SIRT1 Overexpression Antagonizes Cellular Senescence with Activated ERK/S6k1 Signaling in Human Diploid Fibroblasts

Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated β-galactosidase (SA-β-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan.. Western blot results showed that SIRT1 reduced the expression of p16INK4A and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16INK4A and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling

Monomeric C‐reactive protein induces the cellular pathology of Alzheimer's disease

Abstract Introduction Human study shows that elevated C‐reactive protein (CRP) in blood impacts apolipoprotein E (APOE) ε4, but not APOE ε3 or APOE ε2, genotype to increase the risk of Alzheimer's disease (AD). However, whether CRP is directly involved in cellular AD pathogenesis and in which type of neuronal cells of APOE ε4 carriers are unknown. Methods We aimed to use different primary neuronal cells and investigate if CRP induces cellular AD pathology depending on APOE genotypes. Here the different primary neuronal cells from the different APOE genotype knock‐in mice cortex were isolated and used. Results Monomeric CRP (mCRP) increased amyloid beta production and, in parallel, induced tau phosphorylation in addition to their related proteins in the primary neurons in a pattern of APOE ε4 > APOE ε3 > APOE ε2 in a dose‐ and time‐dependent manner. Consistently, mCRP induced the staining of other neurodegenerative biomarkers, including Fluoro‐Jade B stain (FjB), TUNEL and Cleaved Caspase‐3, in primary neurons in a similar pattern of APOE ε4 > APOE ε3 > APOE ε2. In contrast, pentameric CRP (pCRP) had a tendency to induce cellular AD pathology but did not reach statistical significance. On the other hand, it is intriguing that regardless of APOE genotype, mCRP did not influence the expressions of Iba‐1 and CD68 in primary microglia or the expression of glial fibrillary acidic protein in primary astrocytes, and additionally mCRP did not affect the secretions of interleukin (IL)‐1α, IL‐1β, and tumor necrosis factor α from these cells. Discussion This is the first report to demonstrate that mCRP directly induces cellular AD pathogenesis in neurons in an APOE genotype‐dependent pattern, suggesting that mCRP plays a role as a mediator involved in the APOE ε4‐related pathway for AD during chronic inflammation. Highlights Pentameric C‐reactive protein (pCRP) can be dissociated irreversibly to form free subunits or monomeric CRP (mCRP) during and after the acute phase. mCRP increased amyloid beta production in the primary neurons in a pattern of apolipoprotein E (APOE) ε4 > APOE ε3 > APOE ε2 in a dose‐dependent manner. mCRP induced the expression of phosphorylated tau in the primary neurons in a pattern of APOE ε4 > APOE ε3 > APOE ε2 in a dose‐ and time‐dependent manner. mCRP plays an important mediator role in the APOE ε4‐related pathway of Alzheimer's disease risk

Pax6 mediates ß-catenin signaling for self-renewal and neurogenesis by neocortical radial glial stem cells.

The Wnt/ß-catenin pathway is a critical stem cell regulator and plays important roles in neuroepithelial cells during early gestation. However, the role of Wnt/ß-catenin signaling in radial glia, a major neural stem cell population expanded by midgestation, remains poorly understood. This study shows that genetic ablation of ß-catenin with hGFAP-Cre mice inhibits neocortical formation by disrupting radial glial development. Reduced radial glia and intermediate progenitors are found in the ß-catenin-deficient neocortex during late gestation. Increased apoptosis and divergent localization of radial glia in the subventricular zone are also observed in the mutant neocortex. In vivo and in vitro proliferation and neurogenesis as well as oligodendrogenesis by cortical radial glia or by dissociated neural stem cells are significantly defective in the mutants. Neocortical layer patterning is not apparently altered, while astrogliogenesis is ectopically increased in the mutants. At the molecular level, the expression of the transcription factor Pax6 is dramatically diminished in the cortical radial glia and the sphere-forming neural stem cells of ß-catenin-deficient mutants. Chromatin immunoprecipitation and luciferase assays demonstrate that ß-catenin/Tcf complex binds to Pax6 promoter and induces its transcriptional activities. The forced expression of Pax6 through lentiviral transduction partially rescues the defective proliferation and neurogenesis by ß-catenin-deficient neural stem cells. Thus, Pax6 is a novel downstream target of the Wnt/ß-catenin pathway, and ß-catenin/Pax6 signaling plays critical roles in self-renewal and neurogenesis of radial glia/neural stem cells during neocortical development

β-catenin regulates Pax3 and Cdx2 for caudal neural tube closure and elongation.

Non-canonical Wnt/planar cell polarity (PCP) signaling plays a primary role in the convergent extension that drives neural tube closure and body axis elongation. PCP signaling gene mutations cause severe neural tube defects (NTDs). However, the role of canonical Wnt/β-catenin signaling in neural tube closure and NTDs remains poorly understood. This study shows that conditional gene targeting of β-catenin in the dorsal neural folds of mouse embryos represses the expression of the homeobox-containing genes Pax3 and Cdx2 at the dorsal posterior neuropore (PNP), and subsequently diminishes the expression of the Wnt/β-catenin signaling target genes T, Tbx6 and Fgf8 at the tail bud, leading to spina bifida aperta, caudal axis bending and tail truncation. We demonstrate that Pax3 and Cdx2 are novel downstream targets of Wnt/β-catenin signaling. Transgenic activation of Pax3 cDNA can rescue the closure defect in the β-catenin mutants, suggesting that Pax3 is a key downstream effector of β-catenin signaling in the PNP closure process. Cdx2 is known to be crucial in posterior axis elongation and in neural tube closure. We found that Cdx2 expression is also repressed in the dorsal PNPs of Pax3-null embryos. However, the ectopically activated Pax3 in the β-catenin mutants cannot restore Cdx2 mRNA in the dorsal PNP, suggesting that the presence of both β-catenin and Pax3 is required for regional Cdx2 expression. Thus, β-catenin signaling is required for caudal neural tube closure and elongation, acting through the transcriptional regulation of key target genes in the PNP

The alterations of SIRT1 expression and phosphorylation of ERK, AKT and S6K1 were determined in senescent 2BS Cells.

<p>The total cellular lysates from early passage (23 PDs) and late passage of 2BS (63 PDs) were prepared and subjected to western blot analysis by using specific antibodies against SIRT1,β-actin, Phospho-p44/42 ERK(Thr202/Tyr204), p44/42 ERK, Phospho-p70 S6K1 (Thr389), Phospho-p70 S6K1(Thr421/Ser424)and Phospho-AKT (Ser473).</p

SIRT1-dependend phosphorylation of kinase was associated with the cell proliferation.

<p>(A) Transfected 2BS cell containing pcDNA3.1, pcDNA-SIRT1 and pcDNA-H363YSIRT1 were lysated and subjected to western blot analysis by using specific antibodies against Phospho-p44/42 ERK (Thr202/Tyr204), Phospho-AKT (Ser473), Phospho-p70 S6K1(Thr389), Phospho-p70 S6K1 (Thr421/Ser424)and p44/42 ERK. (B, C) 2BS cells and WI38 cells at 40 PDs were treated with solvent alone or 0.2 or 2 µM of resveratrol (Res) for 72 h. Cell lysates analyzed by western blot, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001710#pone-0001710-g004" target="_blank">Figure 4A</a>.</p

Cumulative population doublings of SIRT1, H363YSIRT1, Mock-transfected cells and untransfected cells

<p>Cumulative population doublings of SIRT1, H363YSIRT1, Mock-transfected cells and untransfected cells</p

Changes of senescence-associated features in 2BS cells were induced by enforced expression of SIRT1.

<p>(A) Stable transfected 2BS cells expressing empty Vector, SIRT1 and H363YSIRT1 were passaged until one of them underwent senescence, then stained for SA-β-gal staining, a classical biomarker for senescence. Early passage (23 PDs) 2BS cells were showed as negative control. Late passage (60 PDs) 2BS cells were stained as positive control. (B) Senescence-associated heterochromatic foci (SAHF), another classical marker of senescence, was showed in 2BS cell lines DNA foci accumulate was visualized by DAPI staining in 2BS cells. Scale bars were equal to 20 µm. (C) Enlarged images of DAPI staining are shown in the lower panels. Scale bars were equal to 8 µm.</p