P3‐007: Development of tau proteolytic activity as a target for drug discovery

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). Neurofibrillary tangles, primarily composed of tau protein are a pathological hallmark in Alzheimer's disease (AD). However, the 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 brain 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 to induce neurodegeneration by affecting mitochondrial and synaptic function (Lasagna-Reeves et al. 2012). Here, we describe tau protease activity, screening of inhibitors and the development of HTS assays for drug discovery targeting tau protease. Tau oligomers prepared from recombinant human 4R/2N tau protein were highly purified and used for protease assays. Reverse-phase HPLC was used to assay tau protease activity on peptides from tau and other proteins. An Envision plate reader was used for a peptide-based FRET assay of protease inhibitors. The class of tau protease was determined using protease inhibitors. Autoproteolytic cut sites were characterized by mass spectrometry. Tubulin and peptides from APP and α-endorphin were shown to be cleaved by tau protease. Tau constructs with amino acid substitutions have been produced and are being assayed to determine the active site amino acids. Tau protease activity is dependent on oligomerization causing self-fragmentation, as well as cleavage of other proteins that may be indicative of a direct pathological of tau in AD. The disease relevance of this activity is being studied as this may represent an important mechanism in the development of pathology and a druggable target for the development of therapeutics and biomarkers.