Tauopathy-Associated Tau Fragment Ending at Amino Acid 224 Is Generated by Calpain-2 Cleavage

Article type: Research Article

Authors: Cicognola, Claudia^{a; *} | Satir, Tugce Munise^b | Brinkmalm, Gunnar^a | Matečko-Burmann, Irena^c | Agholme, Lotta^b | Bergström, Petra^b | Becker, Bruno^{a; d} | Zetterberg, Henrik^{a; b; d; e; f} | Blennow, Kaj^{a; d; 1} | Höglund, Kina^{a; d; 1}

Affiliations: [a] Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Mölndal, Sweden | [b] Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden | [c] Department of Psychiatry and Neurochemistry, Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden | [d] Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden | [e] Department of Neurodegenerative Disease, UCL Institute of Neurology Queen Square, London, UK | [f] UK Dementia Research Institute at UCL, London, UK

Correspondence: [*] Correspondence to: Claudia Cicognola, Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, Göteborgsvägen 31, House V3/SU, 43180, Mölndal, Sweden. E-mail: claudia.cicognola@neuro.gu.se.

Note: [1] These authors share senior authorship.

Abstract: Background:Tau aggregation in neurons and glial cells characterizes tauopathies as Alzheimer’s disease (AD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Tau proteolysis has been proposed as a trigger for tau aggregation and tau fragments have been observed in brain and cerebrospinal fluid (CSF). Our group identified a major tau cleavage at amino acid (aa) 224 in CSF; N-terminal tau fragments ending at aa 224 (N-224) were significantly increased in AD and lacked correlation to total tau (t-tau) and phosphorylated tau (p-tau) in PSP and CBD. Objective:Previous studies have shown cleavage from calpain proteases at sites adjacent to aa 224. Our aim was to investigate if calpain-1 or -2 could be responsible for cleavage at aa 224. Methods:Proteolytic activity of calpain-1, calpain-2, and brain protein extract was assessed on a custom tau peptide (aa 220–228), engineered with fluorescence resonance energy transfer (FRET) technology. Findings were confirmed with in-gel trypsination and mass spectrometry (MS) analysis of brain-derived bands with proteolytic activity on the FRET substrate. Finally, knock-down of the calpain-2 catalytic subunit gene (CAPN2) was performed in a neuroblastoma cell line (SH-SY5Y). Results:Calpain-2 and brain protein extract, but not calpain-1, showed proteolytic activity on the FRET substrate. MS analysis of active gel bands revealed presence of calpain-2 subunits, but not calpain-1. Calpain-2 depletion and chemical inhibition suppressed proteolysis of the FRET substrate. CAPN2 knock-down caused a 76.4% reduction of N-224 tau in the cell-conditioned media. Conclusions:Further investigation of the calpain-2 pathway in the pathogenesis of tauopathies is encouraged.

Keywords: Alzheimer’s disease, calpain, fragments, tau, tauopathy

DOI: 10.3233/JAD-191130

Journal: Journal of Alzheimer's Disease, vol. 74, no. 4, pp. 1143-1156, 2020