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• Research Paper Volume 10, Issue 9 pp 2498-2510

  Sirt1 enhances tau exon 10 inclusion and improves spatial memory of Htau mice

  Relevance score: 23.96182

  Shuo Qian, Jianlan Gu, Wufei Dai, Nana Jin, Dandan Chu, Qin Huang, Fei Liu, Wei Qian

  Keywords: Sirt1, resveratrol, 9G8, tau exon 10 splicing, Alzheimer’s disease

  Published in Aging on September 22, 2018

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  Alternative splicing of tau exon 10 generates tau isoforms with three or four microtubule binding repeats, named 3R-tau and 4R-tau, respectively. Dysregulation of tau exon 10 splicing could cause neurofibrillary degeneration. Acetylation is one of the major post-translational protein modifications in the cell by attachment of the acetyl group to either the α-amino group of the N-terminus of proteins or to the ε-amino group of lysine residues. Sirt1, one member in mammalian Sirtuin family, deacetylates protein and is associated closely with age-related diseases including Alzheimer’s disease. However, the role of Sirt1 in tau exon 10 splicing remains elusive. In the present study, we determined the role of Sirt1 in tau exon 10 splicing. We found that activation of Sirt1 by resveratrol enhanced tau exon 10 inclusion, leading to 4R-tau expression. Sirt1 interacted with splicing factor 9G8, deacetylated it at Lys24, and suppressed its function in promoting tau exon 10 exclusion. Moreover, resveratrol improved learning and spatial memory in Htau mice. These findings suggest that Sirt1 may serve as a new drug target for Alzheimer’s Disease related tauopathies and resveratrol may be used to correct dysregulated tau exon 10 with 3R-tau > 4R-tau.

  

  Resveratrol enhances Sirt1 expression and suppresses 4R-tau expression. (A, B) 5-month old Htau mice were treated with resveratrol for 7 months. The levels of 3R-tau, 4R-tau and total tau in the brains were analyzed by western blot developed with anti-Sirt1 (A) and anti-3R-tau and anti-4R-tau (B). The data are presented as mean ± S.D. (n=3-5) and analyzed by student t-test. **, p<0.01. (C) pCI/SI9-SI10 was transfected into HEK-293T cells and then the cells were treated with 0, 25, 50 and 100 μM of resveratrol for 48 h. Total RNA was extracted and alternative splicing of tau exon 10 was analyzed by RT-PCR. Ratio of exon 10 inclusion/exon 10 exclusion was plotted against the resveratrol concentration.

  
  
  

  

  Sirt1 inhibits 9G8-enhanced 3R-tau expression. (A) 9G8 or Sirt1 was overexpressed individually or together into HEK-293T cells transfected with mini-tau gene pCI/SI9-SI10 for 48 h. The splicing products of tau exon 10 were analyzed by RT-PCR. (B) Mini-tau gene was co-transfected into HEK-293T cells with siRNA of Sirt1 only or together with 9G8, and then splicing products were analyzed by RT-PCR 48 h after transfection. (C) pcDNA3.1/SIRT1 or pcDNA/3.1H363Y was transfected only or together with 9G8 into HEK-293T cells. Splicing products were analyzed by RT-PCR after 48 h transfection. The ratios of tau exon 10 inclusion to exclusion are presented as mean ± S.D. and analyzed with two-way ANOVA. *, p < 0.05, **, p < 0.01, ***, p < 0.001, compared with control group; ^## p < 0.01, compared with 9G8 group.

  
  
  

  

  Sirt1 interacts with 9G8 directly. (A) Myc-tagged SIRT1 and HA-tagged 9G8 were co-expressed in HEK-293T cells. After 48 h transfection, cell lysate was incubated with anti-HA antibody coupled onto protein G beads. The proteins bound on beads were analyzed by western blot developed with anti-HA and anti-Myc. (B) Sirt1 tagged with Myc and 9G8 tagged with HA were co-overexpressed in Hela cells. After 48 h transfection, the cells were fixed and immunostained with anti-HA or anti-Myc and followed by TRITC-anti-rabbit IgG or FITC-anti-mouse IgG. Hoechst was used for nuclear staining.

  
  
  

  

  Sirt1 deacetylates 9G8. (A) HA-9G8 was co-transfected with pcDNA3.1/SIRT1 or pcDNA3.1/H363Y or siRNA of Sirt1 into HEK-293T cells. The cell extract was incubated with anti-HA precoupled to protein G beads. The bound proteins were subjected to western blots using anti-HA or anti-acetylated-lysine. The data are presented as mean ± S.D. (n=3) and analyzed with one-way ANOVA. **, p<0.01, *, p<0.05. (B) Immunoprecipitated HA-9G8 as described in A was incubated with or without Sirt1 in the presence of NAD⁺ for 2 h at 37°C. The level of 9G8 acetylation was analyzed by western blot developed with anti-HA and anti-acetylated-lysine and is presented as mean ± S.D. (n=3) and analyzed with student t-test. **, p<0.01.

  
  
  

  

  Sirt1 deacetylates 9G8 at K24. (A) Mass spectra obtained from HA-9G8 immunoprecipitated from HEK-293T cells co-transfected with pcDNA3.1/SIRT1 or control DNA. (B) Sirt1 was expressed alone or co-overexpressed with 9G8_K24R in HEK-293T cells transfected with pCI/SI9-SI10. After 48 h transfection, the alternative splicing products of tau exon 10 were analyzed by RT-PCR. The data are presented as mean ± S.D. (n=3) and analyzed with two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with control group; ^## p < 0.01, compared with 9G8 group.

  
  
  

  

  Resveratrol rescues the impaired spatial memory of Htau mice. (A-E) 5-month old tau-KO and Htau mice were treated with resveratrol for 7 months and subjected to general behavior tests. For elevated plus maze test, the amount of time spent in the open arm and number of open arm (OA) entrances were analyzed (A and B). For open field test, time amount spent in center area and entered into the center area were analyzed (C and D). Locomotor activity by tested rotarod test and fall latency were recorded (E). (F-H) Htau and tau-KO mice were subjected to Morris water maze test. During the training phase, travel latency (F) and swimming speed (G) were recorded and analyzed. In the probe trial, the time spent in each quadrant was recorded (H). The data are presented as mean ± S.D. (n=9-11) and analyzed by two-way ANOVA followed by Bonferroni post-test (n=9-11). *, p<0.05.