14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

<div><p>Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer’s disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated <i>in vitro</i> with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect <i>in vitro</i> tau aggregation (Qureshi <i>et al</i> (2013) Biochemistry 52, 6445–6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. <i>In vitro</i>, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ.</p></div

Microtubule disruption promotes 14-3-3ζ-induced tau aggregation in M17 human neuroblastoma cells–M17 human neuroblastoma cells transfected with Flag-tau with or without Myc-14-3-3ζ were treated with indicated microtubule destabilizing drug and then analyzed for tau phosphorylation, microtubule stability and tau aggregation as in Fig 6.

<p><i>A</i>, Western blot analysis for tau phosphorylation and microtubule stability. The relative amounts of phosphorylated tau at each site and Ac-Tub were determined as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g004" target="_blank">4<i>A</i></a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g006" target="_blank">6</a>. #<i>p</i>< 0.005 against cells transfected with Flag-tau only. <i>B</i>, Tau aggregation. Centrifugation assay for tau aggregation of cells treated with the microtubule destabilizing drug colchicine. <i>C</i>, Centrifugation assay of cells treated with the microtubule destabilizing drug nocodazole. Relative distribution of Flag-tau in various fractions was calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g004" target="_blank">Fig 4<i>B</i></a>. Values in the bar graph are the average ± S.E. of three experiments. *<i>p</i>> 0.04 against pellet of cells transfected with Flag-tau and Myc-14-3-3ζ and treated with vehicle; **<i>p</i>< 0.05 against the pellet of cells transfected with Flag-tau and treated with vehicle; ##<i>p</i> < 0.01 against the pellet of cells transfected with Flag-tau and treated with colchicine or nocodazole.</p

Phosphorylation promotes 14-3-3ζ-induced tau aggregation in M17 human neuroblastoma cells–M17 human neuroblastoma cells co-transfected with Flag-tau, Myc-14-3-3ζ, HA-GSK3β and Myc-PKA in various combinations were analyzed by Western blotting for tau phosphorylation and then by centrifugation assay for tau aggregation.

<p><i>A</i>, Western blot analysis. PHF-1 and pS214 blots represent tau phosphorylated at Ser^396/404 and Ser²¹⁴, respectively. The ser^396/404 site is phosphorylated by GSK3β whereas the ser²¹⁴ site is phosphorylated by PKA. Based on tau band intensities the relative amount of phosphorylated tau was determined. The relative amount of phosphorylated tau was determined by normalizing the phosphorylated tau band intensity by respective band intensity of total tau. Values with ±SE are the average of three determinations. *<i>P</i>< 0.05 with respect to control cells expressing tau alone. <i>B</i>, Centrifugation assay. The centrifugation assay was performed as described in the Materials and Methods. S and P indicate supernatant and pellet, respectively. Based on Flag-tau blot band intensity, the relative distribution of tau and the relative amount of aggregated tau in the indicated fractions were determined as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g002" target="_blank">Fig 2<i>A</i></a>. The relative amount of aggregated tau is expressed as fold of cells expressing tau and 14-3-3ζ. Values in the bar graph are mean ± S.E. and are from three independent experiments. *<i>P</i> < 0.05 with respect to cells expressing tau and 14-3-3ζ.</p

Co-expression of 14-3-3ζ and tau in human neuroblastoma cells causes formation of cytoplasmic thioflavin S positive inclusions.

<p>Cells transfected with either Flag-tau or Flag-tau and Myc-14-3-3ζ were fixed and then stained with thioflavin S (green) followed by either anti-Flag for tau (red) or anti-Myc for 14-3-3ζ (red). <i>A</i>, cells transfected with tau alone stained for tau (red), Thioflavin S (green) and DAPI (nucleus). <i>B</i> and <i>C</i>, cells co-transfected with Flag-tau and Myc-14-3-3ζ. Scale bars 40 μM (<i>A</i>); 100 μM (<i>B</i> and <i>C</i>).</p

Immuno EM of tau aggregates formed in M17 human neuroblastoma cells expressing Flag-tau, Myc-14-3-3ζ, HA-GSK3β and Myc-PKA in various combination.

<p>Pellets obtained by centrifugation assay from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g004" target="_blank">Fig 4<i>B</i></a> were analyzed by Immuno-EM as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g003" target="_blank">Fig 3<i>E</i></a>. Scale bar 100 nm.</p

Inverse correlation between microtubule stability and 14-3-3ζ-induced tau aggregation in M17 human neuroblastoma cells.

<p>Samples from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g004" target="_blank">Fig 4<i>A</i></a> were analyzed by Western blot analysis for microtubules. The relative amount of tubulin was calculated from normalizing the tubulin band of each sample with the corresponding actin band of that sample. Likewise, the relative amounts of Ac-Tub (Ac-tubulin) and Tyr-Tub (Tyr-tubulin) were calculated by normalizing Ac-Tub band or Tyr-Tub band with corresponding β-tubulin band. The relative amount of aggregated tau is from panel <i>B</i> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g004" target="_blank">Fig 4</a>. Values in the bar graph are the average ± S.E. of three independent experiments. *<i>p</i>< 0.05 against the cells transfected with Flag-tau alone. <i>F</i>, Correlation. Data for microtubule stability and aggregated tau are from panel <i>C</i> and <i>E</i>, respectively. Plot was generated by using values from lanes 1, 2, 4 and 6 only.</p

Tau and 14-3-3ζ forms a high molecular weight complex when co-expressed in M17 human neuroblastoma cells.

<p>Cells transfected with either Flag-tau or Myc-14-3-3ζ or co-transfected with Flag-tau and Myc-14-3-3ζ were subjected to OmniPrep density gradient centrifugation. Fractions were collected and analyzed by Western blotting and immunoprecipitation. <i>A</i>, Western blot of fractions corresponding to cells transfected with Flag-tau alone. <i>B</i>, Western blot of fractions corresponding to cells transfected with Myc-14-3-3ζ alone. <i>C</i>, Western blot of samples corresponding to cells co-transfected with Flag-tau and Myc-14-3-3ζ. Based on blot band intensities, the relative amount of Peak 1 (sum of fractions 1–4) and peak 2 (sum of fractions 5–8) was calculated and is expressed as the % of total (sum of fractions 1–8). <i>D</i>, Immunoprecipitation. Immunoprecipitation was carried out using fraction # 6 from panel <i>C</i>.</p

Microtubules protect tau from 14-3-3ζ-induced aggregation <i>in vitro</i>–Microtubules sedimentation assay was performed as described in Materials and Methods.

<p><i>A</i>, Western blots of microtubule sedimentation assay performed in the presence of GTP/Mg²⁺/taxol followed by the addition of GST. <i>B</i>, Western blots of microtubules sedimentation assay in the presence of GTP/ Mg²⁺/taxol followed by the addition of 14-3-3ζ. <i>C</i>, Immuno EM of P2 from microtubule sedimentation assay in the presence of GTP/ Mg²⁺/taxol followed by the addition of 14-3-3ζ. <i>D</i>, Western blots of microtubule sedimentation assay in the absence of GTP/ Mg²⁺/taxol followed by the addition of GST. <i>E</i>, Western blots of microtubule sedimentation assay in the absence of GTP/ Mg²⁺/taxol followed by the addition of 14-3-3ζ. <i>F</i>, Immuno EM of P2 from microtubule sedimentation assay in the absence of GTP/ Mg²⁺/taxol followed by the addition of 14-3-3ζ. Scale bars 100 nm.</p

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

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