Phosphorylation of p66Shc and forkhead proteins mediates Aβ toxicity

Excessive accumulation of amyloid β-peptide (Aβ) plays an early and critical role in synapse and neuronal loss in Alzheimer's Disease (AD). Increased oxidative stress is one of the mechanisms whereby Aβ induces neuronal death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66Shc, we investigated the role of p66Shc in Aβ toxicity. Treatment of cells and primary neuronal cultures with Aβ caused apoptotic death and induced p66Shc phosphorylation at Ser36. Ectopic expression of a dominant-negative SEK1 mutant or chemical JNK inhibition reduced Aβ-induced JNK activation and p66Shc phosphorylation (Ser36), suggesting that JNK phosphorylates p66Shc. Aβ induced the phosphorylation and hence inactivation of forkhead transcription factors in a p66Shc-dependent manner. Ectopic expression of p66ShcS36A or antioxidant treatment protected cells against Aβ-induced death and reduced forkhead phosphorylation, suggesting that p66Shc phosphorylation critically influences the redox regulation of forkhead proteins and underlies Aβ toxicity. These findings underscore the potential usefulness of JNK, p66Shc, and forkhead proteins as therapeutic targets for AD

Classical Mus musculus Igκ Enhancers Support Transcription but not High Level Somatic Hypermutation from a V-Lambda Promoter in Chicken DT40 Cells

Somatic hypermutation (SHM) of immunoglobulin genes is initiated by activation-induced cytidine deaminase (AID) in activated B cells. This process is strictly dependent on transcription. Hence, cis-acting transcriptional control elements have been proposed to target SHM to immunoglobulin loci. The Mus musculus Igκ locus is regulated by the intronic enhancer (iE/MAR) and the 3′ enhancer (3′E), and multiple studies using transgenic and knock-out approaches in mice and cell lines have reported somewhat contradictory results about the function of these enhancers in AID-mediated sequence diversification. Here we show that the M. musculus iE/MAR and 3′E elements are active solely as transcriptional enhancer when placed in the context of the IGL locus in Gallus gallus DT40 cells, but they are very inefficient in targeting AID-mediated mutation events to this locus. This suggests that either key components of the cis-regulatory targeting elements reside outside the murine Igκ transcriptional enhancer sequences, or that the targeting of AID activity to Ig loci occurs by largely species-specific mechanisms

Functional analysis of the 3′E enhancer element.

<p>A) Gene-targeting scheme to generate the 3kE DT40 knock-in cell lines. The 3kE element is represented as an orange oval, the restriction sites used for Southern Blot analyses are marked (B: BamHI, and S: SacI), and the Southern blot probe (red line, P) used for genotyping is shown. For a description of all other elements, see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018955#pone-0018955-g001" target="_blank">Fig. 1</a>. The asterisk in each Southern blot marks the band that originates from the non-rearranged allele. B) Northern Blots were run using 10 µg of total RNA of wild-type (WT) DT40 cells and each of the indicated cell lines, and hybridized sequentially with IGL and GAPDH probes. One representative example is shown. C) Average normalized steady-state transcript levels of IGL measured by Northern blot analyses obtained from at least four independent values per genotype. The value for wild-type (WT) DT40 cells is arbitrarily set to one. D) GCV was quantified by flow cytometry over four weeks starting from single cell clones. The average percentage of IgM+ cells from 12 subclones of each of the two independent 3kE lines is shown. E) Mutation event frequencies in the IGL (filled bars) and IGH (open bars) loci were determined by sequencing from two independent subclones after four weeks of continuous culture. The data for wild type (WT) and ΔME6K were obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018955#pone.0018955-Kothapalli1" target="_blank">[13]</a>. Asterisks indicate p<0.005 in a Student's t-test.</p

Analysis of double Igκ enhancer knock-in DT40 cells.

<p>A) Gene-targeting scheme with the murine iE/MAR and 3kE shown as orange ovals, for a detailed description of all elements see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018955#pone-0018955-g001" target="_blank">Fig. 1</a>. The restriction sites are marked (A: AvrII, and B: BamHI), and the red line above the locus denotes the Southern blot probe (P). The asterisk in each Southern blot marks the band that originates from the non-rearranged allele. B) Northern Blots were run using 10 µg of total RNA of wild-type (WT) DT40 cells and each of the indicated cell lines, and hybridized sequentially with <i>IGL</i> and <i>GAPDH</i> probes. One representative example is shown. C) Normalized steady-state transcript levels of <i>IGL</i> were determined by Northern blots analysis. The level in wild-type cells is arbitrarily set to one, and an average of at least four data points obtained for each genotype is shown. D) Average percentages of IgM⁺ cells (twelve subclones per indicate clone) at each time point are shown for each genotype. E) Mutation event frequencies in the <i>IGL</i> (filled bars) and <i>IGH</i> (open bars) loci were determined by sequencing of the VJ or VDJ exon from two independent subclones of the ieK DT40 cells after four weeks of continuous culture. The data for wild type (WT) and ΔME6K were obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018955#pone.0018955-Kothapalli1" target="_blank">[13]</a>. Asterisks indicate p<0.005 in a Student's t-test.</p

Schematic representation of the <i>IGL</i> loci in individual DT40 lines.

<p>The rearranged allele of the <i>IGL</i> locus in the indicated cell lines is drawn to scale. The leader (L), the VJ exon, constant region (C), and the CR1 retrotransposon (present in the published chicken genome as well as in DT40 cells, S.D.F. unpublished) are shown as filled boxes. The putative matrix attachment region (MAR) and the 467 bp enhancer (Enh) are shown as filled ovals. Elements that are present are shown in black, those that are deleted are in grey. The murine enhancer elements (iE/MAR and 3kE) in the respective knock-in alleles are depicted as orange ovals.</p