This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Cellular/Molecular The Ste20 family kinases MAP4K4, MINK1 and TNIK, converge to regulate stress induced JNK signaling in neurons Martin Larhammar1,3, Sarah Huntwork-Rodriguez1,3, York Rudhard2, Arundhati Sengupta-Ghosh1 and Joseph W. Lewcock1,3 1Department of Neuroscience, Genentech, Inc., 1 DNA Way South San Francisco, California 94080, USA 2In Vitro Pharmacology, Evotec AG, Manfred Eigen Campus, 22419 Hamburg, Germany 3Current Address: Denali Therapeutics Inc., 151 Oyster Point Blvd, South San Francisco, CA 94080, USA, DOI: 10.1523/JNEUROSCI.0905-17.2017 Received: 3 April 2017 Revised: 13 September 2017 Accepted: 2 October 2017 Published: 9 October 2017 Author contributions: M.L., S.H.-R., and J.W.L. designed research; M.L., S.H.-R., A.S.-G., and Y.R. performed research; M.L., S.H.-R., A.S.-G., Y.R., and J.W.L. analyzed data; M.L., S.H.-R., and J.W.L. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. We thank Jeffrey Eastham-Anderson, Hai Ngu, and Oded Foreman for their assistance with image quantification, Weilan Ye and Philip Vitorino for providing Map4k4 conditional knockout mice and helpful discussions, and Chudi Ndubaku for providing MAP4K4 inhibitors and associated information for their use. Corresponding author: Joseph W. Lewcock, PhD, Denali Therapeutics Inc., 151 Oyster Point Blvd, South San Francisco, California 94080 USA, Phone: 650-745-5247, [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.0905-17.2017 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2017 the authors 1 The Ste20 family kinases MAP4K4, MINK1 and TNIK, converge to 2 regulate stress induced JNK signaling in neurons 3 4 Abbreviated title: MAP4K4, TNIK and MINK1, regulate JNK-signaling 5 6 Martin Larhammar1,3,*, Sarah Huntwork-Rodriguez1,3,*, York Rudhard2, Arundhati 7 Sengupta-Ghosh1, and Joseph W. Lewcock1,3,§ 8 9 1 Department of Neuroscience, Genentech, Inc., 1 DNA Way South San Francisco, 10 California 94080, USA, 2 In Vitro Pharmacology, Evotec AG, Manfred Eigen Campus, 11 22419 Hamburg, Germany, 3 Current Address: Denali Therapeutics Inc., 151 Oyster 12 Point Blvd, South San Francisco, CA 94080, USA, *Authors contributed equally to this 13 work. 14 15 § Corresponding author: 16 Joseph W. Lewcock, PhD 17 Denali Therapeutics Inc. 18 151 Oyster Point Blvd 19 South San Francisco, California 94080 USA 20 Phone: 650-745-5247 21 [email protected] 22 23 24 Number of pages: 40 25 Figures: 6 26 Number of words; Abstract: 169, Significance Statement: 96, Introduction: 493, Results: 27 2535 and Discussion: 757 28 29 CONFLICT OF INTEREST 30 M.L., S.H-R., A.S-G., and J.W.L. are current or former employees of Genentech, Inc. 31 Y.R. declares no competing financial interests. 32 33 ACKNOWLEDGMENTS 34 We thank Jeffrey Eastham-Anderson, Hai Ngu, and Oded Foreman for their assistance 35 with image quantification, Weilan Ye and Philip Vitorino for providing Map4k4 36 conditional knockout mice and helpful discussions, and Chudi Ndubaku for providing 37 MAP4K4 inhibitors and associated information for their use. 38 2 39 ABSTRACT 40 The c-Jun-N-terminal Kinase (JNK) signaling pathway regulates nervous system 41 development, axon regeneration, and neuronal degeneration following acute injury or in 42 chronic neurodegenerative disease. Dual Leucine Zipper Kinase (DLK) is required for 43 stress-induced JNK signaling in neurons, yet the factors that initiate DLK/JNK pathway 44 activity remain poorly defined. In the present study, we identify the Ste20 kinases 45 MAP4K4, TNIK, and MINK1 as upstream regulators of DLK/JNK signaling in neurons. 46 Using a trophic factor withdrawal-based model of neurodegeneration in both male and 47 female embryonic mouse dorsal root ganglion neurons, we show that MAP4K4, TNIK, 48 and MINK1 act redundantly to regulate DLK activation and downstream JNK dependent 49 phosphorylation of c-Jun in response to stress. Targeting MAP4K4, TNIK, and MINK1, 50 but not any of these kinases individually, is sufficient to potently protect neurons from 51 degeneration. Pharmacological inhibition of MAP4Ks blocks stabilization and 52 phosphorylation of DLK within in axons and subsequent retrograde translocation of the 53 JNK signaling complex to the nucleus. Together, these results position MAP4Ks as 54 important regulators of the DLK/JNK signaling pathway. 55 56 SIGNIFICANCE STATEMENT 57 Neuronal degeneration occurs in disparate circumstances: during development to refine 58 neuronal connections, following injury to clear damaged neurons, or pathologically 59 during disease. The DLK/JNK pathway represents a conserved regulator of neuronal 60 injury signaling that drives both neurodegeneration and axon regeneration, yet little is 61 known about the factors that initiate DLK activity. Here we uncover a novel role for a 3 62 subfamily of MAP4 kinases, consisting of MAP4K4, TNIK, and MINK1 in regulating 63 DLK/JNK signaling in neurons. Inhibition of these MAP4Ks blocks stress-induced 64 retrograde JNK-signaling and protects from neurodegeneration, suggesting that these 65 kinases may represent attractive therapeutic targets. 66 4 67 INTRODUCTION 68 Post-mitotic mouse neurons express all three of the JNK genes present in vertebrates 69 (JNK1-3, MAPK8-10), which have diverse roles that range from regulation of axonal 70 growth and synaptic stability to directing apoptosis following insult. JNK1 displays high 71 activity under physiological conditions, while JNK2 and JNK3 have low basal levels of 72 activity that are induced by cellular stress (Coffey et al., 2002). Consistent with this, 73 genetic deletion of JNK1 does not affect neuronal injury signaling, whereas animals 74 lacking expression of JNK2 and/or JNK3 display an attenuated response to a broad range 75 of insults (Hunot et al., 2004; Ries et al., 2008; Fernandes et al., 2012; Genabai et al., 76 2015). In the peripheral nervous system, JNK2/3-dependent phosphorylation of the 77 transcription factor c-Jun is required for axon regeneration following injury (Barnat et al., 78 2010; Nix et al., 2011). Conversely, the same signaling events result in axon degeneration 79 and apoptosis during neuronal development, following acute injury in the CNS, and in 80 models of chronic neurodegenerative disease. These findings have generated significant 81 interest in the identification of therapeutic approaches to either directly or indirectly 82 modulate JNK activity. 83 Stress-induced JNK signaling in neurons requires the upstream mixed lineage 84 kinase DLK (Dual Leucine Zipper Kinase, MAP3K12) (Hirai et al., 2005; Miller et al., 85 2009; Ghosh et al., 2011; Pozniak et al., 2013). Following neuronal injury, DLK 86 activation induces retrograde propagation of downstream stress signals, including p-JNK, 87 to the nucleus resulting in c-Jun phosphorylation (Cavalli et al., 2005; Lindwall and 88 Kanje, 2005; Ghosh et al., 2011; Fernandes et al., 2012; Shin et al., 2012; Huntwork- 89 Rodriguez et al., 2013; Simon et al., 2016; Larhammar et al., 2017). This translocation 5 90 event requires the dynein-associated scaffolding protein JIP3, suggesting that 91 translocation of p-JNK occurs via dynein-dependent retrograde transport (Cavalli et al., 92 2005). Genetic deletion or pharmacological inhibition of DLK is sufficient to broadly 93 abrogate stress-induced gene expression changes and results in potent protection from 94 neurodegeneration in many experimental settings and inhibition of axon regeneration in 95 others (Miller et al., 2009; Ghosh et al., 2011; Shin et al., 2012; Watkins et al., 2013; 96 Larhammar et al., 2017; Le Pichon et al., 2017). 97 Recent work has shed light on the mechanisms of DLK/JNK pathway activation 98 (Collins et al., 2006; Huntwork-Rodriguez et al., 2013; Valakh et al., 2013; Wu et al., 99 2015; Simon et al., 2016), but the factors directly upstream of DLK remain poorly 100 defined. Previous studies have demonstrated that certain MAP4 kinases are capable of 101 activating JNK in heterologous systems, with some data to suggest MAP4K4 may also 102 modulate JNK signaling in motor neurons (Machida et al., 2004; Yang et al., 2013b). In 103 the present study, we perform a high content imaging screen using the Nerve Growth 104 Factor (NGF) withdrawal model of developmental neurodegeneration in embryonic 105 dorsal root ganglion (DRG) neurons to demonstrate that the Germinal Center Kinase-IV 106 (GCK-IV) subfamily of Ste20 kinases; MAP4K4, MINK1 (Misshapen-Like Kinase 1, 107 MAP4K6) and TNIK (Traf2- and Nck-interacting Kinase, MAP4K7) act redundantly to 108 regulate retrograde DLK/JNK signaling in neurons. 109 6 110 MATERIALS AND METHODS 111 112 Animal models 113 All experiments were performed under animal protocols approved by the Animal Care 114 and Use Committee at Genentech. CD-1 E12.5 to E13.5 mouse embryos of either sex 115 were used for DRG preparations. Rat E14.5 embryos were used for the small molecule 116 screen. MAP4K4loxp animals were generated as described (Vitorino et al., 2015). 117 MAP4K4loxp animals were crossed to a transgenic pCAGG-Cre/ERT2 line, which contains 118 an insertion of a construct encoding a Cre-estrogen receptor fusion protein behind a 119 CAGGS promoter to give