Figure 1.
Phosphorylation of tau in NGF-exposed PC12 cells.
PC12 cells exposed to NGF for the indicated time points followed by EGTA or P9115 were analyzed for tau phosphorylation by Western blot analysis. (A) Western blots. (B) Relative amount. The relative amount of phosphorylated tau at each site at the indicated time points was determined by normalizing the band intensity of phosphorylated tau with the respective tau band. The relative amount of total tau was determined by normalizing the tau band to the respective actin band. (C) Effects of EGTA and P9115. Band intensity values of tau or phosphorylated tau at indicated sites in cells exposed to NGF for 6 days and treated with EGTA (panel A, lane 7) or P9115 (panel A, lane 8) were normalized as in panel B and are expressed as the % of vehicle-treated control (panel A, lane 6). Values in panels B and C with standard error are the average of three determinations from three cultures. *p < 0.005 with respect to vehicle-treated cells.
Figure 2.
14-3-3ζ promotes PKA-catalyzed Ser262 tau phosphorylation in PC12 and HEK-293 cells.
(A) NGF does not activate PKA in PC12 cells. PC12 cells were treated with forskolin, NGF, or vehicle for 24 hr. Treated cells were lysed and each lysate was assayed for PKA or Cdk5 activity using the respective peptide substrate. Values are the average of three determinations from three cultures, and are expressed as the fold change from vehicle-treated control cells. *p<0.005 with respect to vehicle treated cells. (B) Disruption of 14-3-3ζ function inhibits tau phosphorylation at Ser262 in NGF-exposed PC12 cells. PC12 cells transfected with Myc-14-3-3ζ (K49N) or empty vector were exposed to NGF for 24 hr and then analyzed by Western blotting as in Figure 1. Values with standard error are the average of three determinations from three cultures. **p < 0.001 with respect to vector transfected and NGF-treated cells. (C) 14-3-3ζ promotes PKA-catalyzed tau Ser262 phosphorylation in HEK293 cells. HEK293 cells co-transfected with Flag-tau and Myc-14-3-3ζ were analyzed by Western blotting. The relative amount of Ser262 phosphorylated tau was determined by normalizing the Ser262 band in each lane to the respective Flag-tau band. Values with standard error are the average of three determinations from three cultures. *p < 0.005 with respect to cells transfected with Flag-tau and Myc-PKAc.
Figure 3.
Overexpression of 14-3-3ζ promotes tau phosphorylation at Ser262 in rat hippocampal primary neurons in culture.
Rat hippocampal primary neurons in culture infected with Ln-14-3-3ζ or Ln-vector were analyzed for tau phosphorylation by Western blotting as in Figure 1. (A) Western blots, (B) Relative amounts. Values with standard error are the average of three determinations from three cultures. *p < 0.001 with respect to Ln-vector infected neurons.
Figure 4.
Overexpression of 14-3-3ζ inhibits tau binding to microtubules and reduces the amount of polymerized microtubules in rat hippocampal primary neurons in culture.
Primary neurons infected with Ln-14-3-3ζ or Ln-vector were subjected to a microtubule sedimentation assay. The resulting microtubule pellet (P) and the supernatant (S) were analyzed by Western blotting and the relative distribution of each protein in its respective fraction was determined. The relative distribution value was determined by dividing the band intensity of a protein in the fraction by the total (sum of the intensity value of that protein, both S and P fractions), and is expressed as a % of the total. Values with S.E. are an average of three determinations from three cultures. *p< 0.05 with respect to the P fraction of the Ln-vector control. (B) Relative amount. The relative amounts of polymerized microtubules are relative distribution values from the microtubule pellet in panel (A) and are expressed as the % of Ln-vector control. Likewise, the relative amounts of microtubule-bound tau are the values of total tau in microtubule pellet in panel (A), and are expressed as a % of Ln-vector control. The relative amount of Ser262 phosphorylated tau was determined by normalizing the Ser262 blot by the corresponding tau blot, as in Figure 1. Values are average of three determinations from three cultures. *p<0.05 with respect to the Ln-vector control.
Figure 5.
Overexpression of 14-3-3ζ in primary neurons in culture causes microtubule instability.
Neurons infected with Ln-14-3-3ζ or Ln-vector were analyzed for microtubule instability by Western blotting or immunocytochemistry. (A) Western blot analysis. Western blot analysis for Ac-tubulin (stable microtubules), Tyr-tubulin (unstable microtubules) or β-tubulin (total tubulin) was performed. The Ac-tubulin or Tyr-tubulin band of each sample was normalized against the respective total tubulin band to determine the corresponding relative amount. To determine the relative amount of total tubulin, the tubulin band was normalized against the respective actin band. Values with standard error are the average of three determinations from three cultures. *p< 0.05 with respect to Ln-vector infected controls. (B) Immunocytochemistry. Representative immunofluorescence micrographs of infected neurons immunostained with anti-β-tubulin (total tubulin), anti-Myc (Myc-14-3-3ζ), or anti-Tyr-tubulin. Scale bar. 25 μm.
Figure 6.
Overexpression of 14-3-3ζ downregulates synaptophysin protein level in rat hippocampal primary neurons in culture
– Neurons infected with Ln-14-3-3ζ or Ln-vector were analyzed for synaptophysin protein levels by immunocytochemistry or Western blotting. (A) Immunocytochemistry. Representative immunofluorescent micrographs are of neurons infected with the indicated virus and stained for Myc (14-3-3ζ), synaptophysin, DAPI (nucleus), and merge (co-localization). The corresponding inset is shown in higher magnification in the lower panel. Quantification of synaptophysin puncta from 50 neurites in each group from three different cultures is shown on the right hand side panel. Scale bars: upper 15 μm; lower, 5 μm. *p<0.005 with respect to Ln-vector infected neurons. (B) Western blot analysis. Representative Western blots from extracts of neurons infected with Ln-14-3-3ζ or Ln-vector showing the level of indicated synaptic proteins in each culture. The relative amount of each protein was determined from the blots by normalizing the band intensity of that protein against the respective actin band. Values with standard error are an average of three determinations from three cultures. *p< 0.05 with respect to Ln-vector infected cells.
Figure 7.
Overexpression of 14-3-3ζ in rat primary hippocampal neurons promotes proteosomal degradation of synaptophysin protein
(A) 14-3-3ζ overexpression does not affect synaptophysin protein synthesis in neurons. Ln-14-3-3ζ or Ln-vector infected neurons were treated with cycloheximide or vehicle for the indicated time and analyzed for synaptophysin protein by Western blotting. Synaptophysin band intensity in each lane was normalized against the corresponding actin band and is expressed as the % of 0 hr. Values with ± SE are the average of three determinations. *p<0.005 with respect to Ln-vector infected neurons. (B) Proteosome inhibitors block the downregulation of synaptophysin protein caused by 14-3-3ζ overexpression. Primary neurons infected with Ln-14-3-3ζ or Ln-vector were treated with MG132 or lactacystin for 24 hr. Treated neurons were analyzed by Western blotting and the relative amounts were determined as per Figure 6. Values with standard error are the average of three determinations from three cultures. *p< 0.005 with respect to Ln-14-3-3ζ infected and vehicle treated neurons.
Figure 8.
Microtubule stabilizing drug taxol restores synaptophysin protein levels in 14-3-3ζ overexpressing neurons.
Neurons infected with Ln-14-3-3ζ or Ln-vector were treated with the microtubule stabilizing drug taxol for 24 hr and then analyzed by Western blotting to determine the relative amounts, and then subjected to a microtubule sedimentation assay. (A) Western blot analysis. The relative amount of each protein shown in the lower panel was determined as per Figure 5. Data with standard error are the average of three determinations from three cultures. *p < 00.1 with respect to 14-3-3ζ infected and vehicle treated neurons. (B) Microtubule sedimentation assay. The microtubule sedimentation assay was performed as in Figure 4. The resulting microtubule pellet (P) and the supernatant (S) were analyzed by Western blotting and the relative distribution and relative amounts were determined as in Figure 4. (a) Western blots. (b), relative distribution. Values with S.E. are average of three determinations from three cultures. *p< 0.05 with respect to the P fraction of Ln-vector control. (c) Relative amounts. The relative amount of polymerized microtubules are relative distribution values from the microtubule pellet in panel (b) and are expressed as the % of Ln-vector control. Likewise, the relative amounts of microtubule-bound tau are the values of total tau in the microtubule pellet in panel (b) and are expressed as a % of Ln-vector control. The relative amount of Ser262 phosphorylated tau was determined by normalizing Ser262 blot by corresponding tau blot as in Figure 4. Values are an average of three determinations from three cultures. *p<0.05 with respect to Ln-vector control.