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 neuronal, microtubule-associated protein responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation, but in some neurological diseases it becomes aberrantly hyperphosphorylated, thus contributing to the pathogenesis of several neurological diseases such as Alzheimer's disease. Western Blot (WB) has widely been employed in the assessment of Tau levels and fragmentation in neurological disease models. However, quantification of Tau levels/cleavage by WB should be interpreted with care, as this approach has been recognized as a complex, multi-step technique prone to produce artifactual results if not properly performed. Here, we evaluated the influence of freeze-and-thaw, a common storage procedure preceding WB, to the integrity of Tau in rat brain extracts. We observed that samples submitted to freeze-and-thaw presented a ~25 kDa fragment shown to be derived from Tau. Based on this observation, we strongly recommend that molecular analysis of Tau levels in rodent brain samples in future investigations be performed using fresh extracts. Finally, we discuss the impact of our findings to the translational power of studies involving Tau-induced neurodegeneration.