Accumulation of phosphorylated tau (ptau) protein is a characteristic of tauopathies and many other neurodegenerative diseases. Hyperphosphorylated tau was shown to dissociate from microtubuli, resulting in the breakdown of the axonal flow, and thus impairing neuronal viability and function. Since tau presents a promising drug target, an inducible model of tau phosphorylation provides a quick tool for the analysis of drug effectiveness.
Adult male wild type mice were anesthetized with vaporized isoflurane and either placed on a heating pad and covered to keep body temperature stable or at room temperature to induce hypothermia. Animals were kept under anesthesia for 60 minutes. Animals of the normothermia group showed only a minor reduction of the body temperature while animals of the hypothermia group presented a significantly reduced body temperature starting 10 minutes after anesthesia induction (Figure 1).
Figure 1: Body temperature before and after induction of hypothermia in wild type mice. Animals were anesthetized and kept normothermal or hypothermal for 60 minutes. Body temperature was measured every 10 minutes. Two-way ANOVA with Bonferroni’s post hoc test. n=8 per group. Mean ± SEM. ***p<0.001.
After 60 minutes under anesthesia, all animals were sacrificed, and cortical and hippocampal tissue of all mice was collected and evaluated for total tau by automated western blotting (WES™) and ptau Ser202/Thr205 levels by western blotting. Analysis of the cortex and hippocampus showed unaltered total tau levels in the cortex and hippocampus (Figure 2A, D) but significantly increased ptau Ser202/Thr205 levels of the 3R tau isoform in the cortex (Figure 2B) and the 4R and 3R tau isoform in the hippocampus (Figure 2E, F).
Figure 2: Total tau and ptau Ser202/Thr205 levels in the cortex and hippocampus of wild type mice 1 hour after induction of hypothermia. Cortical (A-C) and hippocampal (D-F) tissue was evaluated for total tau by WES (A, D) and ptau Ser202/Thr205 by western blotting using the AT8 antibody (B, C, E, F). ptau Ser202/Thr205 levels of two tau isoforms, 3R (B, E) and 4R (C, F) were quantified. Unpaired t-test. n=8 per group, Mean + SEM. **p<0.01, ***p<0.001. G: Representative image of western blot by AT8 antibody of cortical tissue; the upper band represents the 4R tau isoform while the lower band represents the 3R tau isoform.
Evaluation of ptau at residue Ser396 by WES™ resulted in significantly increased ptau Ser396 levels in the hippocampus (Figure 3B) but not the cortex (Figure 3A).
Further evaluation of ptau at residue Thr217 by western blotting resulted in significantly increased ptau Thr217 levels of the 4R tau isoform in the hippocampus (Figure 3F) while no changes could be observed for both isoforms in the cortex (Figure 3B, C) and the 3R tau isoform in the hippocampus (Figure 3E).
Figure 3: ptau Ser396 and ptau Thr217 levels in the cortex and hippocampus of wild type mice 1 hour after induction of hypothermia. Cortical (A-C) and hippocampal (D-F) tissue was evaluated for ptau Ser396 (A, D) and ptau Thr217 (B, C, E, F) by automated western blotting (WES™). ptau Thr217 levels of two tau isoforms, 3R (B, E) and 4R (C, F) were quantified. Unpaired t-test. n=3 per group, Mean + SEM. *p<0.05; **p<0.01.
The in vivo hypothermia model allows a rapid evaluation of tau phosphorylation at several residues and across various brain regions. The 3R and 4R tau isoforms can even be quantified separately, no matter if evaluated by standard western blotting or automated western blotting WES™.
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