Abraham’s lab initially stumbled across klotho during a study of brain aging in rhesus monkeys for whom cognitive decline had been established. That study focused on white matter, and a microarray experiment comparing gene expression in young versus old rhesus monkey brain pointed to a 74 percent drop in klotho mRNA in aging white matter (see Duce et al., 2008). Previously, scientists had reported premature aging with cognitive deficits in klotho knockout mice, whereas mice overexpressing klotho lived especially long and expressed copious amounts of antioxidant genes (Kuro-O et al., 1997; Kurosu et al., 2005). A human klotho polymorphism has been associated with longevity and robust health. In short, klotho is a mammalian lifespan gene. It falls into a group with other genes that impair insulin receptor signaling and dramatically increase longevity in worms and fruit flies.

Previous research by others had also shown that klotho gets secreted and acts much like a hormone. In peripheral organs, particularly the kidney, klotho promotes calcium resorption and vitamin D signaling, protecting bones against osteoporosis, for example.

“The question for us is, How does it work in the brain?” Abraham told ARF in San Diego. To that end, her group studied klotho expression and cleavage. On a poster, Abraham showed how biochemical experiments confirmed the microarray data at the protein level, indicating that klotho levels decrease with age in monkey, rat, and mouse brain. Klotho protein was most abundant in the choroid plexus. This capillary/ependymal organ dangling from the brain’s ventricles releases klotho into the CSF. Klotho concentration falls off in samples from AD brain. In the brain itself, klotho expression was highest in the hippocampus, followed by striatum, thalamus, medulla, and spinal cord, Abraham and first author Sonia Podvin reported. What’s more, 12-month-old APP/PS1-transgenic mice with plaques expressed less klotho than did littermates without amyloidosis, the scientist found.

Next, the Boston researchers analyzed klotho cleavage. The gene sequence predicts klotho to be a type 1 transmembrane protein (the type of protein that is cleaved by α-, β-, and γ-secretases), and its cleaved ectodomain appears to be that piece that functions like a hormone. Klotho cleavage generates a long 130 kDa and a short 65 kDa fragment. On a separate poster, as well as in a paper in the December 3 Proceedings of the National Academy of Sciences, first author Ci-Di Chen and colleagues identified the sheddases, aka α-secretases, that cleave klotho. In COS-7 cells and also in rat kidney slices, those enzymes were none other than ADAM 10 and ADAM 17, the metalloproteases thought to cleave APP as well as other AD-relevant proteins such as TNFα. (The human genome contains additional ADAM enzymes, and some of those could cleave klotho, as well, the authors note.)

Thickening the plot, insulin turned out to stimulate this cleavage and subsequent release of the klotho ectodomain, Chen and colleagues reported. Further experiments indicated that insulin promotes klotho cleavage by affecting the proteolytic activity of ADAM 10 and/or 17 rather than change the proteases’ expression (Chen et al., 2007). As insulin receptor signaling stimulates cleavage of klotho, its released ectodomains in turn activate expression of antioxidant enzymes. So do the sirtuin longevity genes. Importantly, elevated klotho might then feed back to inhibit insulin receptor signaling, Abraham proposes. The transgenic klotho overexpressors are insulin-resistant in a way that is clearly beneficial for their health. Klotho’s relationship, if any, to sirtuins and the FoxO transcription factors need to be explored, Abraham said.

Clearly, klotho per se is not specific to AD. It counteracts age-related processes in multiple ways. In San Diego, for example, Dwight German, Kevin Rosenblatt, Makoto Kuro-O, and colleagues at the University of Texas Southwestern Medical Center in Dallas reported data suggesting that klotho protects nigrostriatal dopamine neurons from MPTP toxicity in vivo in mice. Even so, areas of molecular overlap with AD mechanisms deserve study, Abraham said. First, the same sheddases that cleave klotho also cleave APP to generate the protective sAPPα fragment and may preclude Aβ generation. One question to explore here is whether insulin receptor signaling boosts sAPPα release by the same mechanism, i.e., via ADAM 10 and/or 17. Second, one proposed mechanism of Aβ oligomer toxicity focuses on insulin receptor signaling, (see ARF related news story), and indeed, AD is sometimes called “type 3 diabetes.” Abraham’s group is studying whether there are links between klotho and Aβ. The investigators also focus on characterizing the klotho promoter, which they suspect may sustain oxidative damage with age. Theoretically, then, small-molecule drugs that keep klotho expression high could keep Lachesis and Atropos at bay.—Gabrielle Strobel.