The antiaging protein Klotho enhances oligodendrocyte maturation and myelination of the CNS.

We have previously shown that myelin abnormalities characterize the normal aging process of the brain and that an age-associated reduction in Klotho is conserved across species. Predominantly generated in brain and kidney, Klotho overexpression extends life span, whereas loss of Klotho accelerates the development of aging-like phenotypes. Although the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. We found significant effects of Klotho on oligodendrocyte functions, including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein analysis indicated that Klotho's downstream effects involve Akt and ERK signal pathways. Klotho increased OPC maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knock-out mice and control littermates revealed that knock-out mice have a significant reduction in major myelin protein and gene expression. By immunohistochemistry, the number of total and mature oligodendrocytes was significantly lower in Klotho knock-out mice. Strikingly, at the ultrastructural level, Klotho knock-out mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. To decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Together, these studies provide novel evidence for Klotho as a key player in myelin biology, which may thus be a useful therapeutic target in efforts to protect brain myelin against age-dependent changes and promote repair in multiple sclerosis.

Evaluation of Long-Term Cryostorage of Brain Tissue Sections for Quantitative Histochemistry.

Storage of tissue sections for long periods allows multiple samples, acquired over months or years, to be processed together, in the same reagents, for quantitative histochemical studies. Protocols for freezer storage of free-floating frozen sections using sucrose with different additives have been reported and assert that storage has no effect on histochemistry, but no quantitative support has been provided. The present study analyzed the efficacy of long-term storage of brain tissue sections at -80C in buffered 15% glycerol. To determine whether histochemical reactivity is affected, we analyzed 11 datasets from 80 monkey brains that had sections stored for up to 10 years. For processing, sections from multiple cases were removed from storage, thawed, and batch-processed at the same time for different histochemical measures, including IHC for neuronal nuclear antigen, parvalbumin, orexin-A, doublecortin, bromodeoxyuridine, the pro-form of brain-derived neurotrophic factor, and damaged myelin basic protein as well as a histochemical assay for hyaluronic acid. Results were quantified using stereology, optical densitometry, fluorescence intensity, or percent area stained. Multiple regression analyses controlling for age and sex demonstrated the general stability of these antigens for up to a decade when stored in 15% glycerol at -80C.