Controlled Tau Cleavage in Cells Reveals Abnormal Localizations of Tau Fragments. (10th May 2023)

• Record Type: Journal Article
• Authors: Fourest-Lieuvin, Anne; Vinit, Angélique; Blot, Béatrice; Perrot, Anthime; Denarier, Eric; Saudou, Frédéric; Arnal, Isabelle
• Is Part Of: Neuroscience. Volume 518(2023)
• Page Start: 162
• Page End: 177
• Publication Date: 2023-05-10
• Subjects: Tau -- Protein cleavage -- TEV protease -- Microtubule -- Actin -- nucleus; AD Alzheimer's disease -- CTEV C-terminal half of the TEV protease -- CTRL control condition -- CUT cleavage condition -- FKBP FK506-binding protein -- FRB FKBP-rapamycin binding domain of mTOR kinase -- GFP or EGFP enhanced Green fluorescent Protein -- mCh mCherry fluorescent protein -- MEF Mouse Embryonic Fibroblast -- MTT Methylthiazolyldiphenyl-tetrazolium bromide -- NTEV N-terminal half of the TEV protease -- SDS-PAGE sodium dodecyl sulfate–polyacrylamide gel electrophoresis -- tau-TEV tau protein with TEV recognition motif -- TEV Tobacco Etch Virus; Neurochemistry -- Periodicals; Neurophysiology -- Periodicals; Neurology -- Periodicals; Neurochimie -- Périodiques; Neurophysiologie -- Périodiques; Neurochemistry; Neurophysiology; Electronic journals; Periodicals; Electronic journals; 612.8
• DOI: 10.1016/j.neuroscience.2022.08.016
• Languages: English
• ISSNs: 0306-4522

Abstract

Highlights: A TEV-based system was developed to control tau cleavage in cells. The simultaneous effects of the N- and C-terminal fragments can be examined. Tau fragments show aberrant localizations with respect to the cytoskeleton. N-terminal fragments partially relocate to the nucleus. Abstract: In several forms of dementia, such as Alzheimer's disease, the cytoskeleton-associated protein tau undergoes proteolysis, giving rise to fragments that have a toxic impact on neuronal homeostasis. How these fragments interact with cellular structures, in particular with the cytoskeleton, is currently incompletely understood. Here, we developed a method, derived from a Tobacco Etch Virus (TEV) protease system, to induce controlled cleavage of tau at specific sites. Five tau proteins containing specific TEV recognition sites corresponding to pathological proteolytic sites were engineered, and tagged with GFP at one end and mCherry at the other. After a controlled cleavage to produce GFP-N-terminal and C-terminal-mCherry fragments, we followed the fate of tau fragments in cells. Our results showed that whole engineered tau proteins associate with the cytoskeleton similarly to the non-modified tau, whereas tau fragments adopted different localizations with respect to the actin and microtubule cytoskeletons. These distinct localizations were confirmed by expressing each separate fragment in cells. Some cleavages – in particular cleavages at amino-acid positions 124 or 256 – displayed a certain level of cellular toxicity, with an unusual relocalization of the N-terminal fragments to the nucleus. Based on the data presented here, inducible cleavage of tau by the TEV protease appears to be a valuable tool to reproduce tau fragmentation in cells and study the resulting consequences on cell physiology.