Table 1.
Phosphatase, kinase inhibitor and other pharmacological agents used in the study.
Table 2.
Antibodies used in this study.
Fig 1.
OA induced tau hyperphosphorylation and oligomerization at different time points in mouse neuroblastoma N2a cells.
(a). Immunoblots of N2a cells extracted protein (20 μg) using total and phospho-tau antibodies: DA9 (a.a. 102–140), CP13 (pSer202), and PHF-1 (pSer396/pSer404). ⍺II-Spectrin antibody was used to assess neuronal apoptotic pathway activation through monitoring intact spectrin (240 kDa), SBDP150/145 (calpain activation), and SBDP120 (caspase-3 activation). Different tau species are pointed with colored arrows. Blue arrows present monomeric p-tau (48 kDa), and oligomeric p-tau (110 kDa, 170 kDa, and 240 kDa). Red arrows on PHF-1 points on two minor bands of oligomeric p-tau (220 kDa and 260 kDa). Black arrows show non-phospho tau band (46 kDa). SNJ-1945 (abbreviated as S; a calpain inhibitor, 100 μM) and Z-DCB (abbreviated as Z; a caspase-3 inhibitor, 60 μM) were added for all experimental conditions for 1h before the treatment with OA (100 nM) for 6h or 24h, to prevent apoptosis-mediated proteolysis of tau and ⍺II-Spectrin. A reverse-time course followed OA treatment, and all cells were collected at the same time and conditions. (b). Immunoblots quantification. All data are normalized to β-actin and are expressed as a percentage of control. Data are presented as ± SEM for n = 3. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by Bonferroni’s post-hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: non-significant.
Fig 2.
Screening of protein kinase inhibitors on OA-induced Tau hyperphosphorylation and oligomerization in N2a cells.
(a). Immunoblots of N2a cells extracted protein (20 μg) using phospho-tau antibodies (CP13, PHF-1), total tau (DA9), and αII-Spectrin. αII-Spectrin was probed to assess neuronal cell injury monitored with SBDP145/150 and SBDP120. Kinase inhibitors effect on OA-induced tau bands (100 nM) was monitored by evaluating the levels of monomeric (48 kDa) and oligomeric p-tau immunoreactivity (110 kDa, 170 kDa, and 240 kDa; blue arrows), total tau, and non-phospho tau (46 kDa; black arrows). Phosphorylated tau break-down products are shown with PHF-1 immunoblot. For all experimental conditions, S (a calpain inhibitor) and Z (a caspase-3 inhibitor) were added for 1h to before the addition of OA for 24h followed by 6h incubation with the kinase inhibitors. The concentrations used for each protein kinase inhibitor are mentioned in materials and methods, cell treatment section. β-actin was probed as a loading control. All experimental conditions were collected and analyzed at the same time. (b). Immunoblots quantification. All data are normalized to β-actin and are expressed as a percentage of control. Data are presented as ± SEM for n = 3. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by Bonferroni’s post-hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: non-significant.
Fig 3.
Dose-response of TBB on OA-induced tau hyperphosphorylation and oligomerization in N2a cells.
N2a cells were pre-treated with OA for 24h followed by treatment with different concentrations of TBB for 6h, as indicated in the Figure. (a) Immunoblots of cell extracted proteins using phospho-tau antibodies, including CP13 (pSer202), and total tau DA9 (a.a. 102–140). Blue arrows represent monomeric and oligomeric p-tau (48 kDa, 110 kDa, and 170 kDa). αII-Spectrin antibody used to monitor SBDPs with the increasing concentrations of TBB. The β-actin antibody was used as a loading control. All conditions included SNJ-1945 (calpain inhibitor) and Z-DCB (caspase inhibitor). (b) TBB dose-response treatment line chart. TBB concentration (in micromolar) is shown on the X-axis, and the inhibition percentage is presented on the Y-axis. The control sample values were designated as the standard response. The X-axis concentration values are logarithm-transformed to fit a straight line. The half-maximal inhibitory concentration (IC50) was used to measure the effectiveness of TBB in inhibiting OA-induced tau hyperphosphorylation and oligomerization. GraphPad Prism was used to calculate the IC50 (for DA9 and CP13 antibodies) and are presented on the figure. The statistical analysis was performed with one-way ANOVA, followed by Bonferroni's post-hoc test. *p<0.05, **p<0.01, ***p<0.001. Data are presented as ± SEM for n = 3.
Table 3.
Composite effects of kinase inhibitors on OA-induced tau hyperphosphorylation in N2a cells.
Fig 4.
Screening of protein kinase inhibitors on physiologically phosphorylated tau in rat primary cerebrocortical neuronal culture.
Rat primary cerebrocortical neuronal differentiated cultures (CTX) at 15 DIVs, were treated various protein kinases inhibitors, including K252a (30 μM), STS (20 μM), LiCl (10 μM), EGTA (5 mM), Roscovitine (60 μM), Saracatinib (100 μM), TBB (30 μM) and A-107 (20 μM), AR (60 μM) for 6h. Calpain and caspase inhibitors (S+Z) were added to all experimental conditions for 1h before the protein kinase inhibitor treatments. Cell lysates were analyzed on western blots using twenty micrograms of protein. (a) Immunoblots of cell lysates analyzed for phosphorylated tau at the epitopes CP13 (pSer202), PHF-1 (pSer396/404), AT8 (pSer202/pThr205), RZ3 (pThr231), and AT270 (pThr181). Total tau was probed with DA31 (a.a. 150–190) antibody. DA31 blot showed two distinctive tau bands (63 kDa, non-phospho tau, and 67 kDa, p-tau) following kinase inhibitors treatment. SBDP145/150 and SBDP120 were analyzed with the αII-spectrin antibody. Different lanes are numbered at the top of each label in the figure. (b) Immunoblot quantification of basal tau phosphorylation. Ratios of phospho-epitope levels over β-actin ± SD are represented as a percentage. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by Bonferroni’s post-hoc test was performed. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. n = 3 per condition.
Fig 5.
Effect of protein kinase inhibitors on OA-induced tau hyperphosphorylation in rat primary cerebrocortical neuronal culture.
Rat primary cerebrocortical neuronal differentiated cultures (CTX) at 15 DIV were treated with OA (100 nM) for 24h followed by protein kinases inhibitors for 6h. The concentrations of kinase inhibitors are the same as the ones mentioned in Fig 4. CTX cultures were treated with S and Z for 1h before any treatment to prevent apoptotic pathway-mediated tau proteolysis. (a). Immunoblots of cell lysates analyzed for phosphorylated tau at the epitopes CP13, PHF-1, AT8, RZ3, AT270. Total tau was probed with the DA31 antibody. With DA31 blot, the 63 kDa band is referred to as monomeric non-phospho tau and the 67 kDa as monomeric p-tau species. Spectrin Break down products (SBDPs) were monitored with the αII-spectrin antibody. (b) Immunoblot quantification of OA-induced tau phosphorylation. Ratios of phospho-epitope levels over β-actin ± SD are represented as a percentage. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by Bonferroni’s post hoc test was performed. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. n = 3 per condition.
Table 4.
Composite effects of kinase inhibitors on basal and OA-induced tau hyperphosphorylation in rat primary cerebrocortical neuronal cells.
Fig 6.
The tauopathy-model and a proposed mechanism for various protein kinase inhibitors intervention.
Dephosphorylated tau protein binds the microtubules to maintain it in the polymerized state. Phosphorylation of tau protein by a host of different kinases causes tau to dissociate from the microtubules. Dissociation of tau causes the microtubules to depolymerize. Specific phosphatases dephosphorylate tau allowing the microtubule to re-polymerize again, a physiological process that provides structure and shape to the cytoskeleton of neurons. In tauopathies, imbalances between kinases and phosphatases functions lead to tau hyperphosphorylation at particular pathological sites and a higher tendency to dissociate from the microtubules producing soluble tau aggregates and insoluble paired helical filaments (PHF), that could combine to form neurofibrillary tangles (NFT). NFT is known to be the toxic species in AD and CTE, including other tauopathy diseases, and little is known about their active mechanism of neurodegeneration. OA inhibits the function of crucial tau phosphatases (PP1 and PP2A), leading to activation of tau kinases and tau hyperphosphorylation initiating the pathological processes of tauopathies. One pharmaceutical approach to reverse the mechanism of tauopathies is kinase inhibition. The protein kinase inhibitors selected in this study are indicated in this figure. The inhibitors highlighted in blue are ones that showed a promising effect on our OA-induced cell-based tauopathy model. Microsoft PowerPoint was used to create the artwork.