Research ArticleAlzheimer’s Disease

Nitrosylation of GAPDH augments pathological tau acetylation upon exposure to amyloid-β

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Science Signaling  20 Mar 2018:
Vol. 11, Issue 522, eaao6765
DOI: 10.1126/scisignal.aao6765
  • Fig. 1 Nitric oxide regulates tau acetylation at Lys280.

    (A) Coimmunoprecipitation (Co-IP) assay to detect tau acetylation at Lys280 (K280) in primary neurons treated with amyloid-β1–42 (Aβ1–42) for 24 hours in the presence or absence of l-NG-nitroarginine methyl ester (L-NAME). (B) Confocal microscopic analysis of acetylation of tau at Lys280 in primary neurons treated with Aβ1–42 for 24 hours with or without L-NAME. (C) Co-IP assay to detect acetylation of tau in the cortical lysates isolated from neuronal nitric oxide synthase (nNOS)+/+ and nNOS−/− mice after intracortical infusion of Aβ1–42. (D) Confocal microscopy analysis of tau acetylation in the cortex of nNOS+/+ and nNOS−/− mice after infusion of Aβ1–42 into the cortex. The acetylated tau was monitored by red fluorescent signal (quantified), and total tau was determined by green fluorescent signal. Scale bar, 100 μm. (E) Co-IP assay to detect acetylation of tau in primary neuron isolated from nNOS+/+ and nNOS−/− mice. Data are quantified in the Supplementary Materials; all blots and microscopy are representative of three independent experiments from five to seven mice each condition.

  • Fig. 2 Nitrosylated GAPDH activates p300 and acetylates tau.

    (A) S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH-SNO) was measured using the biotin-switch assay in lysates from post-mortem cortical samples from human patients [n = 7 Alzheimer’s disease (AD) patient samples; n = 3 control samples]. (B) Biotin-switch assay to detect GAPDH-SNO in cortex isolated from mice administered with Aβ1–42. (C) Nitrosylation of GAPDH assessed by biotin-switch assay in primary neurons that overexpressed GAPDH and GAPDH C150S and was treated with Aβ1–42. (D) GAPDH or GAPDH C150S was overexpressed in primary neurons isolated from nNOS+/+ mice and treated with Aβ1–42. The interaction between GAPDH and p300 was assessed by co-IP. (E) Co-IP between GAPDH and p300 in cortical lysates isolated from nNOS+/+ and nNOS−/− mice after administration of Aβ1–42. (F) The acetylation of p300 in primary neurons that overexpressed GAPDH or GAPDH C150S and was treated with Aβ1–42, as assessed by co-IP with an antibody to acetyl-lysine and Western blotting with an antibody to acetylated p300. (G) The acetylation of p300, H3, and tau in primary neurons that overexpressed GAPDH or GAPDH K160R and were treated with Aβ1–42, assessed by co-IP with an antibody to acetyl-lysine and subsequent Western blotting. (H) Immunofluorescence signals for acetylated tau (red) and total tau (green) in the cortex from mice overexpressing GAPDH or GAPDH K160R and injected with Aβ1–42. Scale bar, 100 μm. (I) Co-IP assay to assess tau acetylation in primary neurons isolated from nNOS+/+ mice and treated with either control or p300 small interfering RNA (siRNA) before administration of Aβ1–42. Data are quantified in the Supplementary Materials; all blots and microscopy are representative of three independent experiments from five to seven mice each condition. HA, hemagglutinin; RNAi, RNA interference.

  • Fig. 3 Nitrosylation of GAPDH induces SIRT1 nitrosylation and tau acetylation.

    (A) The interaction between deacetylase sirtuin 1 (SIRT1) and tau was monitored by co-IP assay using lysates from cortex isolated from both nNOS+/+ and nNOS−/− mice administered with or without Aβ1–42. (B) Biotin-switch assay was performed to monitor nitrosylation of SIRT1 using the cortical lysates after administration of Aβ1–42. (C) The interaction between GAPDH and SIRT1 was monitored by co-IP assay using cortical lysates after administration of Aβ1–42. (D) Primary neurons isolated from nNOS+/+ mice were overexpressed with either GAPDH or GAPDH C150S construct before administration of Aβ1–42. The interaction between SIRT1 and GAPDH was monitored by co-IP assay. (E) GAPDH or GAPDH C150S was overexpressed in the cortex, and nitrosylation of GAPDH and SIRT1 was monitored by biotin-switch assay. (F) Primary neurons isolated from nNOS+/+ mice were overexpressed with either GAPDH or GAPDH C150S construct before administration of Aβ1–42. The nitrosylation of GAPDH and SIRT1 was then assessed and monitored by biotin-switch assay. (G) Primary neurons isolated from nNOS+/+ mice overexpressed with either SIRT1 or SIRT1 C387/390S were treated with Aβ1–42. Cell lysates were used to do co-IP assay to monitor the interaction between SIRT1 and tau. (H) Primary neurons isolated from nNOS+/+ mice overexpressed with either SIRT1 or SIRT1 C387/390S were treated with Aβ1–42. Acetylation of tau (Lys280) and H3 were assessed in cell lysates using a co-IP assay. The change in the amount of acetylated H3 and tau was quantitated for each sample. (I) Confocal microscopic analysis of total (green) and acetylated (red) tau in the cortex after administration of Aβ1–42. Scale bar, 100 μm. (J) Primary neurons isolated from nNOS+/+ mice were treated with either control or HDAC6 siRNA before administration with Aβ1–42. The tau acetylation at Lys280 was assessed by co-IP assay with antibody to acetyl-lysine and subsequent Western blotting. Data are quantified in the Supplementary Materials; all blots and microscopy are representative of three independent experiments from five to seven mice each condition.

  • Fig. 4 Inhibition of GAPDH nitrosylation results in a reduction in tau acetylation.

    (A) GAPDH or GAPDH C150S was overexpressed in the cortex before administration of Aβ1–42. tau acetylation was assessed by co-IP assay with an antibody to acetyl-lysine and subsequent Western blotting. (B) GAPDH or GAPDH C150S was overexpressed in cortex before administration of Aβ1–42. Confocal microscopic analysis then assessed total (green) and acetylated (red) tau in the cortical tissue. Scale bar, 100 μm. (C) Confocal microscopic analysis then assessed total (green) and acetylated (red) tau in the cortical tissue from mice treated with CGP3466B (top, 0.5 mg/kg; bottom, 2.5 mg/kg) before administration of Aβ1–42 into the cortex (right) compared with Aβ1–42 alone or controls (left). Scale bar, 100 μm. (D and E) Nitrosylation of GAPDH (D) and the acetylation of tau (E) were assessed in primary neurons cultured with Aβ1–42 with or without CGP3466B. Data above are quantified in the Supplementary Materials; all blots and microscopy are representative of three independent experiments from five to seven mice each condition. (F) Total distance calculated in open-field tests in mice treated with Aβ1–42 and with or without CGP3466B. n = 15 to 20 mice; **P < 0.001, Wilcoxon two-sample test (nonparametric version of two sample t test). (G) Latency to find the platform in Morris water maze test was monitored in mice administered with Aβ1–42 with or without CGP3466B treatment. n = 15 to 20 mice; **P < 0.001, Wilcoxon two-sample test. (H) A working model of our findings suggesting that Aβ1–42 leads to an induction in the level of nitric oxide (NO), which, in turn, nitrosylates GAPDH. Nitrosylated GAPDH interacts with an acetyltransferase p300 and a deacetylase SIRT1. The interaction between p300 and GAPDH facilitates an activation of p300 and an induction of tau acetylation. The interaction between SIRT1 and GAPDH results in nitrosylation of SIRT1, which is unable to deacetylate tau. Inhibition of GAPDH-SNO reduces tau acetylation and improves behavioral impairments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/522/eaao6765/DC1

    Fig. S1. Quantitative analysis of tau acetylation in vitro and in vivo.

    Fig. S2. Quantitative analysis of GAPDH nitrosylation after Aβ1–42 administration.

    Fig. S3. Quantitative analysis of transnitrosylation of SIRT1 and its effect on tau acetylation.

    Fig. S4. Quantitative analysis of tau acetylation after inhibition of GAPDH nitrosylation.

  • Supplementary Materials for:

    Nitrosylation of GAPDH augments pathological tau acetylation upon exposure to amyloid-β

    Tanusree Sen, Pampa Saha, Nilkantha Sen*

    *Corresponding author. Email: senn{at}pitt.edu

    This PDF file includes:

    • Fig. S1. Quantitative analysis of tau acetylation in vitro and in vivo.
    • Fig. S2. Quantitative analysis of GAPDH nitrosylation after Aβ1–42 administration.
    • Fig. S3. Quantitative analysis of transnitrosylation of SIRT1 and its effect on tau acetylation.
    • Fig. S4. Quantitative analysis of tau acetylation after inhibition of GAPDH nitrosylation.

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