The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.
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.
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.
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