Clotho, one of the Three Fates of Greek mythology, carried the weighty responsibility of spinning the thread of human life. It seems fitting then that a protein linked to reducing and extending life spans should take its name from this mythic figure. Researchers discovered the klotho protein in 1997, when they found that diminished levels seemed to make the animals age faster. Conversely, mice genetically engineered to maintain elevated klotho levels live 30 percent longer than normal mice. Recent research hints that the protein itself could form the basis of anti-aging therapies.
Many studies have since established klotho as a longevity promoter, including in humans, with numerous protective effects on organs throughout the body. Its levels decline with age but some people with a version of the klotho gene known as KL-VS produce more of the protein and typically live longer. Now a new study, led by physician and neuroscientist Dena Dubal of the University of California, San Francisco, suggests klotho has potential as a therapy against brain aging and the maladies that come with it. The team found beneficial effects in young and aging mice on memory and learning and on some of the motor deficits found in Parkinson’s disease.
These results point to a possible therapeutic to address diseases of brain aging, such as Alzheimer’s, which are on the rise because of demographic trends. “Our life span has nearly doubled since the early 1900s,” Dubal says. “But there’s no effective medical therapy for this insidious problem of losing brain function with age and disease.”
The elusive search for such a therapy prompted Dubal to wonder whether klotho might slow cognitive decline. In a 2014 study she and colleagues tested more than 700 people between ages 52 and 85 on a range of cognitive measures. Some of the study subjects carried the beneficial KL-VS gene variant. The researchers found those with the variant showed the same rate of decline, but they started with an advantage: they had higher levels of functioning on various cognitive tests regardless of age.
Similar effects turned up in genetically engineered mice that produced high levels of klotho. Analysis of the brain tissue of the “smart” mice revealed differences in a type of chemical receptor (NMDA), which is critical for controlling processes that underlie learning and memory. Collectively known as synaptic plasticity, they alter the strength of connections between neurons (the synapses) in response to experience. The mice had double the number of a component, or subunit, of NMDA receptors known as GluN2B in their hippocampi and frontal cortices, two key memory regions. Blocking GluN2B with a drug abolished cognitive benefits. These findings suggested klotho works by increasing the number of GluN2B subunits in NMDA receptors.
That study involved people and mice that had high levels of klotho throughout their lives, which raised another question: Could klotho be administered intermittently like a drug and have similar beneficial effects? In the current study the team used a synthetic klotho fragment, matching the one secreted from cells that circulates in blood as a hormone regulating many biological functions. They tested young mice on a set of tasks in mazes that taxed both spatial memory and learning as well as working memory (the ability to hold information in short-term memory in the face of distraction). Both measures were enhanced in treated mice, and administration of the molecule improved working memory just four hours after injection.
When combined with training tasks to enhance cognitive functioning, the effects continued for at least two weeks, long after the hormone would be cleared from the body, suggesting that a lasting neural reorganization had occurred. “Within hours, in a young adult brain, cognitive function is improved—that is powerful, and has implications for thinking about cognitive enhancement,” Dubal says. “But our laser focus is to harness this toward a brain with dysfunction, with the hope it could help people suffering from disease and improve quality of life.”
With that aim, the researchers tested old mice (18 months—about equivalent to a 65-year-old human) and saw improvements on a task measuring both working and spatial memory after a single injection. “It really improved the aging mouse’s ability to navigate and explore new territory,” Dubal says.
The team then turned to mice engineered to mimic Parkinson’s, which produce high levels of the human alpha-synuclein protein. Treated mice performed significantly better on tests of motor and cognitive function, and analysis of brain tissue showed treatment had not changed levels of alpha-synuclein. Dubal surmises that rather than targeting the toxic proteins directly as other approaches have, klotho enhances the brain’s resilience to toxic insults. The team also measured lasting increases in neuronal connection strength following stimulation in the hippocampus. “Our best speculation, given we see such enhanced synaptic function, is that somehow, by strengthening the synapse, that’s countering the effects of aging and diseases related to alpha-synuclein,” Dubal says.
To explore how klotho exerts its effects, the team analyzed levels of GluN2B in the hippocampus of treated mice, expecting to find increased numbers of the receptor subunits, as they had in the previous study. “We looked carefully at levels of GluN2B, and they just weren’t different; it didn’t fit our hypothesis,” Dubal says. It gradually dawned on one of the researchers, Julio Leon, that the protein was not increasing the numbers of GluN2B but rather activating the receptor components that were already in place. The team then blocked GluN2B with a drug, and again found this abolished klotho's beneficial effects. Finally, the team analyzed changes in around 4,000 proteins after treatment, which pointed to glutamate signaling—involving the neurotransmitter used by NMDA receptors—as the main process affected by klotho.
The anti-aging hormone has these effects without entering the brain, because it is too big to cross the protective blood–brain barrier. The researchers found no evidence of the protein fragment entering the brain or altering levels of the brain’s own klotho. “Since the fragment doesn’t cross the blood–brain barrier, it suggests it must be interacting with another factor that’s able to get into the brain,” says neuroscientist David Holtzman of Washington University in Saint Louis, who was not involved in the study. “Whether it’s an immune system–related factor or something else will be important to understand in future studies as the effects could ultimately turn out to be useful from a clinical standpoint.”
Most studies in mice fail to translate to success in human trials, but there are two reasons for optimism: Klotho is naturally produced in human kidneys and brains, and we experience widely varying levels over our lifetimes, so tolerability and safety is less of a concern. It is also already linked to enhanced human cognition. “It’s really important to me, as a physician [and] scientist, that what we study in mice has relevance in humans,” Dubal says. “But we have strong evidence that klotho is linked to better brain health in humans, at least by association.”
Neuroscientist Carmela Abraham of Boston University is investigating ways to increase the brain’s klotho supply. “What's amazing here is that something you inject into the belly has such effects on cognition without increasing levels of klotho in the brain,” she says. Abraham collaborated with Dubal and Lennart Mucke of the Gladstone Institute of Neurological Disease on both the 2014 work as well as a 2015 study, which showed adding the gene that elevates klotho levels to mice engineered to mimic Alzheimer’s rescues the cognitive impairments usually seen in such animals. Although excited about klotho's potential, Abraham has two reservations: First, the effects of the protein may involve many steps in a chain of molecular events by which it ultimately affects GluN2B. “We don't know the mechanism,” Abraham says. “It's important to find all the molecules on the pathway, before we do anything with humans.” Second, she says many groups are struggling to make klotho in a consistent manner because of the size and complexity of the protein. Her group is trying to identify molecules that interact with klotho. “If it’s a hormone, it must have a receptor,” she says. “Once we find such a receptor, we can design small molecules that activate it, which would be much easier to produce than a huge protein like klotho.”
For her part, Dubal is interested in partnering with biotech and pharma companies with a view to moving toward human trials of klotho itself. “Whether our discovery leads to pathways for effective small molecules or klotho-based biologics to enhance brain function and resilience, either or both would be a remarkable advance to potentially improve quality of life,” she says.