Shuji Wakatsuki

Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.

Roles of meltrin beta/ADAM19 in the processing of neuregulin

Meltrin β/ADAM19 is a member of ADAMs (a d isintegrin andmetalloproteases), which are a family of membrane-anchored glycoproteins that play important roles in fertilization, myoblast fusion, neurogenesis, and proteolytic processing of several membrane-anchored proteins. The expression pattern ofmeltrin β during mouse development coincided well with that of neuregulin-1 (NRG), a member of the epidermal growth factor family. Then we examined whether meltrin β participates in the proteolytic processing of membrane-anchored NRGs. When NRG-β1 was expressed in mouse L929 cells, its extracellular domain was constitutively processed and released into the culture medium. This basal processing activity was remarkably potentiated by overexpression of wild-type meltrin β, which lead to the significant decrease in the cell surface exposure of extracellular domains of NRG-β1. Furthermore, expression of protease-deficient mutants of meltrin β exerted dominant negative effects on the basal processing of NRG-β1. These results indicate that meltrin β participates in the processing of NRG-β1. Since meltrin β affected the processing of NRG-β4 but not that of NRG-α2, meltrin β was considered to have a preference for β-type NRGs as substrate. Furthermore, the effects of the secretory pathway inhibitors suggested that meltrin β participates in the intracellular processing of NRGs rather than the cleavage on the cell surface.

ZNRF1 promotes Wallerian degeneration by degrading AKT to induce GSK3B-dependent CRMP2 phosphorylation.

Wallerian degeneration is observed in many neurological disorders, and it is therefore important to elucidate the axonal degeneration mechanism to prevent, and further develop treatment for, such diseases. The ubiquitin–proteasome system (UPS) has been implicated in Wallerian degeneration, but the underlying molecular mechanism remains unclear. Here we show that ZNRF1, an E3 ligase, promotes Wallerian degeneration by targeting AKT to degrade through the UPS. AKT phosphorylates glycogen synthase kinase-3β (GSK3B), and thereby inactivates it in axons. AKT overexpression significantly delays axonal degeneration. Overexpression of the active (non-phosphorylated) form of GSK3B induces CRMP2 phosphorylation, which is required for the microtubule reorganization observed in the degenerating axon. The inhibition of GSK3B and the overexpression of non-phosphorylated CRMP2 both protected axons from Wallerian degeneration. These findings indicate that the ZNRF1–AKT–GSK3B–CRMP2 pathway plays an important role in controlling Wallerian degeneration.

Meltrin β/ADAM19 mediates ectodomain shedding of Neuregulin β1 in the Golgi apparatus : fluorescence correlation spectroscopic observation of the dynamics of ectodomain shedding in living cells

Membrane‐anchored Neuregulin β1 sheds its ectodomain as soluble factors. Two proteases that belong to a disintegrin and metalloprotease (ADAM) family are known to cleave Neuregulin β1. One is tumor necrosis factor‐α converting enzyme (TACE/ADAM17). The other is Meltrin β (ADAM19). Against our expectation that shedding by ADAM proteases occurs at the cell surface, here we found that Meltrin β mediates the ectodomain shedding of Neuregulin β1 in the Golgi apparatus. Meltrin β was localized in and around the Golgi apparatus in developing sensory neurons. Subcellular fractionation revealed that Meltrin β generated soluble Neuregulin β1 in Golgi‐enriched fractions while TACE‐cleaved Neuregulin β1 was recovered in lighter fractions. To examine whether Meltrin β‐mediated ectodomain shedding occurs in the Golgi apparatus in living cells, we took advantage of different diffusion properties of cleavage products from those of membrane‐anchored precursor proteins. Fluorescence correlation spectroscopy (FCS) is the most sensitive method to determine milli∼submillisecond diffusion in vivo. Protease‐active Meltrin β caused a shift in autocorrelation function in FCS of green fluorescent protein (GFP)‐tagged Neuregulin β1 in the Golgi apparatus, suggesting a conversion of Neuregulin β1 molecules from membrane‐anchored to soluble forms in that organelle. The Golgi apparatus is a site of processing Neuregulin β1 by Meltrin β.

Lipid rafts identified as locations of ectodomain shedding mediated by meltrin β/ADAM19

Meltrin β (Mel β, also called ADAM19) is a member of the ADAM (adisintegrin and metalloprotease) family, which are membrane‐anchored glycoproteins that play crucial roles in various biological processes. Many intercellular signaling molecules are membrane‐anchored proteins, which are proteolytically processed after becoming membrane‐bound, to liberate their extracellular domains (ectodomain shedding). Genetic and biochemical studies have shown that some ADAMs participate in these events. We found previously that Mel β can cleave the extracellular region of the membrane‐anchored β‐exon‐containing Neuregulin‐1 (NRG β1) protein, which is one of the main ligands for the neural ErbB receptor. Mel β‐deficient mice showed developmental defects in the nervous system. These observations raise the possibility that the NRG ectodomain shedding mediated by Mel β is closely related to the neural development. Here we show that Mel β‐mediated ectodomain shedding of NRG β1 takes place in the lipid rafts of neurons. The lipid rafts localization of Mel β requires its membrane‐anchoring region, and NRG β1 ectodomain shedding is not enhanced if Mel β cannot reach the lipid rafts. These results indicate that localization of Mel β in lipid rafts is critical for its ectodomain shedding.

Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction

Background Development of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously. Principal Findings We report here that Meltrin β, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor α mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin β–deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin β–deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin β and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin β and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin β and EphA4 strongly blocked vesicular internalization of ephrin-A5–EphA4 complexes without requiring the protease activity of Meltrin β, suggesting a regulatory role of Meltrin β in ephrin-A5-Eph signaling. Conclusion Meltrin β plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin β stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

Roles of Meltrin-β/ADAM19 in Progression of Schwann Cell Differentiation and Myelination during Sciatic Nerve Regeneration

Remyelination is an important aspect of nerve regeneration after nerve injury, but the underlying mechanisms are not fully understood. Here, we show that meltrin-β (ADAM19), a member of the ADAM (a disintegrin and metalloprotease) family, plays crucial roles in nerve regeneration after a crush injury to the sciatic nerves. The expression of meltrin-β was up-regulated in neurons after the crush injury. Morphometrical analysis revealed a delay in remyelination in meltrin-β-deficient nerves, whereas no significant defects were observed in their axon elongation. The activation of Krox-20, an indispensable transcription factor for myelination, was delayed in meltrin-β-deficient nerves and was accompanied by the retarded expression of myelin-related proteins. Expression of Krox-20 in Schwann cells was mediated by Akt. Phosphorylation of Akt but not that of Erks was reduced in regenerating nerves of meltrin-β-deficient mice. The cell membrane fraction prepared from meltrin-β-deficient nerves showed a defective activation of Akt in the membrane-loaded Schwann cells. Meltrin-β-deficient mice exhibited delayed sciatic functional recovery after the nerve crush. Altogether, these results reveal a role of meltrin-β in Schwann cell differentiation and re-myelination in nerve regeneration. Moreover, this study suggests that meltrin-β functions as a modulator of juxtacrine signaling from axons that activate the Akt pathway and the Krox-20 expression, which is the prerequisite for Schwann cell differentiation.

Meltrin β mini, a new ADAM19 isoform lacking metalloprotease and disintegrin domains, induces morphological changes in neuronal cells1

Meltrin β (ADAM19) is a metalloprotease‐disintegrin expressed in the peripheral nervous system and other organs during embryogenesis. We report here an alternatively spliced isoform, meltrin β mini, that lacks the prodomain, metalloprotease and disintegrin domains. A comparison of the cDNA and genomic sequences suggested the existence of a new exon. This isoform was detected in murine dorsal root ganglion and neuronal cell lines by RT‐PCR. Overexpression of meltrin β mini but not meltrin β induced neurite outgrowth in neuronal cells. These studies suggest that the novel meltrin β isoform has a distinct function related to neurogenesis.

Neuregulin-1/glial growth factor stimulates Schwann cell migration by inducing α5 β1 integrin-ErbB2-focal adhesion kinase complex formation.

After peripheral nerve injury, Schwann cells gain a migratory phenotype and remodel their extracellular matrix to provide a supportive environment for axonal regeneration. The soluble neuregulin‐1 isoform, that is, glial growth factor (GGF), is expressed in regenerating axons of injured peripheral nerves and regulates Schwann cell motility by activating the ErbB family of tyrosine kinase receptors, but how GGF/ErbB signaling contributes to Schwann cell motility remains unclear. Here, we show that GGF stimulates Schwann cell migration by inducing the formation of a protein complex containing the fibronectin receptor α5β1 integrin, ErbB2, and focal adhesion kinase (FAK). ErbB2 co‐localizes and co‐immunoprecipitates with the focal complex members including α5β1 integrin and FAK after GGF treatment. These effects of GGF appear to involve FAK activation, which occurs downstream of ErbB2 stimulation. RNAi‐mediated down‐regulation of α5 integrin expression in primary cultured Schwann cells resulted in significantly decreased interaction between FAK and ErbB2, as well as decreased GGF‐induced migration. An increase in the α5β1 integrin–ErbB2–FAK complex formation was observed in injured nerve Schwann cells, but not uninjured control. Taken together, these data suggest that GGF plays an important modulatory role in Schwann cell migration after nerve crush by inducing α5β1 integrin–ErbB2–FAK complex formation.

GSK3B-mediated phosphorylation of MCL1 regulates axonal autophagy to promote Wallerian degeneration.

Macroautophagy is a catabolic process, in which portions of cytoplasm or organelles are delivered to lysosomes for degradation. Emerging evidence has indicated a pathological connection between axonal degeneration and autophagy. However, the physiological function and induction mechanism of autophagy in axons remain elusive. We herein show that, through activation of BECLIN1, glycogen synthase kinase 3B (GSK3B)–mediated phosphorylation of BCL2 family member MCL1 induces axonal autophagy and axonal degeneration. Phosphorylated MCL1 is ubiquitinated by the FBXW7 ubiquitin ligase and degraded by the proteasome, thereby releasing BECLIN1 to induce axonal autophagy. Axonal autophagy contributes to local adenosine triphosphate production in degenerating axons and the exposure of phosphatidylserine—an “eat-me” signal for phagocytes—on transected axons and is required for normal recruitment of phagocytes to axonal debris in vivo. These results suggest that GSK3B–MCL1 signaling to regulate autophagy might be important for the successful completion of Wallerian degeneration.