Grb2-associated binder-1 is required for neuregulin-1-induced peripheral nerve myelination.

Grb2-associated binders (Gabs) are scaffolding proteins implicated in cell signaling via receptor tyrosine kinases including neuregulin-1(NRG1)-ErbB receptor signaling, which is essential for peripheral nerve myelination. Here, we show that the conditional removal of Gab1 from Schwann cells resulted in hypomyelination and abnormal development of Remak bundles. In contrast, hypomyelination was not observed in conventional Gab2 knock-out mice. Tyrosine phosphorylation of Gab1, but not Gab2, in sciatic nerves was upregulated during the myelination period and was found to be suppressed in NRG1-type III(+/-) mice, which display a hypomyelinated phenotype similar to that observed in Gab1 knock-out mice. Gab1 knock-out and NRG1-type III(+/-) mice both exhibited reduced extracellular signal-regulated kinase activity in myelinating nerves. In addition, Krox20, a transcription factor that is critical for myelination, has been identified as a target of the NRG1-Gab1 pathway during the myelination process. Our findings suggest that Gab1 is an essential component of NRG1-type III signaling during peripheral nerve development.

The Neuregulin-Rac-MKK7 pathway regulates antagonistic c-jun/Krox20 expression in Schwann cell dedifferentiation.

Schwann cells respond to nerve injury by dedifferentiating into immature states and producing neurotrophic factors, two actions that facilitate successful regeneration of axons. Previous reports have implicated the Raf-ERK cascade and the expression of c-jun in these Schwann cell responses. Here we used cultured primary Schwann cells to demonstrate that active Rac1 GTPase (Rac) functions as a negative regulator of Schwann cell differentiation by upregulating c-jun and downregulating Krox20 through the MKK7-JNK pathway, but not through the Raf-ERK pathway. The activation of MKK7 and induction of c-jun in sciatic nerves after axotomy was blocked by Rac inhibition. Microarray experiments revealed that the expression of regeneration-associated genes, such as glial cell line-derived neurotrophic factor and p75 neurotrophin receptor, after nerve injury was dependent on Rac but not on ERK. Finally, the inhibition of ErbB2 signaling prevented MKK7 activation, c-jun induction, and Rac-dependent gene expression in sciatic nerve explant cultures. Taken together, our results indicate that the neuregulin-Rac-MKK7-JNK/c-jun pathway regulates Schwann cell dedifferentiation following nerve injury.

Actin polymerization is essential for myelin sheath fragmentation during Wallerian degeneration.

The mechanisms that trigger Wallerian degeneration (WD) of peripheral nerves after injury are not well understood. During the early period of WD, fragmentation of myelin into ovoid structures occurs near the Schmidt-Lantermann incisures (SLI), a noncompact region of the myelin sheath containing autotypical adherens junction. In this study, we found that new filamentous actin polymerization occurs in the SLI of mouse sciatic nerves after injury and that its inhibition prevented not only the degradation of E-cadherin in the SLI but also myelin ovoid formation. However, the inhibition of actin polymerization could not block Schwann cell dedifferentiation. The activation of Rac GTPase was observed in the distal stump of the injured nerves, and a specific Rac inhibitor, a dominant-negative Rac, and Rac1-RNA interference blocked myelin ovoid formation. Together, these findings suggest that dynamic changes in actin in the SLI are essential for initiation of demyelination after peripheral nerve injury.

Local production of serum amyloid a is implicated in the induction of macrophage chemoattractants in Schwann cells during wallerian degeneration of peripheral nerves.

The elevation of serum levels of serum amyloid A (SAA) has been regarded as an acute reactive response following inflammation and various types of injuries. SAA from the liver and extrahepatic tissues plays an immunomodulatory role in a variety of pathophysiological conditions. Inflammatory cytokines in the peripheral nerves have been implicated in the Wallerian degeneration of peripheral nerves after injury and in certain types of inflammatory neuropathies. In the present study, we found that a sciatic nerve axotomy could induce an increase of SAA1 and SAA3 mRNA expression in sciatic nerves. Immunohistochemical staining showed that Schwann cells are the primary sources of SAA production after nerve injury. In addition, interleukin-6-null mice, but not tumor necrosis factor-α-null mice showed a defect in the production of SAA1 in sciatic nerve following injury. Dexamethasone treatment enhanced the expression and secretion of SAA1 and SAA3 in sciatic nerve explants cultures, suggesting that interleukin-6 and corticosteroids might be major regulators for SAA production in Schwann cells following injury. Moreover, the stimulation of Schwann cells with SAA1 elicited the production of the macrophage chemoattractants, Ccl2 and Ccl3, in part through a G-protein coupled receptor. Our findings suggest that locally produced SAA might play an important role in Wallerian degeneration after peripheral nerve injury.

Pathological adaptive responses of Schwann cells to endoplasmic reticulum stress in bortezomib-induced peripheral neuropathy.

Bortezomib, a proteasome inhibitor, has been considered as a promising anticancer drug in the treatment of recurrent multiple myeloma and some solid tumors. The bortezomib-induced peripheral neuropathy (BIPN) is a prominent cause of dose-limiting toxicities after bortezomib treatment. In this study, we found that BIPN in a mouse model is characterized by acute but transient endoplasmic reticulum (ER) damages to Schwann cells. These damaged Schwann cells exhibit abnormal outcomes from healing processes such as the myelination of Remak bundles. A morphometric analysis of polymyelinated Remak bundles revealed that the pathological myelination was not related to the axonal parameters that regulate the normal myelination process during development. In addition, demyelinating macrophages were focally infiltrated within endoneurium of the sciatic nerve. To identify the mechanism underlying these pathologies, we applied a gene microarray analysis to bortezomib-treated primary Schwann cells and verified the changes of several gene expression in bortezomib-treated sciatic nerves. The analysis showed that bortezomib-induced ER stress was accompanied by the activation of several protective molecular chaperones and the down-regulation of myelin gene expression. ER stress inducers such as thapsigargin and bredelfin A also suppressed the mRNA expression of myelin gene P0 at transcriptional levels. In addition, the expression of chemokines such as the macrophage chemoattractants Ccl3 and Cxcl2 was significantly increased in Schwann cells in response to bortezomib and ER stress inducers. Taken together, these observations suggest that the pathological adaptive responses of Schwann cells to bortezomib-induced ER stress may, in part, participate in the development of BIPN.

A novel mechanism of methylglyoxal cytotoxicity in neuroglial cells.

Methylglyoxal (MGO) is an endogenous dicarbonyl compound that is highly produced in hyperglycemic conditions. It forms advanced glycation endproducts that are believed to contribute, as etiological factors, to the pathophysiology of diabetic complications. In addition, MGO suppresses cell viability through the induction of apoptosis in vitro. In this study, we have, for the first time, demonstrated the effect of MGO on the gp130 cytokine-induced signal transducer and activator of transcription 3 (STAT3) responses in RT4 schwannoma, PC12 pheochromocytoma and U87MG glioma cells. At dose that very mildly affects cell viability, MGO rapidly induces endocytotic degradation of gp130, which involves the di-leucine internalization motif in the cytoplasmic domain of gp130, without affecting other growth factor receptors. Concomitant inhibition of basal and interleukin-6-induced STAT3 activation was observed following pre-treatment with MGO. The inhibitory effect of MGO on the gp130/STAT3 signaling was prevented by the pre-treatment with an advanced glycation endproduct scavenger aminoguanidine. Finally, these deleterious effects of MGO on STAT3 signaling led to down-regulation of a STAT3 target gene, Bcl-2, and sensitized cellular toxicity induced by H(2)O(2) and etoposide. Our data indicate that MGO affects cell viability via desensitization of gp130/STAT3 signaling, which is the key signaling pathway for cell survival, and thereby promotes cytotoxicity.

Interleukin-6 is required for the early induction of glial fibrillary acidic protein in Schwann cells during Wallerian degeneration.

Signal transducer and activator of transcription 3 (STAT3) regulates gene transcription in response to cytokines and growth factors. In the central nervous system, STAT3 plays a role in neuroprotection and reactive gliosis after lesions. During peripheral nerve regeneration, a nerve injury-induced up-regulation of cytokines and growth factors accompanies STAT3 activation in sensory neurons and Schwann cells (SCs) even though its molecular details and functions are unknown. We then analyzed the ligands and functions of STAT3 activation in RT4 schwannoma cells and adult SCs in vitro and in vivo. We have identified that interleukin-6 (IL-6), but not ciliary neurotrophic factor, leukemia inhibitory factor, or ligands for receptor tyrosine kinases, activates STAT3 in SCs. The IL-6/STAT3 signaling in primary SCs and RT4 cells induced the gene expression of glial fibrillary acidic protein (GFAP), which is known to be required for the proper regeneration of the injured nerves. Finally, the GFAP induction in the sciatic nerves after injury was significantly delayed in IL-6-deficient mice. These findings indicate that IL-6 plays an important role in STAT3-dependent GFAP induction in SCs during peripheral nerve regeneration.

Interleukin-6 induces proinflammatory signaling in Schwann cells: a high-throughput analysis.

Interleukin-6 plays an important role in peripheral nerve regeneration. We recently reported that IL-6 targets Schwann cells in the peripheral nerve for its function. In this study, we analyzed genes whose expression is regulated by IL-6 in a cell line derived from Schwann cells, the peripheral glia, using the Illumina gene microarray. At measurements 3 and 12h after IL-6 treatment, 35 genes were found to be upregulated by IL-6. Most upregulated genes were proinflammatory genes that are known to be induced in inflammatory conditions. Interestingly, the expression of immunoproteasome subunits was upregulated by IL-6 in Schwann cells. Treatment with forskolin, an agent that mimics axonal signaling, suppressed the expression of IL-6-inducible genes. Finally, we found for the first time that sciatic nerve injury induced immunoproteasome expression in vivo. These findings indicate that IL-6 is involved in peripheral nerve regeneration by regulating proinflammatory signaling in Schwann cells.

Capsaicin inhibits the IL-6/STAT3 pathway by depleting intracellular gp130 pools through endoplasmic reticulum stress.

Capsaicin has been shown to have anti-carcinogenic effects on various tumor cells through multiple mechanisms. It was recently reported that capsaicin inhibited interleukin-6 (IL-6)-induced activation of signal transducer and activator of transcription 3 (STAT3), an anti-apoptotic transcription factor. Here we demonstrate that capsaicin induced downregulation of the IL-6 receptor gp130 within 2h in glial tumors. The downregulation of gp130 was not caused by enhanced degradation of gp130 or by inhibition of mRNA transcription. The downregulation was attributed to translation inhibition of gp130, which was associated with activation of endoplasmic reticulum (ER) stress. The depletion of the intracellular pool of gp130 by capsaicin and an ER stress inducer led to an immediate loss of the IL-6 response due to the short half-life of membrane localized gp130. These results suggest a novel mechanism for the anti-tumor effect of capsaicin.

Nidogen plays a role in the regenerative axon growth of adult sensory neurons through Schwann cells.

We previously reported that nidogen is an extracellular matrix protein regulating Schwann cell proliferation and migration. Since Schwann cells play a critical role in peripheral nerve regeneration, nidogen may play a role in it via regulation of Schwann cells. Here, we demonstrate direct evidence that nidogen induces elongation of regenerative axon growth of adult sensory neurons, and that the effect is Schwann cell dependent. Continuous infusion of recombinant ectodomain of tumor endothelial marker 7, which specifically blocks nidogen function in Schwann cells, suppressed regenerative neurite growth in a sciatic nerve axotomy model. Taken together, it is likely that nidogen is required for proper regeneration of peripheral nerves after injury.

Cloning, characterization and neuronal expression profiles of tumor endothelial marker 7 in the rat brain.

Tumor endothelial marker7 (TEM7) is a putative transmembrane protein that is highly expressed in the tumor endothelium. In the present study, the expression profile of TEM7 was investigated in TEM7-transfected human embryonic kidney (HEK) 293 cells and the rat brain. The extracellular secretion of the recombinant N-terminal ectodomain of TEM7, not full-length TEM7, was observed in the transiently transfected HEK 293 cells. The full-length TEM7 was found inside and membrane part of cells as demonstrated by confocal microscopy. In situ hybridization study revealed that TEM7 mRNA expressions were localized to specific neuronal areas, such as cerebellar Purkinje cells, the layer IV and V of cerebral cortex, hippocampal pyramidal cells and hypothalamic magnocellular nuclei. Immunohistochemical investigation of TEM expression with specific antibodies against TEM7 further supported the spatial expression patterns of TEM7 mRNA. The temporal expression of TEM7 mRNA in the cerebellar Purkinje cells demonstrated a postnatal developmental regulation of TEM7 expression. Our results indicate that TEM7 plays a role as a transmembrane receptor in some neuronal populations of the vertebrate brains.

[Students' perception of the educational environment of medical schools in Korea: findings from a nationwide survey].

PURPOSE: The purpose of this study was to examine students' perception of the educational environment of medical schools in Korea. METHODS: A total of 9,096 of 12,035 students (75.6%) responded to our questionnaire. This study was conducted at the end of the 2013 academic year using the Dundee Ready Education Environment Measure (DREEM) at 40 medical schools in Korea. DREEM comprises five domains: students' perceptions of learning (SPL); students' perceptions of teachers (SPT); students' academic self-perceptions; students' perceptions of atmosphere; and students' social self-perception. The data were analyzed using descriptive statistics, independent t-test, and one-way analysis of variance. RESULTS: The overall mean DREEM score was 113.97 (of 200), and the scores for the 40 medical schools ranged from 100.24 to 134.32. The overall mean and domains scores of the DREEM differed significantly between educational systems, grades, genders, and academic achievement levels. Graduate-level medical students had higher scores for the DREEM and its five domains than undergraduate medical students. The scores were lowest in second-year students (mean, 111.80). Male students' perceptions were significantly higher than those of female students except for SPL and SPT. High academic achievers' perceptions were also greater versus low academic achievers. CONCLUSION: Students' perceptions of their educational environment are positive in Korea. The learning environment should be evaluated by curriculum planners and administrators of medical schools to improve its quality.

Calcium-dependent proteasome activation is required for axonal neurofilament degradation.

Even though many studies have identified roles of proteasomes in axonal degeneration, the molecular mechanisms by which axonal injury regulates proteasome activity are still unclear. In the present study, we found evidence indicating that extracellular calcium influx is an upstream regulator of proteasome activity during axonal degeneration in injured peripheral nerves. In degenerating axons, the increase in proteasome activity and the degradation of ubiquitinated proteins were significantly suppressed by extracellular calcium chelation. In addition, electron microscopic findings revealed selective inhibition of neurofilament degradation, but not microtubule depolymerization or mitochondrial swelling, by the inhibition of calpain and proteasomes. Taken together, our findings suggest that calcium increase and subsequent proteasome activation are an essential initiator of neurofilament degradation in Wallerian degeneration.

Complementary and alternative medicine in the undergraduate medical curriculum: a survey of Korean medical schools.

BACKGROUND: The current status of complementary and alternative medicine (CAM) education in Korean medical schools is still largely unknown, despite a growing need for a CAM component in medical education. The prevalence, scope, and diversity of CAM courses in Korean medical school education were evaluated. DESIGN: Participants included academic or curriculum deans and faculty at each of the 41 Korean medical schools. A mail survey was conducted from 2007 to 2010. Replies were received from all 41 schools. RESULTS: CAM was officially taught at 35 schools (85.4%), and 32 schools (91.4%) provided academic credit for CAM courses. The most common courses were introduction to CAM or integrative medicine (88.6%), traditional Korean medicine (57.1%), homeopathy and naturopathy (31.4%), and acupuncture (28.6%). Educational formats included lectures by professors and lectures and/or demonstrations by practitioners. The value order of core competencies was attitude (40/41), knowledge (32/41), and skill (6/41). Reasons for not initiating a CAM curriculum were a non-evidence-based approach in assessing the efficacy of CAM, insufficiently reliable reference resources, and insufficient time to educate students in CAM. CONCLUSIONS: This survey reveals heterogeneity in the content, format, and requirements among CAM courses at Korean medical schools. Korean medical school students should be instructed in CAM with a more consistent educational approach to help patients who participate in or demand CAM.

Transient lysosomal activation is essential for p75 nerve growth factor receptor expression in myelinated Schwann cells during Wallerian degeneration.

Myelinated Schwann cells in the peripheral nervous system express the p75 nerve growth factor receptor (p75NGFR) as a consequence of Schwann cell dedifferentiation during Wallerian degeneration. p75NGFR has been implicated in the remyelination of regenerating nerves. Although many studies have shown various mechanisms underlying Schwann cell dedifferentiation, the molecular mechanism contributing to the re-expression of p75NGFR in differentiated Schwann cells is largely unknown. In the present study, we found that lysosomes were transiently activated in Schwann cells after nerve injury and that the inhibition of lysosomal activation by chloroquine or lysosomal acidification inhibitors prevented p75NGFR expression at the mRNA transcriptional level in an ex vivo Wallerian degeneration model. Lysosomal acidification inhibitors suppressed demyelination, but not axonal degeneration, thereby suggesting that demyelination mediated by lysosomes may be an important signal for inducing p75NGFR expression. Tumor necrosis factor-α (TNF-α) has been suggested to be involved in regulating p75NGFR expression in Schwann cells. In this study, we found that removing TNF-α in vivo did not significantly suppress the induction of both lysosomes and p75NGFR. Thus, these findings suggest that lysosomal activation is tightly correlated with the induction of p75NGFR in demyelinating Schwann cells during Wallerian degeneration.

Injury-induced CRMP4 expression in adult sensory neurons; a possible target gene for ciliary neurotrophic factor.

Neurotrophic cytokines, such as ciliary neurotrophic factor (CNTF) play an important role in the development and regeneration of the nervous system. In the present study, we screened gene expression induced by CNTF in adult dorsal root ganglion (DRG) neurons using the Illumina microarray. We found that the expression of both short and long forms of collapsin response-mediator protein 4 (CRMP4) was increased in cultured primary sensory neurons by CNTF. In addition, sciatic nerve injury induced the expression of CRMP4 mRNA and protein in DRG neurons. Finally, the increased CRMP4 protein was transported into peripheral axons following nerve injury. These findings indicate that CRMP4 may be a target gene for CNTF in the regenerative axon growth of DRG neurons after injury.

Extracellular Signal-regulated Kinase Activation Is Required for Serine 727 Phosphorylation of STAT3 in Schwann Cells in vitro and in vivo.

In the peripheral nerves, injury-induced cytokines and growth factors perform critical functions in the activation of both the MEK/ERK and JAK/STAT3 pathways. In this study, we determined that nerve injury-induced ERK activation was temporally correlated with STAT3 phosphorylation at the serine 727 residue. In cultured Schwann cells, we noted that ERK activation is required for the serine phosphorylation of STAT3 by neuropoietic cytokine interleukin-6 (IL-6). Serine phosphorylated STAT3 by IL-6 was transported into Schwann cell nuclei, thereby indicating that ERK may regulate the transcriptional activity of STAT3 via the induction of serine phosphorylation of STAT3. Neuregulin-1 (NRG) also induced the serine phosphorylation of STAT3 in an ERK-dependent fashion. In contrast with the IL-6 response, serine phosphorylated STAT3 induced by NRG was not detected in the nucleus, thus indicating the non-nuclear function of serine phosphorylated STAT3 in response to NRG. Finally, we determined that the inhibition of ERK prevented injury-induced serine phosphorylation of STAT3 in an ex-vivo explants culture of the sciatic nerves. Collectively, the results of this study show that ERK may be an upstream kinase for the serine phosphorylation of STAT3 induced by multiple stimuli in Schwann cells after peripheral nerve injury.

Netrin-1 specifically enhances cell spreading on fibronectin in human glioblastoma cells.

Netrins are secreted molecules and involved in axon guidance, cell migration and tumor development. Recent studies revealed that netrins perform novel functions in such processes as epithelial development and angiogenesis without operating through the classical netrin receptors, DCC (Deleted in Colorectal Cancer) and Unc5h. In the present study, we investigated the roles of netrin-1 and its receptors in cell spreading of human glioblastoma cells, and found that netrin-1 haptotactically enhanced fibronectin-induced cell spreading and focal adhesion formation in U373 glioblastoma cells. Netrin-1 binding to the U373 cell membrane was blocked by an antibody against alphav integrin subunit, but not by an anti-DCC or anti-Unc5h antibody. In addition, enhancement of the fibronectin response by netrin-1 was abrogated by a function blocking antibody against integrin alphavbeta3. Since the alphav subunit of the integrin family plays an important role in the pathophysiological aspects of cell migration, including tumor angiogenesis and metastasis, our data provide important insight into the molecular mechanism of netrin function.

Netrin induces down-regulation of its receptor, Deleted in Colorectal Cancer, through the ubiquitin-proteasome pathway in the embryonic cortical neuron.

The proper regulation of temporal and spatial expression of the axon guidance cues and their receptors is critical for the normal wiring of nervous system during development. Netrins, a family of secreted guidance cues, are involved in the midline crossing of spinal commissural axons and in the guidance of cortical efferents. Axons normally lose the responsiveness to their attractants when they arrive at their targets, where the attractant is produced. However the molecular mechanism is still unknown. We investigated the molecular mechanism of down-regulation of netrin-1 signaling in the embryonic cortical neurons. Netrin-1 induced the ubiquitination and proteolytic cleavage of Deleted in Colorectal Cancer (DCC), a transmembrane receptor for netrin, in dissociated cortical neurons. A dramatic decrease of DCC level particularly on the cell surface was also observed after netrin-1 stimulation. Specific ubiquitin-proteasome inhibitors prevented the netrin-induced DCC cleavage and decrease of cell surface DCC. We suggest that the ligand-mediated down-regulation of DCC might participate in the loss of netrin-responsiveness in the developing nervous system.