors found that at 10 to 12 months of age, KL mice remembered the location of a hidden platform in the Morris water maze test better than non-transgenic mice. Young KL mice also outperformed age-matched controls on this test. They were more apt to explore, and froze more often in a fear-conditioning context, suggesting better working and contextual memory, respectively. As in humans, the results suggested that klotho benefits cognition in old and young animals.

Analysis of the brains of these mice clued the researchers in to how klotho operates. Knowing that NMDA and AMPA glutamate receptors are crucial for learning and memory, the researchers looked to see if they changed. Synapses in the frontal cortex and hippocampus in KL mice contained nearly twice as many NMDA receptors with the GluN2B subunit. Immunohistochemistry suggested that learning activated downstream gene targets of NMDARs more strongly in KL mice than controls, further suggesting that these receptors had a hand in klotho-induced changes. Hippocampal slices showed enhanced long-term potentiation. When Dubal blocked GluN2B-containing NMDA receptors in KL mice with ifenprodil, a selective inhibitor of these channels, she found that they performed no better than controls.

The results suggest that an abundance of klotho improves cognitive function by jacking up the GluN2B-containing NMDARs (see image below). Mucke noted that since klotho declines with age, raising it might maintain cognition. How this might help someone with Alzheimer's is unclear. “Whether increased klotho would be strong enough to counteract the cast of detrimental players in AD, including Aβ, ApoE4, or tau, we do not know,” said Mucke. The researchers will explore whether klotho-enhancing compounds developed by co-author Carmela Abraham, Boston University School of Medicine (see King et al., 2012), prevent disease phenotypes in animal models. “Our preliminary findings are quite encouraging,” Mucke said. 

Boosting klotho expression in humans or mice leads to better cognition by elevating GluN2B NMDARs in synapses. [Image courtesy of Arturo Moreno.]

“This work provides a clear indication that klotho has something to do with cognitive improvement,” said Christoph Kaether, Leibniz Institute for Age Research in Jena, Germany. Both Kaether and Zheng wondered why this cognitive benefit occurred only in heterozygotes, not in people who have two copies of the gene. The authors are puzzled as well. They proposed that decades of high klotho levels could be detrimental. That might have therapeutic implications, Zheng said. Alternatively, the variant itself could produce less klotho, leaving homozygotes with insufficient levels, the authors suggested. Heterozyogtes might benefit, the theory goes, because their one wild-type allele picks up the slack and overcompensates, resulting in more klotho than in people with two normal copies of the gene. Mucke and colleagues are now collecting blood samples from more people with the rare homozygote genotype to determine their serum levels of klotho. 

Apart from raising or lowering the level of klotho in the blood, KL-VS could modify cognition by changing the function of the protein itself, Mucke said. Work from Abraham's group suggests that KL-VS forms dimers less readily than wild-type does (see Tucker et al., 2013). Mucke pointed out that this finding offers no explanation for the cognitive improvements seen in mice that overexpress the normal klotho. However, he is interested to see whether a change in dimerization affects cognition in people with a single copy of the variant. 

Klotho may exert its cognitive effects by acting outside of the brain, too. Hyoung-Chun Kim, Kangwon National University, South Korea, pointed out that klotho expression declines in people with chronic kidney disease, and these people become cognitively impaired (see Madero et al., 2008). He proposed that klotho could act through the kidney to augment cognition in humans. 

Another recent paper, published April 28 in PNAS Early Edition, suggests that klotho exerts its effects through altered proteolytic activity. Scientists led by Douglas Vaughan, Northwestern University Feinberg School of Medicine, Chicago, found that mice lacking klotho lived longer if they lacked plasminogen activator inhibitor-1 (PAI-1). This serine protease inhibitor suppresses proteolytic activity outside the cell and is upregulated in klotho knockout mice. The findings suggest that a lack of klotho raises PAI-1 levels outside the cell and reduces proteolytic activity.—Gwyneth Dickey Zakaib

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  1. The klotho gene is predominantly expressed in the kidney and, to a lesser extent, in the brain and reproductive and endocrine organs, and it has been identified as a novel anti-aging factor (Kuro-o et al., 1997; Yang et al., 2010). We have, for the first time, reported memory impairment induced by klotho gene knockout in mice (Nagai et al., 2003). Surprisingly, Dubal et al. demonstrate here a very novel finding (using transgenic mice with systemic overexpression of klotho): that klotho enhances cognition even early in life. Similar to our recent report using klotho mutant mice (Park et al., 2013), they clearly suggest a causal role for NMDARs and their GluN2B subtype in klotho-mediated cognitive enhancement.

    Even though the present article shows unexpected findings, we think that klotho mutant mice should still be considered a disease model for premature aging syndromes. The process of premature aging in this model, however, is clearly different from the natural aging process, which is influenced by many factors. Indeed, klotho mutant mice do not reveal some phenotypes usually seen in aged humans, such as brain atrophy with deposition of amyloid or senile plaques (Kuro-o et al., 1997; Anamizu et al., 2005; Nixon et al., 2005). Therefore, it might also be of great importance to better understand the fundamental molecular mechanisms underlying the pleiotropic actions of klotho for possible therapeutics against premature aging- and natural aging-associated complications.

    Importantly, the klotho gene has a remarkable renoprotective effect in response to ICR-derived glomerulonephritis in a mouse model, via attenuating mitochondrial oxidative stress (Haruna et al., 2007). In addition, klotho gene expression is decreased in patients suffering chronic kidney disease (Kuro-o, 2009). Clinical data suggests that individuals of all ages with chronic kidney disease are at higher risk for developing dementia and cognitive impairment (Madero et al., 2008). Therefore, it is possible that renopathological changes (in the absence of klotho) are responsible for cognitive impairments in humans.

    Nonetheless, we think that multifactorial approaches the authors used to analyze the cognitive function of the klotho gene help us understand the role of this gene in the aging-dependent and aging-independent complications.

    View all comments by Hyoung-Chun Kim

References

Paper Citations

  1. . Association of human aging with a functional variant of klotho. Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):856-61. Epub 2002 Jan 15 PubMed.
  2. . Association between a functional variant of the KLOTHO gene and high-density lipoprotein cholesterol, blood pressure, stroke, and longevity. Circ Res. 2005 Mar 4;96(4):412-8. Epub 2005 Jan 27 PubMed.
  3. . Klotho in the cerebrospinal fluid of adults with and without Alzheimer's disease. Neurosci Lett. 2014 Jan 13;558:37-40. Epub 2013 Nov 7 PubMed.
  4. . Cognition impairment in the genetic model of aging klotho gene mutant mice: a role of oxidative stress. FASEB J. 2003 Jan;17(1):50-2. PubMed.
  5. . Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997 Nov 6;390(6655):45-51. PubMed.
  6. . Suppression of aging in mice by the hormone Klotho. Science. 2005 Sep 16;309(5742):1829-33. PubMed.
  7. . Identification of novel small molecules that elevate Klotho expression. Biochem J. 2012 Jan 1;441(1):453-61. PubMed.
  8. . Biochemical and functional characterization of the klotho-VS polymorphism implicated in aging and disease risk. J Biol Chem. 2013 Dec 20;288(51):36302-11. Epub 2013 Nov 11 PubMed.
  9. . Cognitive function in chronic kidney disease. Semin Dial. 2008 Jan-Feb;21(1):29-37. PubMed.

Further Reading

Papers

  1. . Identification of novel small molecules that elevate Klotho expression. Biochem J. 2012 Jan 1;441(1):453-61. PubMed.
  2. . Association of Klotho polymorphisms with healthy aging: a systematic review and meta-analysis. Rejuvenation Res. 2014 Apr;17(2):212-6. Epub 2014 Apr 11 PubMed.

Primary Papers

  1. . PAI-1-regulated extracellular proteolysis governs senescence and survival in Klotho mice. Proc Natl Acad Sci U S A. 2014 May 13;111(19):7090-5. Epub 2014 Apr 28 PubMed.
  2. . Life extension factor klotho enhances cognition. Cell Rep. 2014 May 22;7(4):1065-76. Epub 2014 May 10 PubMed.