Tau proteolysis mechanisms and relevance for tauopathies

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Abstract

Neurodegenerative diseases with tau inclusions in the brain are classified as tauopathies; 28 of these exist and typically present with dementia or parkinsonism symptoms. During tauopathy pathogenesis, proteases cleave the microtubule-associated protein tau into fragments which can have neurotoxic properties and an increased propensity to be phosphorylated, secreted, to aggregate and propagate aggregation. However, many of these fragments related to tauopathy pathogenesis have been poorly characterised and the protease responsible for their generation has yet to be identified. One such tau fragment without a protease identified to be responsible for its generation, was found to be specific to the brains of patients with tauopathies, the tau35 fragment. Tau35 induces progressive cognitive and motor deficits when expressed in a mouse. In this thesis, tau35-like C-terminal tau fragments and novel N-terminal tau fragments were identified in different fractions generated from corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) tauopathy patient brains that were not identified in age-matched controls. The cleavage sites at P179-T180, K240-S241 and L243-Q244 of tau1-441 that may cause the production of tau fragments between 33-37kDa were identified in an extract of a PSP patient brain. The cleavage site within tau to produce the tau35 fragment was also analysed using bioinformatics and shown to potentially be cleaved at R194-S195 by granzyme A. Intraneuronal granzyme A expression is quantified, however, no differences in the percentage of granzyme A-positive neurons were seen between CBD and PSP patients and age-matched controls. Using recombinant and cell-based assays, granzyme A was shown to cleave tau1-441 at R194-S195, R209-S210 and potentially K240-S241. Granzyme A-cleaved tau fragments corresponding to cleavage of tau1-441 at R194-S195 and R209-S210 were generated and expressed in cells and differences were seen in their phosphorylation status, subcellular localisation, propensity to aggregate and propagate tau aggregation but not in their mechanism of secretion. In collaboration with the Stoller Biomarker Discovery Centre at the University of Manchester, we examined whether Sequential Window Acquisition of all Theoretical fragment ions-Mass Spectrometry (SWATH-MS) could be used to detect tau peptides in Alzheimers disease (AD) patients and age-matched control plasma samples. Tau peptides were identified that matched those previously detected by other groups in the cerebrospinal fluid of AD patients and age-matched control samples. However, the presence of tau peptides in the plasma was too low to allow discrimination between AD patients and age-matched controls; further work is ongoing to improve the tau peptide coverage detected by this technique. Data generated in this thesis highlight that granzyme A cleaves tau to potentially generate tauopathy-specific tau fragments, including tau35. Overall, this work unravels mechanisms in which tau fragmentation alters the function of tau, generates new therapeutic targets for tauopathies and highlights potential novel biomarkers of tauopathies.

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Keyword(s)

Biomarker; Dementia; Granzyme A; Neurodegeneration; Proteolysis; Proteolytic cleavage; Tau; Tauopathies

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