Symposia

Symposia The signaling of JNK as a positive signal for neuronal development and regeneration／神経発生及び正の再生シグナルとしてのJNKシグナリング

2S6-1 Stress-activated protein kinase MKK7 regulates neuronal development and maintenance Hiroshi Nishina Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University The c-Jun NH(2)-terminal protein kinase (JNK), which belongs to the mitogen-activated protein kinase family, plays important roles in a broad range of physiological processes. JNK is controlled by two upstream regulators, mitogen-activated protein kinase kinase (MKK) 7 and MKK4. To elucidate the physiological functions of MKK7, we used Nestin-Cre to generate a novel mouse model in which the mkk7gene was specifically deleted in the nervous system (Mkk7(flox/flox) Nestin-Cre mice). These mice were indistinguishable from their control littermates in gross appearance during embryogenesis but died immediately after birth without breathing. Histological examination showed that the mutants had severe defects in brain development, including enlarged ventricles, reduced striatum, and minimal axon tracts. Moreover, MKK7 regulated axon elongationin a cell-autonomous manner in vivo, a finding confirmed in vitro. These results indicate that MKK7-mediated regulation of JNK plays an important regulatory role in neural development.In this talk, I will provide post-developmental functions of MKK7 in the nervous system. We generated another neuron-specific Mkk7 knockout mice (Mkk7(flox/flox) Synapsin-Cre mice), which displayed impaired circadian behavioral rhythms and decreased locomotor activity.		2S6-2 DLK-dependent JNK activation facilitates the axon formation in cortical neurons Syu-ichi Hirai Dept Biol, Sch Med, Wakayama Med Univ In embryonic cerebral cortex, newborn pyramidal neurons sprout axon first and dendrite elaboration starts thereafter. Well-grown multiple dendrites will be incompatible with the radial or tangential migration of neurons, which is essential for cortical layer formation. On the other hand, a single axon like a trailing process will be compatible with the migration. Therefore, the sequential order of axon formation and dendritic elaboration is well suited for cortical layer formation. The sequential formation of axons and dendrites has been reproduced in cultured neurons, indicating the presence of an intrinsic mechanism regulating the timing of axon formation and dendritic elaboration; however, this mechanism remains to be elucidated. c-Jun N-terminal kinase (JNK) and its upstream activating kinase, dual leucine zipper kinase (DLK), play critical roles in the development of cortical neurons in vivo as well as in vitro (Hirai et al, J Neurosci, 2011). JNK phosphorylates a variety of cytoplasmic proteins including superior cervical ganglion 10 (SCG10), in addition to transcription factors such as c-Jun. SCG10 is a member of the stathmin family, which is known to destabilize microtubules by binding to the tubulin tetramer. SCG10 is widely expressed in different kinds of neurons, and is required for the formation of neurites including axons and dendrites. Our recent observations suggest that the protein level of SCG10 in developing cortical neurons is regulated through the DLK-JNK pathway and that the changes in the SCG10 protein level can be a molecular base supporting the orderly progression of axon formation and dendritic elaboration.

2S6-3 Regulation of axon regeneration by JNK signaling in Caenorhabditis elegans Naoki Hisamoto，Strahil Pastuhov，Kunihiro Matsumoto Dept Biol Sci, Grad Sch Sci, Nagoya Univ Axon regeneration is an important process to recover from the motor and sensory disorders caused by a nerve injury. Previous studies have revealed that axon regeneration occurs not only in vertebrates such as humans but also in invertebrates. The nematode Caenorhabditis elegans has recently emerged as a genetically tractable model for studying regenerative responses in neurons. Extensive studies over several years using this organism have revealed a number of intrinsic and extrinsic signal transduction cascades that regulate axon regeneration, many of which are conserved from worms to humans. Further studies have demonstrated that these cascades consist of several signaling networks that ultimately merge into the JNK signaling. In this symposium, I will talk about our recent findings about the regulation of axon regeneration by JNK signaling in C. elegans.		2S6-4 Critical role of JSAP in axonal transport to prevent neuronal degeneration Katsuji Yoshioka，Tokiharu Sato Div Mol Cell Signaling, Cancer Res Inst, Kanazawa Univ Axonal transport is critical for neuronal development and function, and defective axonal transport has been implicated in neurodegenerative diseases. However, how axonal transport is regulated, or how defective transport leads to neuronal degeneration, remains unclear. Here we show that JSAP1 (also known as JIP3) and JSAP2 (also known as JLP and SPAG9), both known scaffolding proteins for MAP kinase signaling pathways, are essential for postnatal brain development. JSAP1 and JSAP2 are structurally related proteins that are highly expressed in the brain. Mice with a double-knockout (DKO) in Jsap1 and Jsap2 in the dorsal telencephalon developed progressive neuronal loss. Using a primary neuron culture system with induced disruption of targeted genes, combined with gene rescue experiments, we show that JSAP1 and JSAP2 regulate kinesin-1-dependent axonal transport with functional redundancy. We also show that the binding of JSAP1 and JSAP2 to kinesin-1 heavy chain is crucial for interactions between kinesin-1 and microtubules. In this symposium, we will discuss a molecular mechanism by which defective kinesin-1-dependent axonal transport in Jsap1:Jsap2 DKO neurons causes axonal degeneration and subsequent neuronal death.

2S6-5 JNK substrates involved in axonal growth, identified by phosphoproteomics of the growth cone Asami Kawasaki^1,2，Masayasu Okada^1,2,3，Atsushi Tamada^1,2，Michihiro Igarashi^1,2 ¹Department of Neurochemistry and Molecular Cell Biology, Niigata University，²Trans-disciplinary Research Program, Niigata University，³Departments of Neurosurgery, Institute for Brain Research, Niigata University Axon growth and pathfinding for the accurate synaptogenesis are believed to be performed through the complex signaling pathways, most of which are not well understood. In this study, we carried out unbiased phosphoproteomics of axonal growth cone membranes (GCM), and identified over 6,000 phosphorylation sites on 1,900 proteins. A large number of these phosphorylation sites were proline-directed Ser/Thr (Ser/Thr-Pro), in which the highly phosphorylated sites are vertebrate-specific. The most frequently phosphorylated site was Ser96 of growth associated protein 43 (GAP-43/neuromodulin), a classical marker of axon growth. Biochemical analysis demonstrated that c-Jun N-terminal kinase (JNK), one of the proline-directed protein kinases, phosphorylates not only Ser96, but also Thr172 and Ser142 of this protein. Other JNK substrates involved in axon growth were also identified in the GCM, suggesting an important role of JNK in this event. The phospho-specific antibody against Ser96 of GAP-43 strongly recognized the growing and the regenerating axons. Intriguingly, cultured hippocampal neurons prepared from the GAP-43 Ser96Ala knock-in mice exhibited shorter axon lengths than those from wild-type mice. These results suggest that JNK regulates axonal growth via phosphorylation of a number of GCM proteins, including GAP-43.