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[1, 2].  However, one cannot control where the DNA inserts and in one line of mice, an interesting and unrelated phenotpye 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[2]. What had occurred in these ko-mouse-alonemice 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[2, 3]. 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[4]. And thus the struggle began to determine how it was that klotho modified the aging process.

Klotho expression is limited to only a few organs with highest expression in kidney[2]. 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[5]. However, klotho can also be shed from the cell surface and is detected in both serum and cerebrospinal fluid (CSF)[6]. 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[4, 7-9]).

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[3, 14, 15].   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[3, 10-14]

cp-web-divicesExpression of the klotho protein in the brain is contested. Although it is clear and reproducible that klotho is expressed by the choroid plexus[16] and shed into the CSF, the amazing number of nonspecific antibodies on the market has caused disparity across labs in detecting it in the brain. Although by Western blot, expression in brain (even when choroid plexus is absent) is clear, anatomical localization studies conflict. 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 immunocytochemical detection of klotho that showed expression in wild-type mouse kidney but not in brain[17]. After identifying the one antibody able to do so, we confirmed Western blot findings of brain parenchymal expression[17, 18]. 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).

Klotho is age-downregulated in the brain[11, 18]. Its absence promotes an environment where cognitive impairment can develop very rapidly. As well its absence induces a phenotype consistent with premature aging and mild neurodegeneration. It is expressed in multiple forms such that it could act in cell autonomous or non-autonomous processes in response to normal brain function and/or physiological signals from the rest of the body. Understanding what it does in the normal brain and how this function changes with age will yield a wealth of information about brain aging. Studies presently ongoing in the lab seek to specifically understand 1) whether klotho is involved in synaptic function of the brain and 2) whether klotho is involved in the adult neurogenic process.

1.         Kuro-o, M., Klotho and aging. Biochim Biophys Acta, 2009. 1790(10): p. 1049-58.

2.         Kuro-o, M., et al., Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature, 1997. 390(6655): p. 45-51.

3.         Nagai, T., et al., Cognition impairment in the genetic model of aging klotho gene mutant mice: a role of oxidative stress. FASEB J, 2003. 17(1): p. 50-2.

4.         Kurosu, H., et al., Suppression of aging in mice by the hormone Klotho. Science, 2005. 309(5742): p. 1829-33.

5.         Kurosu, H., et al., Regulation of fibroblast growth factor-23 signaling by klotho. J Biol Chem, 2006. 281(10): p. 6120-3.

6.         Imura, A., et al., Secreted Klotho protein in sera and CSF: implication for post-translational cleavage in release of Klotho protein from cell membrane. FEBS Lett, 2004. 565(1-3): p. 143-7.

7.         Doi, S., et al., Klotho inhibits transforming growth factor-beta1 (TGF-beta1) signaling and suppresses renal fibrosis and cancer metastasis in mice. J Biol Chem, 2011. 286(10): p. 8655-65.

8.         Liu, H., et al., Augmented Wnt signaling in a mammalian model of accelerated aging. Science, 2007. 317(5839): p. 803-6.

9.         Wolf, I., et al., Klotho: a tumor suppressor and a modulator of the IGF-1 and FGF pathways in human breast cancer. Oncogene, 2008. 27(56): p. 7094-105.

10.       Chen, C.D., et al., The antiaging protein Klotho enhances oligodendrocyte maturation and myelination of the CNS. J Neurosci, 2013. 33(5): p. 1927-39.

11.       Duce, J.A., et al., Gene profile analysis implicates Klotho as an important contributor to aging changes in brain white matter of the rhesus monkey. Glia, 2008. 56(1): p. 106-17.

12.       Park, S.J., et al., Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging. Neuropsychopharmacology, 2013.

13.       Shiozaki, M., et al., Morphological and biochemical signs of age-related neurodegenerative changes in klotho mutant mice. Neuroscience, 2008. 152(4): p. 924-41.

14.       Uchida, A., et al., Neurofilaments of Klotho, the mutant mouse prematurely displaying symptoms resembling human aging. J Neurosci Res, 2001. 64(4): p. 364-70.

15.       Shiraki-Iida, T., et al., Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein. FEBS Lett, 1998. 424(1-2): p. 6-10.

16.       Li, S.A., et al., Immunohistochemical localization of Klotho protein in brain, kidney, and reproductive organs of mice. Cell Struct Funct, 2004. 29(4): p. 91-9.

17.       Clinton, S.M., et al., Expression of klotho mRNA and protein in rat brain parenchyma from early postnatal development into adulthood. Brain research, 2013. 1527: p. 1-14.

18.       King, G.D., D.L. Rosene, and C.R. Abraham, Promoter methylation and age-related downregulation of Klotho in rhesus monkey. Age (Dordr), 2011.