Overexpression of 14-3-3ζ Promotes Tau Phosphorylation at Ser²⁶² and Accelerates Proteosomal Degradation of Synaptophysin in Rat Primary Hippocampal Neurons

<div><p>β-amyloid peptide accumulation, tau hyperphosphorylation, and synapse loss are characteristic neuropathological symptoms of Alzheimer’s disease (AD). Tau hyperphosphorylation is suggested to inhibit the association of tau with microtubules, making microtubules unstable and causing neurodegeneration. The mechanism of tau phosphorylation in AD brain, therefore, is of considerable significance. Although PHF-tau is phosphorylated at over 40 Ser/Thr sites, Ser²⁶² phosphorylation was shown to mediate β-amyloid neurotoxicity and formation of toxic tau lesions in the brain. <i>In vitro</i>, PKA is one of the kinases that phosphorylates tau at Ser²⁶², but the mechanism by which it phosphorylates tau in AD brain is not very clear. 14-3-3ζ is associated with neurofibrillary tangles and is upregulated in AD brain. In this study, we show that 14-3-3ζ promotes tau phosphorylation at Ser²⁶² by PKA in differentiating neurons. When overexpressed in rat hippocampal primary neurons, 14-3-3ζ causes an increase in Ser²⁶² phosphorylation, a decrease in the amount of microtubule-bound tau, a reduction in the amount of polymerized microtubules, as well as microtubule instability. More importantly, the level of pre-synaptic protein synaptophysin was significantly reduced. Downregulation of synaptophysin in 14-3-3ζ overexpressing neurons was mitigated by inhibiting the proteosome, indicating that 14-3-3ζ promotes proteosomal degradation of synaptophysin. When 14-3-3ζ overexpressing neurons were treated with the microtubule stabilizing drug taxol, tau Ser²⁶² phosphorylation decreased and synaptophysin level was restored. Our data demonstrate that overexpression of 14-3-3ζ accelerates proteosomal turnover of synaptophysin by promoting the destabilization of microtubules. Synaptophysin is involved in synapse formation and neurotransmitter release. Our results suggest that 14-3-3ζ may cause synaptic pathology by reducing synaptophysin levels in the brains of patients suffering from AD. </p> </div

14-3-3ζ promotes PKA-catalyzed Ser²⁶² tau phosphorylation in PC12 and HEK-293 cells.

<p>(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. *<i>p</i><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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084615#pone-0084615-g001" target="_blank">Figure 1</a>. Values with standard error are the average of three determinations from three cultures. **<i>p</i> < 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. *<i>p</i> < 0.005 with respect to cells transfected with Flag-tau and Myc-PKAc. </p

Phosphorylation of tau in NGF-exposed PC12 cells.

<p>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. *<i>p</i> < 0.005 with respect to vehicle-treated cells.</p

Overexpression of 14-3-3ζ in primary neurons in culture causes microtubule instability.

<p>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. *<i>p</i>< 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.</p

Overexpression of 14-3-3ζ promotes tau phosphorylation at Ser²⁶² in rat hippocampal primary neurons in culture.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084615#pone-0084615-g001" target="_blank">Figure 1</a>. (A) Western blots, (B) Relative amounts. Values with standard error are the average of three determinations from three cultures. *<i>p</i> < 0.001 with respect to Ln-vector infected neurons.</p

Overexpression of 14-3-3ζ in rat primary hippocampal neurons promotes proteosomal degradation of synaptophysin protein

<p>(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. *<i>p</i><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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084615#pone-0084615-g006" target="_blank">Figure 6</a>. Values with standard error are the average of three determinations from three cultures. *<i>p</i>< 0.005 with respect to Ln-14-3-3ζ infected and vehicle treated neurons. </p

Microtubule stabilizing drug taxol restores synaptophysin protein levels in 14-3-3ζ overexpressing neurons.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084615#pone-0084615-g005" target="_blank">Figure 5</a>. Data with standard error are the average of three determinations from three cultures. *<i>p</i> < 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084615#pone-0084615-g004" target="_blank">Figure 4</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084615#pone-0084615-g004" target="_blank">Figure 4</a>. (a) Western blots. (b), relative distribution. Values with S.E. are average of three determinations from three cultures. *<i>p</i>< 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084615#pone-0084615-g004" target="_blank">Figure 4</a>. Values are an average of three determinations from three cultures. *<i>p</i><0.05 with respect to Ln-vector control. </p

Interaction of 14-3-3ζ with Microtubule-Associated Protein Tau within Alzheimer’s Disease Neurofibrillary Tangles

Alzheimer’s disease (AD) is characterized by the presence of abnormal, straight filaments and paired helical filaments (PHFs) that are coated with amorphous aggregates. When PHFs are treated with alkali, they untwist and form filaments with a ribbonlike morphology. Tau protein is the major component of all of these ultrastructures. 14-3-3ζ is present in NFTs and is significantly upregulated in AD brain. The molecular basis of the association of 14-3-3ζ within NFTs and the pathological significance of its association are not known. In this study, we have found that 14-3-3ζ is copurified and co-immunoprecipitates with tau from NFTs of AD brain extract. <i>In vitro</i>, tau binds to both phosphorylated and nonphosphorylated tau. When incubated with 14-3-3ζ, tau forms amorphous aggregates, single-stranded, straight filaments, ribbonlike filaments, and PHF-like filaments, all of which resemble the corresponding ultrastructures found in AD brain. Immuno-electron microscopy determined that both tau and 14-3-3ζ are present in these ultrastructures and that they are formed in an incubation time-dependent manner. Amorphous aggregates are formed first. As the incubation time increases, the size of amorphous aggregates increases and they are incorporated into single-stranded filaments. Single-stranded filaments laterally associate to form double-stranded, ribbonlike, and PHF-like filaments. Both tau and phosphorylated tau aggregate in a similar manner when they are incubated with 14-3-3ζ. Our data suggest that 14-3-3ζ has a role in the fibrillization of tau in AD brain, and that tau phosphorylation does not affect 14-3-3ζ-induced tau aggregation