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\u27s 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

Fingolimod modulates multiple neuroinflammatory markers in a mouse model of Alzheimer’s disease

Sphingosine 1-phosphate (SP1) receptors may be attractive targets for modulation of inflammatory processes in neurodegenerative diseases. Recently fingolimod, a functional S1P1 receptor antagonist, was introduced for treatment of multiple sclerosis. We postulated that anti-inflammatory mechanisms of fingolimod might also be protective in Alzheimer’s disease (AD). Therefore, we treated a mouse model of AD, the 5xFAD model, with two doses of fingolimod (1 and 5 mg/kg/day) and measured the response of numerous markers of Aβ pathology as well as inflammatory markers and neurochemistry using biochemical, immunohistochemistry and high resolution magic angle spinning magnetic resonance spectroscopy (MRS). In mice at 3 months of age, we found that fingolimod decreased plaque density as well as soluble plus insoluble Aβ measured by ELISA. Fingolimod also decreased GFAP staining and the number of activated microglia. Taurine has been demonstrated to play a role as an endogenous anti-inflammatory molecule. Taurine levels, measured using MRS, showed a very strong inverse correlation with GFAP levels and ELISA measurements of Aβ, but not with plaque density or activated microglia levels. MRS also showed an effect of fingolimod on glutamate levels. Fingolimod at 1 mg/kg/day provided better neuroprotection than 5 mg/kg/day. Together, these data suggest a potential therapeutic role for fingolimod in AD

Moderate Exercise Delays The Motor Performance Decline In A Transgenic Model Of Als

The relationship between exercise and amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by motor neuron loss, rapidly progressive weakness and early death has been controversial. We studied the effect of a high (HEX and moderate-level exercise (MEX) on body weight, motor performance and motor neuron counts in the ventral horn of spinal cords in a transgenic mouse model of ALS (G93A-SOD1) that overexpresses a mutated form of the human SOD1 gene that is a cause of familial ALS. These transgenic mice show several similarities to the human disease, including rapid progressive motor weakness from 100 days of age and premature death at around 135 days of age. Mice were exposed to high or mid-level exercise of left sedentary (SED). At 70, 95 and 120 days of age, spinal cords were processed following euthanasia. Motor neurons larger than 15 mu m in diameter were counted with a design-based stereological protocol using an optical fractionator probe in the ventral horn of different regions of the cord and compared to wild-type littermates. Moderate exercise delayed the onset of motor deficit by over a week. High exercise slightly but significantly hastened the onset of motor performance deficits. Motor neuron density in the lumbar cord was significantly higher in MEX group compared to SED at 95 days of age. These results show the beneficial effects of moderate exercise on the preservation of motor performance that correlates with higher motor neuron density in the ventral horn of the lumbar spinal cord in G93A mice. Published by Elsevier B.V.Wo

Depletion of BIK led to decreased mitochondrial cell death priming in HCT-116 wt cells.

<p>(A) HCT-116 wt and (B) HCT-116 p53 -/- cells were untreated (upper panels) or transiently transfected with BIK siRNA (lower panels) and mitochondrial depolarization was measured following incubation of cells with DMSO, BIM, BID, BAD, PUMA, BMF and NOXA peptides at 100 μM and FCCP at 10 μM. Graphs are shown as mean±SEM, n = 3 and sample mitochondrial potential (ΔΨm) kinetic tracings are provided. (C) Microplate-based BH3 profiles were confirmed by ELISA-based cytochrome c release assays following incubation of isolated mitochondria with DMSO, BIM, BID, BAD, PUMA, BMF and NOXA peptides at 100 μM (mean±SEM, n = 3).</p

BIK mediates cisplatin-induced cell death in HCT-116 wt cellular spheroids, but not in HCT-116 p53 -/- spheroids.

<p>(A) HCT-116 wt and HCT-116 p53 -/- cells were transfected with BIK siRNA or scrambled siRNA for 24h. Cells were subsequently grown in 24-well 3D Algimatrix plates in the presence of RNAi duplexes. Microscopic evaluation of spheroids was done to verify that siRNA treatments did not interfere with the 3D growth of HCT-116 cells. (B) The efficiency of BIK depletion by RNA interference in HCT-116 wt and HCT-116 p53 -/- was determined by immunoblot analysis. Actin was probed as loading control. (C) Spheroids were treated with cisplatin (200 μM) for 48h and cell viability was assessed by using alamarBlue assay (mean±SEM, n = 3, *P<0.05).</p

Cisplatin induces early LMP in HCT-116 p53 -/- cells.

<p>(A) HCT-116 wt and HCT-116 p53-/- cells were treated with cisplatin (20 μM) for 1h. HCT-116 p53 -/- cells were TIRON (10 mM) for 2h and then treated with cisplatin for 1h to examine the effect of TIRON on cisplatin-induced LMP. Cells were stained for galectin-3 to evaluate the lysosomal membrane permeabilization. (B) HCT-116 wt and HCT-116 p53 -/- cells were transfected with BIK siRNA or scrambled siRNA for 24h. Cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 1h and the Cathepsin B/L activity was measured in untransfected and transfected cells (mean RFU±SEM, n = 3). (C) HCT-116 p53 -/- cells were pretreated with CA-074Me (100 μM) or Z-FA-FMK (10 μM) for 2h and then treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 48h. Cell viability was determined by CellTiterGlo assay and expressed as % of untreated control (mean±SEM, n = 3, *P<0.05, **P<0.01).</p

BIK/BCL-2 and BIK/BCL-XL interactions were increased in HCT-116 wt cells in response to DNA damage.

<p>HCT-116 wt and HCT-116 p53 -/- cells were treated with cisplatin (20 μM) or UV (100 mJ/cm²) for 4h and the interaction of BIK with BCL-2, BCL-XL, and MCL-1 was detected by coimmunoprecipitation assays. Inputs for coimmunoprecipitation experiments were also subjected to immunoblot analysis and actin was probed as loading control.</p