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The Loss of C-Jun N-Terminal Protein Kinase Activity Prevents the Amyloidogenic Cleavage of Amyloid Precursor Protein and the Formation of Amyloid Plaques in Vivo

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The Loss of C-Jun N-Terminal Protein Kinase Activity Prevents the Amyloidogenic Cleavage of Amyloid Precursor Protein and the Formation of Amyloid Plaques in Vivo University of Massachusetts Medical School eScholarship@UMMS Davis Lab Publications Program in Molecular Medicine 2011-11-23 The loss of c-Jun N-terminal protein kinase activity prevents the amyloidogenic cleavage of amyloid precursor protein and the formation of amyloid plaques in vivo Sonia Mazzitelli University of Manchester Et al. Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/davis Part of the Biochemistry Commons, Cell Biology Commons, Cellular and Molecular Physiology Commons, and the Molecular Biology Commons Repository Citation Mazzitelli S, Xu P, Ferrer I, Davis RJ, Tournier C. (2011). The loss of c-Jun N-terminal protein kinase activity prevents the amyloidogenic cleavage of amyloid precursor protein and the formation of amyloid plaques in vivo. Davis Lab Publications. https://doi.org/10.1523/JNEUROSCI.4491-11.2011. Retrieved from https://escholarship.umassmed.edu/davis/40 Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in Davis Lab Publications by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. The Journal of Neuroscience, November 23, 2011 • 31(47):16969–16976 • 16969 Cellular/Molecular The Loss of c-Jun N-Terminal Protein Kinase Activity Prevents the Amyloidogenic Cleavage of Amyloid Precursor Protein and the Formation of Amyloid Plaques In Vivo Sonia Mazzitelli,1 Ping Xu,2 Isidre Ferrer,3 Roger J. Davis,2 and Cathy Tournier1 1Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom, 2Howard Hughes Medical Institute and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, and 3Institut de Neuropatologia, Servei Anatomia Patolo`gica, Instituto de Investigacio´n Biome´dica de Bellvitge–Hospital Universitari de Bellvitge, 08907 Hospitalet de Llobregat, Spain Phosphorylation plays a central role in the dynamic regulation of the processing of the amyloid precursor protein (APP) and the production of amyloid-␤ (A␤), one of the clinically most important factors that determine the onset of Alzheimer’s disease (AD). This has led to the hypothesis that aberrant A␤ production associated with AD results from regulatory defects in signal transduction. However, conflicting findings have raised a debate over the identity of the signaling pathway that controls APP metabolism. Here, we demonstrate that activation of the c-Jun N-terminal protein kinase (JNK) is essential for mediating the apoptotic response of neurons to A␤. Further- more, we discovered that the functional loss of JNK signaling in neurons significantly decreased the number of amyloid plaques present in the brain of mice carrying familial AD-linked mutant genes. This correlated with a reduction in A␤ production. Biochemical analyses indicate that the phosphorylation of APP at threonine 668 by JNK is required for ␥-mediated cleavage of the C-terminal fragment of APP producedby␤-secretase.Overall,thisstudyprovidesgeneticevidencethatJNKsignalingisrequiredfortheformationofamyloidplaques in vivo. Therefore, inhibition of increased JNK activity associated with aging or with a pathological condition constitutes a potential strategy for the treatment of AD. Introduction Activation of the proapoptotic c-Jun N-terminal protein ki- Alzheimer’s disease (AD) is a neurodegenerative disorder char- nase (JNK) signaling pathway constitutes one possible mecha- acterized by the presence of extracellular amyloid plaques in the nism by which ROS produced by the accumulation of A␤ impairs brain formed by the progressive deposition of amyloid-␤ (A␤). synaptic function and leads to neuronal loss. This is mostly dem- A␤ is a 4 kDa peptide that derives from the sequential cleavage of onstrated by the observation that A␤-induced neuronal death is the amyloid precursor protein (APP) by ␤- and ␥-secretases significantly reduced in cortical neurons lacking JNK expression (Mattson, 2004). Mutations in genes associated with familial (Morishima et al., 2001). JNK is a member of the mitogen- forms of AD (FAD) result in elevated production of A␤ that activated protein kinase (MAPK) family that phosphorylates and precedes disease pathology (Hardy, 1997; Bertram et al., 2010). activates transcription factors of the AP-1 (activator protein-1) These findings have led to the idea that the accumulation of A␤ is family, including c-Jun and ATF2, in response to various stresses a primary cause of AD. A␤ neurotoxicity is mediated, at least in (Davis, 2000). Analogous to other MAPKs, JNK is activated by part, by enhancing the levels of reactive oxygen species (ROS) phosphorylation at threonine (Thr) and tyrosine (Tyr) residues (Behl et al., 1994). Furthermore, a positive-feedback loop by by two dual-specificity MAPK kinases (MKK4 and MKK7) which oxidative stress produced following the interaction of A␤ (Wang et al., 2007a). with the receptor for advanced glycation endproducts (RAGE) Consistent with a role of JNK signaling in mediating A␤ tox- increases RAGE expression, thereby enhancing A␤ neurotoxicity, icity (Morishima et al., 2001), recent studies have demonstrated has been suggested (Yan et al., 1996). the neuroprotective effect of inhibiting JNK activity in rodent AD models (Braithwaite et al., 2010; Ramin et al., 2011). Further- more, JNK-mediated APP phosphorylation at Thr (T) 668 has Received Sept. 2, 2011; accepted Sept. 30, 2011. been proposed as a mechanism to prevent APP degradation and Author contributions: S.M. and C.T. designed research; S.M. and I.F. performed research; P.X. and R.J.D. contrib- increase A␤ production in a neuroglioma cell line (Colombo et uted unpublished reagents/analytic tools; I.F. and C.T. analyzed data; C.T. wrote the paper. This work was supported by a scholarship from the Medical Research Council (S.M.) and by a grant from the al., 2009). The importance of this finding is highlighted by evi- Alzheimer’sResearchUnitedKingdom(C.T.).WethankA.Whitmarsh(UniversityofManchester,Manchester,UK)for dence that T668 phosphorylation is increased in human AD brain critically reviewing this work, and the technicians at the animal facility for looking after the mice. (Lee et al., 2003). However, the demonstration that the cyclin- The authors declare no competing financial interests. dependent kinase 5 (Cdk5) is responsible for phosphorylating Correspondence should be addressed to Cathy Tournier, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK. E-mail: [email protected]. APP at T668 in naturally degenerating CAD cells, a CNS-derived DOI:10.1523/JNEUROSCI.4491-11.2011 neuronal cell line used for studying neuronal cell biology and Copyright © 2011 the authors 0270-6474/11/3116969-08$15.00/0 pathology, has challenged the idea that JNK is implicated in con- 16970 • J. Neurosci., November 23, 2011 • 31(47):16969–16976 Mazzitelli et al. • Role of JNK Signaling in Alzheimer’s Disease trolling APP metabolism under pathologi- cal conditions (Muresan and Muresan, 2007). In contrast, this study provided ev- idence that JNK phosphorylates APP to control its transport in distal neurites un- der normal conditions. Conflicting findings regarding the role of JNK signaling in regulating APP pro- cessing may be attributed to differences in experimental conditions and emphasize the importance of using animal models to identify physiologically relevant regula- tory mechanisms. Therefore, we decided to test the effect of the loss of JNK activ- ity in the brain of mice that overexpress FAD-linked mutant genes to elucidate the requirement of JNK signaling in AD pathology. Materials and Methods Mice. The mouse strains were maintained in a pathogen-free facility at the University of Manchester. All animal procedures were per- formed under license in accordance with the United Kingdom Home Office Animals (Sci- entific Procedures) Act (1986) and institu- tional guidelines. Genotype determination of mice and tissues. Offspring carrying flox alleles, creER T2, APP, Figure 1. The loss of MKK4 and MKK7 protects neurons against A␤42-induced apoptosis. Cortical neurons displaying homozy- T2 and PS1 transgenes were identified by PCR on gous fl mutation for the mkk4 and/or mkk7 alleles and expressing (ϩ) or not (Ϫ) CreER were incubated for 72 h with 0.1 ␮M tail and brain tissue DNA, as previously de- 4-OHT before being treated with A␤42 (200 nM) for the indicated times. Where indicated, the cells were pretreated with zVAD (10 scribed (Jankowsky et al., 2004; Wang et al., ␮M). A, Protein lysates were analyzed by immunoblot with antibodies against MKK4, MKK7, and JNK. B, E, Immunofluorescence 2007b). was performed with a specific antibody to MAP2. The immune complex was detected with a secondary antibody conjugated to Tissue culture. Primary cultures of cortical fluorescein (green). DNA was stained with DAPI (blue). Scale bar, 10 ␮m. C, Caspase 3 activity was measured by caspase assay. D, neurons were prepared from the cerebral cor- Cell survival was measured by MTT assay. The data correspond to the mean Ϯ SE (N ϭ 3). tices of 17-d-old embryos (E17) and cultured on poly-ornithine-precoated six-well plates in S-transferase (GST)-c-Jun (Wang et al., 2007b). The radioactivity incorpo- Neurobasal media containing B27 supplement, 1% penicillin/strepto- rated into the recombinant protein
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