Active c-jun N-terminal kinase induces caspase cleavage of tau and additional phosphorylation by GSK-3b is required for tau aggregation Naruhiko Sahara, 1 Miyuki Murayama, 1 Boyoung Lee, 1 Jung-Mi Park, 1 Sarita Lagalwar, 2 Lester I. Binder 2 and Akihiko Takashima 1 1 Laboratory for Alzheimer’s Disease, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, Japan 2 Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA Keywords: caspase cleavage, caspase-3 activity, c-jun N-terminal kinase, human tau, tau phosphorylation Abstract Neurofibrillary tangles (NFTs), comprising human intracellular microtubule-associated protein tau, are one of the hallmarks of tauopathies, including Alzheimer’s disease. Recently, a report that caspase-cleaved tau is present in NFTs has led to the hypothesis that the mechanisms underlying NFT formation may involve the apoptosis cascade. Here, we show that adenoviral infection of tau into COS-7 cells induces activation of c-jun N-terminal kinase (JNK), followed by excessive phosphorylation of tau and its cleavage by caspase. However, JNK activation alone was insufficient to induce sodium dodecyl sulfate (SDS)-insoluble tau aggregation and additional phosphorylation by GSK-3b was required. In SH-SY5Y neuroblastoma cells, overexpression of active JNK and GSK-3b increased caspase-3 activation and cytotoxicity more than overexpression of tau alone. Taken together, these results indicate that, although JNK activation may be a primary inducing factor, further phosphorylation of tau is required for neuronal death and NFT formation in neurodegenerative diseases, including those characterized by tauopathy. Introduction Neurofibrillary tangles (NFTs) and neuronal loss are commonly observed in neurological disorders, including Alzheimer’s disease and other tauopathies (Lee et al., 2001; Swaab et al., 2002; Binder et al., 2005). Brain regions showing dysfunction overlap those displaying NFTs and neuronal loss (Gomez-Isla et al., 1997), suggesting that mechanisms of NFT formation and neuronal loss may underlie the neuronal dysfunction of affected brain areas. Hence, understanding the mechanisms underlying NFT formation and neuronal death may point to a promising therapy for tauopathies. In frontotemporal dementia with Parkinsonism linked to chromo- some 17 (FTDP-17), tau gene mutations induce NFT formation and neuronal loss (Goedert & Spillantini, 2000; Hutton, 2000; Spillantini et al., 2000; Reed et al., 2001), suggesting that dysregulation of tau may be a cause of NFT formation and neuronal death. This notion is supported by some reports showing that overexpression of FTDP-17 mutant tau induces NFT formation, neuronal loss and behavioral abnormalities. In a mouse model overexpressing P301L mutant tau under the regulation of tetracycline, the inhibition of mutant tau overexpression in the disease state blocked neuronal death but still induced NFT formation (Santacruz et al., 2005), suggesting that NFTs themselves are not toxic but the mechanism of neuronal death may underlie NFT formation. The NFTs are composed of microtubule-associated protein tau. The tau in NFTs is modified by phosphorylation and ubiquitination and forms fibrils, so-called paired helical filaments, and straight filaments. However, it is still unclear which processes activate the mechanism underlying neuronal death. One possibility is that tau may form a toxic aggregate that precedes NFT formation. Alternatively, tau overaccu- mulation may indirectly activate a mechanism of neuronal death. The presence of truncated tau in paired helical filaments was first reported by Wischik et al. (1988). More recently, caspase-cleaved tau was identified in Alzheimer’s disease brain (Gamblin et al., 2003; Rissman et al., 2004; Guillozet-Bongaarts et al., 2005; Newman et al., 2005). One consequence of this observation is that the NFT formation process might activate the apoptotic cascade. Active caspase could cleave tau at Asp421, as well as other caspase substrates, leading to neuronal apoptosis. Furthermore, cleaved tau shows a preference for forming tau aggregates in vitro (Berry et al., 2003), suggesting that cleaved tau may act as ‘seeds’ for tau fibrils, accelerating NFT formation. Taken together, these findings indicate that the processes underlying NFT formation and neuronal death may diverge after tau abnormalities are induced and caspase is activated. As NFTs consist of hyperphosphorylated tau, one might presume that caspase activation and tau phosphorylation occur simultaneously in neurons before reaching a point at which the cascades directing NFT formation and neuronal death diverge, ultimately leading to tauopathy. To understand the divergent mechanisms underlying NFT formation and neuronal death, we used an adenovirus-mediated gene expression system and investigated how tau overexpression affects the generation of caspase-cleaved tau and how it affects kinase activity. We found Correspondence: Dr Akihiko Takashima, as above. E-mail: kenneth@brain.riken.jp Received 6 January 2008, revised 28 March 2008, accepted 9 April 2008 European Journal of Neuroscience, Vol. 27, pp. 2897–2906, 2008 doi:10.1111/j.1460-9568.2008.06258.x ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience