Tau cleavage by calpain: The intermediate at Mr 17kD / MW 11kD is metastable but not toxic

Abstract

The amyloid cascade hypothesis of AD posits that Aß triggers the neurofibrillary pathology of Tau. It was proposed that a toxic “17 kD” fragment of Tau, generated by the protease calpain, was induced by exposure of neurons to Aß. Its size (residues 45-230, MW=18.7kD) was estimated on the basis of potential cleavage sites in Tau by calpain (Park & Ferreira, J. Neurosci. 2005). In the current study, we sought to identify the exact nature of the “17 kD” fragment of Tau generated by calpain and its mode of toxicity. The limited proteolysis of Tau by calpain in vitro or in cultured neurons caused by treatment with Aß oligomers was analysed by western blotting. Calpain-induced fragments of Tau were identified by protein sequencing, mass spectrometry, and antibody labelling. Cytotoxicity of Tau fragments was monitored by LDH release assay or apoptotic assay. The presence of fragments in Alzheimer or control brains was assessed by immunoblotting. The “Mr=17 kD” fragment is actually much smaller than assumed so far, containing only residues 125-230 (MW=10.7kD). On SDS gels it runs at Mr∼17kD because of the anomalous behavior of Tau. Inducing Tau hyperphosphorylation by okadaic acid or mimicking phosphorylation by multiple Glu mutations did not prevent the generation of this fragment. The fragment was induced not only by Aß oligomers, but also by other cell stressors, e.g. thapsigargin or glutamate in cortical neurons. However, overexpression of neither Tau45-230 nor Tau125-230 fragment was toxic to neurons. Finally, the calpain-induced fragment was observed both in AD brains and in control normal human brains. The Mr=17 kD Tau fragment is a metastable cleavage product of calpain and comprises residues 125-230. The fragment is induced by cell stress, including exposure to Aß, but it is not a mediator of Aß-induced toxicity. This suggests that events upstream of calpain activation might cause both Tau fragmentation and toxicity. - Supported by: EU/Memosad, BMBF/KNDD, Breuer Fund, Metlife Fund.