The discovery of klotho is a truly classic story of the power of scientific observation. In this story the careful observation of a scientist, even when his experiment had gone “awry”, yielded amazing new knowledge. Dr. Kuroo and colleagues were attempting to generate a transgenic mouse, a fairly straightforward process by which DNA expressing a gene of interest is inserted into the genome. However, one cannot control where the DNA inserts and in one line of mice, an interesting and unrelated phenotype developed. The mice were fine until adolescence but rapidly developed an array of disorders consistent with premature aging and died having lived no more than 4 months. What had occurred in these mice was insertion of the transgene into the promoter of the klotho gene. Beyond alterations of lifespan, the knockout mice developed an array of disorders typically only observed in advanced human age. These include physical attributes like thinning skin and hair loss that in the overall scope of life are rather trivial. If the symptoms had stopped there, may not have been so interesting, however in addition to the minor phenotypes the mice developed system wide, lethal dysfunction including arteriosclerosis, osteoporosis, emphysema, and cognitive impairment. The confluence of shortened lifespan and development of age-related disorders resulted in the klotho protein being touted as an age-regulating protein.
However, it is fairly easy to modify the genetic makeup of a mouse such that lifespan is shortened without actually doing something to modify the aging process. Thus to know whether klotho is involved in aging, overexpression of the protein should extend lifespan. And it did by ~30% with more of an effect observed in male mice than female mice. And thus the struggle began to determine how it was that klotho affects aging.
Klotho expression is limited to only a few organs with highest expression in kidney. Subsequent research revealed that klotho was a single pass transmembrane protein. As a transmembrane protein, klotho functions as a co-receptor with FGFR to transduce FGF23 signaling. However, klotho can also be shed from the cell surface and is detected in both serum and cerebrospinal fluid (CSF). This likely accounts for effects observed in places like the lung, where klotho is not produced but an emphysemic phenotype develops in the knockout mouse. As a shed protein, klotho is most noted for its effects as a sialidase and as a signal pathway inhibitor (inhibiting insulin/IGF1, wnt, and TGFβ).
While the kidney is clearly important, our lab’s interest lie in understanding the aging process as it applies to the brain. So what is known about klotho in the brain? The knockout develops cognitive impairment in only 8 weeks of life. Its brain is mildly neurodegenerative with altered expression level of proteins involved in synaptic function, structure, and axonal transport. Although several fantastic studies have been done to describe the brain of the knockout, our understanding of klotho in the brain mechanistically is fairly limited and generally restricted to observation at a single time point near the end of life.
Expression of the klotho protein in the brain is expressed by the choroid plexus and shed into the CSF. Although by Western blot, expression in brain (even when choroid plexus is absent) is clear, anatomical localization studies conflict, largely because of the lack of highly specific klotho antibodies. Use of antibodies claiming to be klotho specific must be validated before assessment of expression can be clear. Thus we undertook a study to identify antibodies that could be used for immunohistochemical detection of klotho that showed expression in wild-type mouse kidney but not in brain. After identifying the one antibody able to do so, we confirmed Western blot findings of brain parenchymal expression. Thus, like the kidney, in the brain, klotho is observed in its transmembrane form (neurons and oligodendrocytes) and in its shed form (from CSF shedding). We then went on to develop our own highly-specific klotho antibodies!
In the last few years our knowledge of the importance of klotho protein in the brain expanded. Importantly, numerous groups demonstrate that klotho expression is disease modifying in a way that would indicate klotho is powerfully neuroprotective. Models of Multiple Sclerosis, Alzheimer’s disease, Parkinson’s Disease, and normal brain aging all show improvement when klotho is increased. Unfortunately, klotho is age-downregulated in the brain. Its absence promotes an environment where cognitive impairment can develop very rapidly. Our work has shown that klotho is critically important for normal hippocampal adult neurogenesis and for synaptic plasticity indicating that the normal function of klotho in the brain is required for activities we associated with the brain. Our lab seeks to understand the molecular mechanisms behind klotho action in the brain to enable thoughtful and well informed therapeutic development to prevent and treat neurodegenerative disease.