Characterization of a novel monoclonal antibody specific for a tau autoproteolytic cut site that differentiates between Alzheimer's disease and non-Alzheimer's specimens

Abstract

Tau is a microtubule binding protein that normally functions in axons to maintain microtubule structure but is hyperphosphorylated in AD facilitating its dissociation from microtubules and accumulation in the somatodendritic compartment where it can aggregate (Iqbal et al. 2010). Oligomeric forms of tau appear to be most closely associated with neuronal loss and memory impairment in mouse models of tauopathy (Berger et al. 2007; Yoshiyama et al. 2007). Tau oligomers were also found to accumulate in human AD specimens (Maeda et al. 2006; Patterson et al. 2011; Lasagna-Reeves et al. 2012). Importantly, extracellular tau oligomers have been shown to cause memory impairment and toxicity in mice inhibiting formation of long-term potentiation in hippocampal slices and formation of associative fear memory (Fá et al. 2010) and induce neurodegeneration by affecting mitochondrial and synaptic function (Lasagna-Reeves et al. 2012). However, the particular structure and mechanism by which tau oligomers exert their deleterious effects has not been determined. We have found that upon the formation of oligomeric structures tau acquires the additional function of a protease leading to self-cleavage and the ability to cut other proteins. Here, we present initial characterization of a novel monoclonal antibody specific for an end of a tau fragment generated by the autoproteolytic activity of tau oligomers. N-terminal sequencing of autoproteolytic fragments of tau oligomers was performed using mass spectrometry. A cut site was used to generate peptides to produce polyclonal antibodies specific to the cut ends. A rabbit was also chosen for the production of hybridomas to select a clone producing monoclonal antibodies specific for the cut end of a fragment that has tau oligomer protease activity. ELISAs and immunoblots were performed using peptides, recombinant tau and brain specimens for selection and characterization. A hybridoma line showing specificity was selected and monoclonal antibody was purified. Immunoblot analysis of AD and non-AD brain specimens using this antibody showed oligomeric and fragment bands that were specific to the AD specimens. This approach was successful in producing a monoclonal antibody that demonstrates the disease-relevance of a tau oligomer autoproteolytic cut site. This antibody may also have biomarker and immunotherapeutic potential.