A freeze-and-thaw-induced fragment of the microtubule-associated protein tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity

Abstract

Tau is a microtubule-associated protein (MAP) responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation. However, in some neurological diseases Tau becomes aberrantly hyperphosphorylated, which contributes to the pathogenesis of neurological diseases, known as tauopathies. Western blotting (WB) has been widely employed to determine Tau levels in neurological disease models. However, Tau quantification by WB should be interpreted with care, as this approach has been recognized as prone to produce artifactual results if not properly performed. In the present study, our goal was to evaluate the influence of a freeze-and-thaw cycle, a common procedure preceding WB, to the integrity of Tau in brain homogenates from rats, 3xTg-AD mice and human samples. Homogenates were prepared in ice-cold RIPA buffer supplemented with protease/phosphatase inhibitors. Immediately after centrifugation, an aliquot of the extracts was analyzed via WB to quantify total and phosphorylated Tau levels. The remaining aliquots of the same extracts were stored for at least 2 weeks at either -20 or -80°C and then subjected to WB. Extracts from rodent brains submitted to freeze-and-thaw presented a ∼25 kDa fragment immunoreactive to anti-Tau antibodies. An in-gel digestion followed by mass spectrometry (MS) analysis in excised bands revealed this ∼25 kDa species corresponds to a Tau fragment. Freeze-and-thaw-induced Tau proteolysis was detected even when extracts were stored at -80°C. This phenomenon was not observed in human samples at any storage condition tested. Based on these findings, we strongly recommend the use of fresh extracts of brain samples in molecular analysis of Tau levels in rodents.

Keywords: Alzheimer's disease; disease models; neurodegeneration; tau protein; tauopathy; western blot.

Figure 1. Presence of a ∼25 kDa anti-Tau immunoreactive band in freeze-and-thawed brain extracts of 12-month-old Wistar and WAR rats

Extracts were analyzed by WB after a freeze-and-thaw cycle at −20°C (−20°C F/T; A,B) or immediately after preparation (fresh; C,D). Membranes were probed with either anti-Tau phospho S396 (pTau; A–D,F) or anti-Tau [E178] (Total Tau; A–D) antibodies. Extracts were prepared from dorsal hippocampus (A,C) or frontal cortex (B,D) of Wistar (WT) and WAR rats, as discriminated above each lane. (E) pTau/25kDa band ratio. A representative WB comparing extracts from frontal cortex stored at different temperatures is shown (n=3 per group; *P<0.05). Some extracts were additionally probed for β-actin (F). The arrow-head highlights the ∼25 kDa regions on the membranes.

Figure 2. Localization of Tau-derived peptides identified by MS in full-length Tau sequence

All the peptides identified in the MS fragment analysis are shown in gray (a unique peptide is highlighted in orange). All the tryptic peptides are located after the Ser³⁸⁸ (human analog of human Ser³⁹⁶) and near to the C-terminus of full-length Tau from rat. The total Tau antibody binding site, indicated in the C-terminus, refers to the region where the proprietary sequence is located.

Figure 3. Evaluation of freeze-and-thaw-induced Tau degradation in extracts from experimental models of neurodegeneration

(A) Representative WB images of 3xTg mice frontal cortex extracts probed for pTau and β-actin. Extracts were either analyzed fresh or after storage at −20°C F/T. The arrow-head indicates the presence of a band near 25 kDa in both fresh and −20°C F/T. (B) Full length/fragment ratio obtained from the quantification of 3xTg frontal cortex WB analysis (n=6 for each group; t test, P<0.05). (C) WB analysis using human brain slices extracts obtained from two donors.

Figure 4. Sequence comparison of Tau residues around phospho Ser³⁹⁶ epitope in rodents and humans

Comparison between Tau sequences from rat, mouse and human around the serine-phosphorylation site (gray arrow) probed by the anti-Tau phospho S396 antibody (Ser³⁹⁶ analog in rat is Ser³⁸⁸; in mouse is S³⁶⁹). Considering the differences in reactivity and biochemical nature of side chains, the positions where significant substitutions are present comparing rodent versus human sequences are indicated by yellow arrows. The default color scheme of AliView was used in the alignment.

Figure 5. Freeze-and-thaw-induced Tau fragmentation in rat brain extracts is diminished when extracts are frozen in SDS/PAGE loading buffer

Representative Western blot of fresh and freeze/thawed extracts from frontal cortex of Wistar (WT) rats probed for pTau. Where indicated, extracts were diluted in SDS/PAGE loading buffer and boiled prior to freezing. Membranes were additionally probed for β-actin.