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. 2019 May 1;319:60-68.
doi: 10.1016/j.jneumeth.2018.09.030. Epub 2018 Sep 29.

Human Neuroblastoma SH-SY5Y Cells Treated With Okadaic Acid Express Phosphorylated High Molecular Weight Tau-Immunoreactive Protein Species

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Free PMC article

Human Neuroblastoma SH-SY5Y Cells Treated With Okadaic Acid Express Phosphorylated High Molecular Weight Tau-Immunoreactive Protein Species

Mirta Boban et al. J Neurosci Methods. .
Free PMC article

Abstract

Background: Early stages of Alzheimer's disease (AD) are characterized by high phosphorylation of microtubule-associated protein tau, which may result from the downregulation of protein phosphatases.

New method: In order to model phosphatase downregulation and analyze its effect on tau aggregation in vitro, we treated neuroblastoma SH-SY5Y cells with okadaic acid (OA), a protein phosphatase inhibitor, and examined high molecular weight phospho-tau species.

Results and comparison with existing methods: OA treatment led to the appearance of heat-stable protein species with apparent molecular weight around 100 kDa, which were immunoreactive to anti-tau antibodies against phosphorylated Ser202 and Ser396. As these high molecular weight tau-immunoreactive proteins (HMW-TIPs) corresponded to the predicted size of two tau monomers, we considered the possibility that they represent phosphorylation-induced tau oligomers. We attempted to dissociate HMW-TIPs by urea and guanidine, as well as by alkaline phosphatase treatment, but HMW-TIPs were stable under all conditions tested. These characteristics resemble properties of certain sodium dodecyl sulfate (SDS)-resistant tau oligomers from AD brains. The absence of HMW-TIPs detection by anti-total tau antibodies Tau46, CP27 and Tau13 may be a consequence of epitope masking and protein truncation. Alternatively, HMW-TIPs may represent previously unreported phosphoproteins cross-reacting with tau.

Conclusions: Taken together, our data provide a novel characterization of an OA-based cell culture model in which OA induces the appearance of HMW-TIPs. These findings have implications for further studies of tau under the conditions of protein phosphatase downregulation, aiming to explain mechanisms involved in early events leading to AD.

Keywords: Alzheimer’s disease; Cell culture; Immunoblot; Neurodegeneration; Okadaic acid; Oligomerization; Phosphorylation; Protein phosphatase; SH-SY5Y; Tau protein.

Conflict of interest statement

Competing interests. All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare no financial relationships with any organizations that might have an interest in the submitted work in the previous three years. All authors declare no other relationships or activities that could appear to have influenced the submitted work.

Figures

Figure 1.
Figure 1.. OA treatment of SH-SY5Y cells induces expression of a high molecular weight protein species immunoreactive to phospho-tau.
Undifferentiated SH-SY5Y cells were treated with indicated concentrations of OA for an indicated time period. Around 25 μg protein in total cells lysates were analyzed by immunoblotting using antibodies that detect tau-pS396 or tau-pS202 (CP13). GAPDH served as a loading control (A-C). Note a strong band migrating around 100-kDa in lysates of OA-treated cells (A-C). (D) Viability of cells treated with 30 nM OA for 8 h (number of independent samples n = 8) and control cells (n = 4) was tested using MTT assay. Percent viability of control cells is shown. No significant difference in viability was found (t = 0.372, df = 10, p = 0.125). (E) Nitrocellulose membrane with transferred proteins was cut vertically in the middle of the lane (scissors) and the halves were incubated with CP13 or anti-tau-pS396 antibodies. Membranes were placed side-by-side and imaged simultaneously.
Figure 2.
Figure 2.. HMW-TIP is detected in OA-treated SH-SY5Y cells differentiated into neuron-like cells.
Differentiated SH-SY5Y cells visualized using phase contrast microscopy (A) were treated with 100 nM OA for two hours. Note the differentiated cells with visible neuritic processes. Scale bar = 50 μm. Total cell lysates were analyzed by immunoblot using anti-tau-pS202 (CP13), anti-tau-pS396 and anti-total tau (Tau46) antibodies (B). Note a strong band migrating around 100-kDa in lysates of OA-treated cells. GAPDH is used as a loading control.
Figure 3.
Figure 3.. HMW-TIPs are not detected by anti-total tau antibodies.
Where indicated, cells were treated with 100 nM (A, D) or 150 nM (B, C, E) OA for 2 – 2.5 hours. Total cell lysates were analyzed by immunoblotting using anti-total tau antibodies Tau46 (A), CP27 (B), Tau13 (C) and rabbit polyclonal antibody (D), and antibody DC11 against truncated tau (E). To visualize HMW-TIPs, same samples were analyzed by antibody CP13 (A-C) or anti-tau-pS396 (D, E). Note a CP13- and anti-tau-pS396- reactive band migrating around 100-kDa in lysates of OA-treated cells (A-E). Note that all anti-total antibodies detect a band migrating above 50 kDa that represents monomeric tau, but do not detect 100 kDa protein (A-D). Tau46 antibody additionally detects a 70 kDa band that presumably represents MAP2 isoform (A). GAPDH served as a loading control (C, D). Control and OA-treated samples in (D) were present on the same membrane. Note that the rabbit polyclonal anti-total antibody (right panel) detects a band migrating above 100 kDa tau (red arrow), which is clearly distinct from the OA-induced anti-tau-pS396-reactive band (left panel, OA+) migrating at 100 kDa (black arrow).
Figure 4.
Figure 4.. HMW-TIP is present in a heat-stable fraction.
SH-SY5Y cells were treated with 100 nM (OA+) or DMSO (OA-) for 2:15 h. (A) Heat-stable (HS) and pellet fractions were analyzed by immunoblot using anti-total tau antibody Tau46 (upper panels) and anti-β-actin (lower panels). Heat-stable fraction and pellet samples were analyzed on the same nitrocellulose membrane and imaged simultaneously. Note that 50 kDa tau and 70 kDa proteins (70 kDa protein presumably being a MAP2 isoform) detected by Tau46 antibody are enriched in the heat-stable fraction compared to the pellet. In contrast, β-actin is enriched in the pellet compared to heat-stable fraction. (B) The same samples of heat-stable (HS) fraction as in (A) were analyzed by immunoblot using anti-tau-pS396 and CP13 (anti-tau-pS202) antibodies. Note the presence of pS396- and CP13-immunoreactive 100 kDa bands in the heat stable fraction.
Figure 5.
Figure 5.. HMW-TIP is stable under denaturing conditions.
Cells were treated with 150 nM (A) or 100 nM (B) OA for 2 h. Cell lysates were prepared in the absence or presence of 8 M urea (A) or with 6 M guanidine-hydrochloride (B). Immunoblotting was performed using anti-tau-pS202 antibody (CP13). GAPDH was used as a loading control. Note that the 100 kDa band is present in samples from OA-treated cells under all tested conditions. The graph shows signal intensity of HMW-TIP relative to GAPDH levels of the samples shown in A. Average values and standard deviation of replicate samples are shown.
Figure 6.
Figure 6.. HMW-TIPs stability upon protein dephosphorylation.
Cells were incubated with 100 nM OA for 2 h. Heat-stable fraction was treated with alkaline phosphatase (AP) where indicated and analyzed by immunoblot using anti-tau-pS396 antibody. Note the persistent antibody detection of protein migrating around 100 kDa and changed electrophoretic mobility of proteins treated with alkaline phosphatase.

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