Background:Klotho is an age suppressor protein whose brain function is unknown. Results: Klotho protects hippocampal neurons from glutamate and amyloid ␤-induced oxidative damage through the induction of the thioredoxin/peroxiredoxin system. Conclusion: Klotho is neuroprotective via the regulation of the redox system. Significance: Understanding the mechanism underlying Klotho-induced neuroprotection may lead to the development of novel therapeutic approaches against neurodegeneration.

We have previously shown that myelin abnormalities and loss 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. While the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. In the present study, we found significant effects of Klotho on oligodendrocyte functions including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein Western analysis indicated Klotho's downstream effects involve Akt and ERK signal pathways. Klotho increased OPCs maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knockout mice and their control littermates revealed that knockout 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 knockout mice. Strikingly, at the ultrastructural level, Klotho knockout mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. In order to decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Taken 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.

Several antidepressants, mainly selective serotonin-reuptake inhibitors (SSRIs) and some tricyclic antidepressants (TCAs), have been shown to possess potent apoptotic activity in different cell lines. Our aim was to screen and select those agents with significant activity and elucidate the molecular pathway underlying this process in rat glioma and human neuroblastoma cell lines. We studied the effect of different antidepressants on apoptotic markers, including: cell viability, DNA fragmentation, cytochrome c (Cyt c) release from mitochondria, and caspase-3- like activity. In addition, the involvement of MAPK genes, c-Jun, and ERK was determined. Paroxetine and fluoxetine, SSRIs, clomipramine, a TCA, but not imipramine or mianserin (an atypical antidepressant), caused apoptosis in both cell lines, as assessed by flow cytometry of propidium iodide-stained C6 cells and typical fluorescence microscopy in glioma cells. These apoptotic changes were preceded by rapid increase in p-c-Jun levels, Cyt c release from mitochondria, and increased caspase-3-like activity. Assessment of paroxetine cytotoxicity in primary mouse brain and neuronal cultures showed significantly lower sensitivity to the drug's proapoptotic activity. These results strongly suggest that selected antidepressants induce apoptosis in neuronal and glial cell lines. Activation of p-c-Jun and subsequent increased Cyt c mitochondrial release participate in the apoptotic mechanism of the antidepressant. The high sensitivity to these drugs of the cancer cell, compared with primary brain tissue, suggests the potential use of these agents in the treatment of brain-derived tumors.

Membrane protein shedding is a critical step in many normal and pathological processes. The anti-aging protein klotho (KL), mainly expressed in kidney and brain, is secreted into the serum and CSF, respectively. KL is proteolytically released, or shed, from the cell surface by ADAM10 and ADAM17, which are the α-secretases that also cleave the amyloid precursor protein and other proteins. The transmembrane KL is a coreceptor with the FGF receptor for FGF23, whereas the shed form acts as a circulating hormone. However, the precise cleavage sites in KL are unknown. KL contains two major cleavage sites: one close to the juxtamembrane region and another between the KL1 and KL2 domains. We identified the cleavage site involved in KL release by mutating potential sheddase(s) recognition sequences and examining the production of the KL extracellular fragments in transfected COS-7 cells. Deletion of amino acids T958 and L959 results in a 50–60% reduction in KL shedding, and an additional P954E mutation results in further reduction of KL shedding by 70–80%. Deletion of amino acids 954–962 resulted in a 94% reduction in KL shedding. This mutant also had moderately decreased cell surface expression, yet had overall similar subcellular localization as that of WT KL, as demonstrated by immunofluorescence. Cleavage-resistant mutants could function as a FGFR coreceptor for FGF23, but they lost activity as a soluble form of KL in proliferation and transcriptional reporter assays. Cleavage between the KL1 and KL2 domains is dependent on juxtamembrane cleavage. Our results shed light onto mechanisms underlying KL release from the cell membrane and provide a target for potential pharmacologic interventions aimed at regulating KL secretion.

Emdogain, a formulation of Enamel Matrix Proteins, is used clinically for periodontal regeneration to stimulate PDL (periodontal ligament), cementum, and bone formation. Its effects on gingival fibroblasts and tissue have not been thoroughly studied. Therefore, we investigated the mechanisms by which Emdogain affects the cell cycle of human gingival fibroblasts. Without serum, Emdogain (50 microg/mL) induced human gingival fibroblast entry into the S phase and DNA synthesis, but not completion of the cell cycle. With low serum concentrations (0.2-0.5%), Emdogain synergistically induced completion of the cell cycle, resulting in increased cell numbers. The mitogenic response to Emdogain depended on Extracellular Regulated Kinase (ERK) activation, which occurred in two waves, peaking after 15 min and 4 to 6 hrs, since it was abolished by U0126, a specific MAPK inhibitor. Inhibition of the second wave was sufficient to abrogate mitogenesis. This study characterized the mitogenic effect of Emdogain on primary human gingival fibroblasts, its cooperation with serum growth factors, and the key mediatory role of the ERK cascade.

Objective Much of the genetic basis for Alzheimer disease (AD) is unexplained. We sought to identify novel AD loci using a unique family-based approach that can detect robust associations with infrequent variants (minor allele frequency <0.10). Methods We conducted a genome-wide association study in the Framingham Heart Study (FHS) (discovery) and NIA-LOAD (replication) family-based cohorts using an approach that accounts for family structure and calculates a risk score for AD as the outcome. Links between the most promising gene candidate and AD pathogenesis were explored in silico as well as experimentally in cell-based models and in human brain. Results Genome-wide significant association was identified with a PLXNA4 SNP (rs277470) located in a region encoding the semaphorin-3A (SEMA3A) binding domain (meta-analysis p value [meta-P]=4.1×10−8). A test for association with the entire region was also significant (meta-P=3.2×10−4). Transfection of SH-SY5Y cells or primary rat neurons with full-length PLXNA4 (TS1) increased tau phosphorylation when stimulated by SEMA3A. The opposite effect was observed when transfected with shorter isoforms (TS2 and TS3). However, transfection of any isoform into HEK293 cells stably expressing APP did not result in differential effects on APP processing or Aβ production. Late-stage AD cases (n=9) compared to controls (n=5) had 1.9-fold increased expression of TS1 in cortical brain tissue (P=1.6×10−4). Expression of TS1 was significantly correlated with the Clinical Dementia Rating score (ρ=0.75, P=2.2×10−4), plaque density (ρ=0.56, P=0.01) and Braak stage (ρ=0.54, P=0.02). Interpretation Our results indicate that PLXNA4 has a role in AD pathogenesis through isoform-specific effects on tau phosphorylation.

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In this study we aimed to (1). screen phenothiazines for cytotoxic activity in glioma, neuroblastoma, and primary mouse brain tissue; and (2). determine the mechanism of the cytotoxic effect (apoptosis, necrosis) and the roles of calmodulin inhibition and sigma receptor modulation. Rat glioma (C6) and human neuroblastoma (SHSY-5Y) cell lines were treated with different phenothiazines. All agents induced a dose-dependent decrease in viability and proliferation, with the highest activity elicited by thioridazine. Sensitivity to thioridazine of glioma and neuroblastoma cells was significantly higher (p < 0.05) than that of primary mouse brain culture (IC50 11.2 and 15.1 microM vs 41.3 microM, respectively). The N-mustard fluphenazine induced significantly lower cytotoxicity in glioma cells, compared to fluphenazine. The sigma receptor selective ligand (+)-SK&F10047 increased viability slightly while combined with fluphenazine; SK&F10047 did not alter fluphenazine activity. Flow cytometry of propidium iodide (PI)-stained glioma cells treated with thioridazine, fluphenazine, or perphenazine (6-50 microM) resulted in a concentration-dependent increase of fragmented DNA up to 94% vs 3% in controls by all agents. Thioridazine (12.5 microM)-treated glioma cells costained with PI and Hoechst 33342 revealed a red fluorescence of fragmented nuclei in treated cells and a blue fluorescence of intact control nuclei. After 4-h exposure to thioridazine (25 and 50 microM), a 25- to 30-fold increase in caspase-3 activity in neuroblastoma cells was noted. Overall, the marked apoptotic effect of phenothiazines in brain-derived cancer cells, and the low sensitivity of primary brain tissue suggest the potential use of selected agents as therapeutic modalities in brain cancer.