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 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 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. Here, 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 humans. 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°C 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 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.

NMDA glutamate receptor antagonist MK‐801 induces proteome changes in adult human brain slices which are partially counteracted by haloperidol and clozapine

“…We demonstrate that human brain slices preserve the human CNS complexity in terms of cell types found in vivo, validating our previous observations (Almeida et al, 2021;Crunfli et al, 2022;Figueiredo et al, 2019;Mendes et al, 2018;Sebollela et al, 2012;Vasconcelos et al, 2021). After identifying the proteome changes induced by MK-801 treatment, we evaluated and compared the responses to either a first-generation or typical antipsychotic (haloperidol) or to a second-generation or atypical drug (clozapine).…”

“…In all donors, the tissue collected was mapped as excluded from the epileptogenic zone. Our group has previously employed this experimental model to model molecular events associated with Alzheimer's disease (Mendes et al., 2018; Sebollela et al., 2012; Vasconcelos et al., 2021) and viral neuroinfection (Almeida et al., 2021; Crunfli et al., 2022; Figueiredo et al., 2019). We demonstrate that human brain slices preserve the human CNS complexity in terms of cell types found in vivo, validating our previous observations (Almeida et al., 2021; Crunfli et al., 2022; Figueiredo et al., 2019; Mendes et al., 2018; Sebollela et al., 2012; Vasconcelos et al., 2021).…”

“…In all donors, the tissue collected was mapped as excluded from the epileptogenic zone. Our group has previously employed this experimental model to model molecular events associated with Alzheimer's disease (Mendes et al, 2018;Sebollela et al, 2012;Vasconcelos et al, 2021) and viral neuroinfection (Almeida et al, 2021;Crunfli et al, 2022;Figueiredo et al, 2019).…”

“…Our group has previously employed this experimental model to model molecular events associated with Alzheimer's disease (Mendes et al., 2018; Sebollela et al., 2012; Vasconcelos et al., 2021) and viral neuroinfection (Almeida et al., 2021; Crunfli et al., 2022; Figueiredo et al., 2019). We demonstrate that human brain slices preserve the human CNS complexity in terms of cell types found in vivo, validating our previous observations (Almeida et al., 2021; Crunfli et al., 2022; Figueiredo et al., 2019; Mendes et al., 2018; Sebollela et al., 2012; Vasconcelos et al., 2021). After identifying the proteome changes induced by MK‐801 treatment, we evaluated and compared the responses to either a first‐generation or typical antipsychotic (haloperidol) or to a second‐generation or atypical drug (clozapine).…”