miR-381 Attenuates Peripheral Neuropathic Phenotype Caused by Overexpression of PMP22

Charcot-Marie Tooth disease type 1A (CMT1A), the major type of CMT, is caused by duplication of peripheral myelin protein 22 (PMP22) gene whose overexpression causes structural and functional abnormalities in myelination. We investigated whether miRNA-mediated regulation of PMP22 expression could reduce the expression level of PMP22, thereby alleviating the demyelinating neuropathic phenotype of CMT1A. We found that several miRNAs were down-regulated in C22 mouse, a CMT1A mouse model. Among them, miR-381 could target 3′ untranslated region (3′UTR) of PMP22 in vitro based on Western botting and quantitative Real Time-PCR (qRT-PCR) results. In vivo efficacy of miR-381 was assessed by administration of LV-miR-381, an miR-381 expressing lentiviral vector, into the sciatic nerve of C22 mice by a single injection at postnatal day 6 (p6). Administration of LV-miR-381 reduced expression level of PMP22 along with elevated level of miR-381 in the sciatic nerve. Rotarod performance analysis revealed that locomotor coordination of LV-miR-381 administered C22 mice was significantly enhanced from 8 weeks post administration. Electrophysiologically, increased motor nerve conduction velocity was observed in treated mice. Histologically, toluidine blue staining and electron microscopy revealed that structural abnormalities of myelination were improved in sciatic nerves of LV-miR-381 treated mice. Therefore, delivery of miR-381 ameliorated the phenotype of peripheral neuropathy in CMT1A mouse model by down-regulating PMP22 expression. These data suggest that miRNA can be used as a potent therapeutic strategy to control diseases with copy number variations such as CMT1A.

Pattern of Extraocular Muscle Involvements in Miller Fisher Syndrome

BACKGROUND AND PURPOSE: The most-common initial manifestation of Miller Fisher syndrome (MFS) is diplopia due to acute ophthalmoplegia. However, few studies have focused on ocular motility findings in MFS. This study aimed to determine the pattern of extraocular muscle (EOM) paresis in MFS patients. METHODS: We consecutively recruited MFS patients who presented with ophthalmoplegia between 2010 and 2015. The involved EOMs and the strabismus pattern in the primary position were analyzed. Antecedent infections, other involved cranial nerves, and laboratory findings were also reviewed. We compared the characteristics of the patients according to the severity of ophthalmoplegia between complete ophthalmoplegia (CO) and incomplete ophthalmoplegia (IO). RESULTS: Twenty-five patients (15 males and 10 females) with bilateral ophthalmoplegia were included in the study. The most-involved and last-to-recover EOM was the lateral rectus muscle. CO and IO were observed in 11 and 14 patients, respectively. The patients were aged 59.0±18.4 years (mean±SD) in the CO group and 24.9±7.4 years in the IO group (p<0.01), and comprised 63.6% and 21.4% females, respectively (p=0.049). Elevated cerebrospinal fluid protein was identified in 60.0% of patients with CO and 7.7% of patients with IO (p=0.019) for a mean follow-up time from the initial symptom onset of 3.7 days. CONCLUSIONS: The lateral rectus muscle is the most-involved and last-to-recover EOM in ophthalmoplegia. The CO patients were much older and were more likely to be female and have an elevation of cerebrospinal fluid protein than the IO patients.

A De Novo RAPGEF2 Variant Identified in a Sporadic Amyotrophic Lateral Sclerosis Patient Impairs Microtubule Stability and Axonal Mitochondria Distribution

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is frequently linked to microtubule abnormalities and mitochondrial trafficking defects. Whole exome sequencing (WES) of patient-parent trios has proven to be an efficient strategy for identifying rare de novo genetic variants responsible for sporadic ALS (sALS). Using a trio-WES approach, we identified a de novo RAPGEF2 variant (c.4069G>A, p.E1357K) in a patient with early-onset sALS. To assess the pathogenic effects of this variant, we have used patient-derived skin fibroblasts and motor neuron-specific overexpression of the RAPGEF2-E1357K mutant protein in Drosophila. Patient fibroblasts display reduced microtubule stability and defective microtubule network morphology. The intracellular distribution, ultrastructure, and function of mitochondria are also impaired in patient cells. Overexpression of the RAPGEF2 mutant in Drosophila motor neurons reduces the stability of axonal microtubules and disrupts the distribution of mitochondria to distal axons and neuromuscular junction (NMJ) synapses. We also show that the recruitment of the pro-apoptotic protein BCL2-associated X (BAX) to mitochondria is significantly increased in patient fibroblasts compared with control cells. Finally, increasing microtubule stability through pharmacological inhibition of histone deacetylase 6 (HDAC6) rescues defects in the intracellular distribution of mitochondria and BAX. Overall, our data suggest that the RAPGEF2 variant identified in this study can drive ALS-related pathogenic effects through microtubule dysregulation.

The Scaffolding Protein, Grb2-associated Binder-1, in Skeletal Muscles and Terminal Schwann Cells Regulates Postnatal Neuromuscular Synapse Maturation

The vertebrate neuromuscular junction (NMJ) is considered as a “tripartite synapse” consisting of a motor axon terminal, a muscle endplate, and terminal Schwann cells that envelope the motor axon terminal. The neuregulin 1 (NRG1)-ErbB2 signaling pathway plays an important role in the development of the NMJ. We previously showed that Grb2-associated binder 1 (Gab1), a scaffolding mediator of receptor tyrosine kinase signaling, is required for NRG1-induced peripheral nerve myelination. Here, we determined the role of Gab1 in the development of the NMJ using muscle-specific conditional Gab1 knockout mice. The mutant mice showed delayed postnatal maturation of the NMJ. Furthermore, the selective loss of the gab1 gene in terminal Schwann cells produced delayed synaptic elimination with abnormal morphology of the motor endplate, suggesting that Gab1 in both muscles and terminal Schwann cells is required for proper NMJ development. Gab1 in terminal Schwann cells appeared to regulate the number and process elongation of terminal Schwann cells during synaptic elimination. However, Gab2 knockout mice did not show any defects in the development of the NMJ. Considering the role of Gab1 in postnatal peripheral nerve myelination, our findings suggest that Gab1 is a pleiotropic and important component of NRG1 signals during postnatal development of the peripheral neuromuscular system.

Mitogen Activated Protein Kinase Family Proteins and c-jun Signaling in Injury-induced Schwann Cell Plasticity

Schwann cells (SCs) in the peripheral nerves myelinate axons during postnatal development to allow saltatory conduction of nerve impulses. Well-organized structures of myelin sheathes are maintained throughout life unless nerves are insulted. After peripheral nerve injury, unidentified signals from injured nerves drive SC dedifferentiation into an immature state. Dedifferentiated SCs participate in axonal regeneration by producing neurotrophic factors and removing degenerating nerve debris. In this review, we focus on the role of mitogen activated protein kinase family proteins (MAP kinases) in SC dedifferentiation. In addition, we will highlight neuregulin 1 and the transcription factor c-jun as upstream and downstream signals for MAP kinases in SC responses to nerve injury.

Expression Profile of Fas-Fas Ligand in Spiral Ganglion Cells During Apoptosis

OBJECTIVES: To examine the expression profile of Fas-Fas ligand (FasL) during glutamate (Glu)-induced spiral ganglion cell (SGC) apoptosis. METHODS: Cultured SGCs were treated with 10-mM, 25-mM, and 50-mM concentrations of Glu and incubated for 24 or 48 hours. The expression intensity of FasL, Fas, caspase 3, and morphology of single SGC were evaluated using immunofluorescence staining. RESULTS: In semiquantitative analysis of the Glu-treated SGC, FasL, and caspase 3 expression intensity were increased with concentration- and time-dependent manner. Fas expression intensity did not change with different concentration at 48 hours. In morphologic analysis of the Glu-treated SGC, number of apoptotic cells were increased with concentration- and time-dependent manner. CONCLUSION: FasL was expressed in apoptotic SGCs, suggesting that the Fas-FasL signaling pathway may be involved in the Glu-induced apoptosis of dissociated SGCs.

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

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-alpha (TNF-alpha) has been suggested to be involved in regulating p75NGFR expression in Schwann cells. In this study, we found that removing TNF-alpha 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.

Janus Kinase 2 Inhibitor AG490 Inhibits the STAT3 Signaling Pathway by Suppressing Protein Translation of gp130

The binding of interleukin-6 (IL-6) cytokine family ligands to the gp130 receptor complex activates the Janus kinase (JAK)/ signal transducer and activator of transcription 3 (STAT3) signal transduction pathway, where STAT3 plays an important role in cell survival and tumorigenesis. Constitutive activation of STAT3 has been frequently observed in many cancer tissues, and thus, blocking of the gp130 signaling pathway, at the JAK level, might be a useful therapeutic approach for the suppression of STAT3 activity, as anticancer therapy. AG490 is a tyrphostin tyrosine kinase inhibitor that has been extensively used for inhibiting JAK2 in vitro and in vivo. In this study, we demonstrate a novel mechanism associated with AG490 that inhibits the JAK/STAT3 pathway. AG490 induced downregulation of gp130, a common receptor for the IL-6 cytokine family compounds, but not JAK2 or STAT3, within three hours of exposure. The downregulation of gp130 was not caused by enhanced degradation of gp130 or by inhibition of mRNA transcription. It most likely occurred by translation inhibition of gp130 in association with phosphorylation of the eukaryotic initiation factor-2alpha. The inhibition of protein synthesis of gp130 by AG490 led to immediate loss of mature gp130 in cell membranes, due to its short half-life, thereby resulting in reduction in the STAT3 response to IL-6. Taken together, these results suggest that AG490 blocks the STAT3 activation pathway via a novel pathway.

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.

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.

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 alpha v 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 alpha v beta 3. Since the alpha v 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.

Tyrphostin ErbB2 Inhibitors AG825 and AG879 Have Non-specific Suppressive Effects on gp130/ STAT3 Signaling

Although the interaction between gp130 and the ErbB family has frequently been shown in cancer cells, the mechanism of this interaction remains unclear and controversial. In the present study, we found that specific tyrphostin inhibitors of ErbB2 (AG825 and AG879), but not ErbB1 inhibitor (AG1478), suppressed IL-6-induced tyrosine phosphorylation of STAT3 in schwannoma cells. However, biochemical evidence for transactivation of ErbB2 by IL-6 was not observed. Additionally, the inhibition of ErbB2 expression, with either a specific RNAi or transfection of an ErbB2 mutant lacking the intracellular domain did not inhibit the IL-6-induced tyrosine phosphorylation of STAT3. Thus, it seems that tyrphostins, which are known as specific inhibitors of the ErbB2 kinase, may have non-specific suppressive effects on the IL-6/STAT3 pathway.

Netrin Inhibits Regenerative Axon Growth of Adult Dorsal Root Ganglion Neurons in Vitro

Netrin is a neuronal guidance molecule implicated in the development of spinal commissural neurons and cortical neurons. The attractive function of netrin requires the receptor, Deleted in Colorectal Cancer (DCC), while the receptor Unc5h is involved in the repulsive action of netrin during embryonic development. Although the expression of netrin and its receptor has been demonstrated in the adult nervous system, the function of netrin in adult neurons has not yet been elucidated. Here, we show that netrin treatment inhibited neurite outgrowth of adult dorsal root ganglion (DRG) neurons in explant and dissociated cultures. In addition, unc5h1-3 mRNAs, but not the dcc mRNA, are abundantly expressed in the adult DRG. An in situ hybridization study demonstrated that unc5h mRNAs were expressed in DRG neurons. This finding indicates that netrin/Unc5h signaling may play a role in the neurite outgrowth of adult DRG neurons and that netrin may be involved in the regulation of peripheral nerve regeneration.

Expression Profile of Tumor Endothelial Marker 7 and a Putative Ligand in the Rat Spinal Cord and Dorsal Root Ganglion

STUDY DESIGN: To analyze the expression profile of tumor endothelial marker 7 (TEM7) in the spinal cord and dorsal root ganglion (DRG). PURPOSE: To investigate the expression profile of TEM7 in the spinal cord and DRG of adult and developing rats. OVERVIEW OF LITERATURE: Tumor endothelial marker 7 (TEM7) is a putative transmembrane protein that is highly expressed in the tumor endothelium and in cerebellar neurons. METHODS: In the present study, the expression profile of TEM7 in the spinal cord and DRG of the rat was investigated using in situ hybridization and immunohistochemical analysis. In addition, the secreted recombinant ectodomain of TEM7 was employed to label the expression of a putative ligand of TEM7 in the spinal cord and DRG. RESULTS: Specific TEM7 mRNA localization was observed in the motor neurons of the spinal cord and sensory neurons of the DRG. Glial cells and vascular endothelial cells did not show hybridization signals. Immunohistochemical analysis with a specific polyclonal antibody revealed a similar localization profile for TEM7 mRNA expression. In the spinal cord, weak labeling was observed in the gray matter. The TEM7 ectodomain localized the expression of a putative ligand of TEM7 in the neurilemmal structures and perineurium of the spinal nerve roots. In the DRG, ligand labeling was observed in the endoneurium and perineurium of the spinal nerves, and extracellular matrix around the sensory neurons. A developmental study has shown that TEM7 mRNA expression in the motor neurons of the spinal cord and DRG increased with age during postnatal development. Conclusion: These findings indicate that TEM7 plays a role as a transmembrane receptor in neuronal populations of the spinal cord and DRG.

zVAD-fmk, unlike BocD-fmk, does not inhibit caspase-6 acting on 14-3-3/Bad pathway in apoptosis of p815 mastocytoma cells

In a preliminary study, we found that benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD- fmk), unlike Boc-aspartyl(OMe)-fluoromethylketone (BocD-fmk), at usual dosage could not prevent genistein-induced apoptosis of p815 mastocytoma cells. This study was undertaken to reveal the mechanism underlying the incapability of zVAD-fmk in preventing this type of apoptosis. We observed that 14-3-3 protein level was reduced in genistein-treated cells and that BocD-fmk but not zVAD-fmk prevented the reduction of 14-3-3 protein level and the release of Bad from 14-3-3. We also demonstrated that truncated Bad to Bcl-xL interaction in genistein- treated cells was prevented by BocD-fmk but not by zVAD-fmk treatment. Our data indicate that BocD- fmk, compared to zVAD-fmk, has a certain preference for inhibiting 14-3-3/Bad signalling pathway. We also elucidated that this differential efficacy of BocD-fmk and zVAD-fmk resulted from the different effect in inhibiting caspase-6 and that co-treatment of zVAD-fmk and caspase-6 specific inhibitor substantially prevented genistein-induced apoptosis. Our data shows that caspase-6 plays a role on Bad/14-3-3 pathway in genistein-induced apoptosis of p815 cells, and that the usual dose of zVAD-fmk, in contrast to BocD-fmk, did not prevent caspase-6 acting on 14-3-3/Bad-mediated event.

Postnatal Developmental Expression of TEM7 mRNA and its Putative Ligand in the Rat Cerebellum / 대한해부학회지

Tumor endothelial marker 7 (TEM7) is a putative transmembrane protein that is highly expressed in the tumor endothelium and cerebellar neurons. In the present study, the expression profile of TEM7 mRNA and its putative ligand in the developing cerebellum of the rat was investigated using in situ hybridization and ligand binding assay. The secreted recombinant ectodomain of TEM7 was employed to label the expression of putative ligand of TEM7 in the cerebellum. The expression of a putative ligand of TEM7 demonstrated by using TEM7 ectodomain was found in the molecular layer of the cerebellum, where the dendritic trees of Purkinje cells are present. A developmental study has shown that TEM7 mRNA expression in the Purkinje neurons was increased with age during postnatal development, whereas the putative ligand labeling in the molecular layer was observed throughout the developmental period. These findings indicate that TEM7-ligand interaction plays a role in the differentiation of Purkinje cells during postnatal development.

Immunohistochemical Study on the Distribution of Tumor Endothelial Marker 7 in the Rat Forebrain / 대한해부학회지

Tumor endothelial marker 7 (TEM7) is a putative transmembrane protein that is highly expressed in the tumor endothelium. In the present study, the expression profile of TEM7 in the rat forebrain was investigated using immunohistochemistry with a specific polyclonal antibody against the extracellular region of TEM7. The immunohistochemical research revealed that TEM7 expressions were localized to specific neuronal areas such as cerebral cortex, hippocampus and hypothalamic magnocellular nuclei. The TEM7 protein was mainly present in the dendrite and cell body of the projection neurons. However, glial cells, vascular endothelial cells and meningeal cells did not show the expression of TEM7, indicating the specific roles of TEM7 in the neuronal cells in the vertebrate nervous system.

Fyn Tyrosine Kinase-mediated Tyrosine Phosphorylation of Roundabout (Robo), the Slit Receptor / 대한해부학회지

In this study, the molecular mechanism of tyrosine phosphorylation of Roundabout (Robo), the transmembrane receptor for slits, was investigated. The tyrosine phosphorylation of intracellular portion of Robo was increased by the treatment of tyrosine phosphatase inhibitors in human embryonic kidney cells transfected with Robo. The Robo tyrosine phosphorylation was inhibited by the treatment of Src family kinase inhibitor, PP2. The co-transfection of constitutively active form of Fyn, not the dominant negative form of Fyn, and Robo dramatically enhanced the tyrosine phosphorylation of Robo. Furthermore, the SH2 domain of Fyn, which binds to phosphorylated tyrosine residues, interact with Robo, and the interaction was increased by the inhibition of tyrosine phosphatases. These findings indicate that the tyrosine phosphorylation of Robo is regulated by Fyn.

Multiple Regulation of Roundabout (Robo) Phosphorylation in a Heterologous Cell System

Roundabout (Robo) is the transmembrane receptor for slit, the neuronal guidance molecule. In this study, the tyrosine phosphorylation of Robo was observed in Robo-transfected human embryonic kidney cells and developing rat brains, and found to be increased by the treatment with protein kinase A activator, forskolin. In contrast, protein kinase C activation by phorbol-12-myristate-13-acetate decreased the phosphorylation of Robo. Intracellular calcium was required for the tyrosine phosphorylation. Furthermore, the transfection of an Eph receptor tyrosine kinase dramatically enhanced the tyrosine phosphorylation. These findings indicate that the tyrosine phosphorylation of Robo is regulated by multiple mechanisms, and that Eph receptor kinases may play a role in the regulation of tyrosine phosphorylation of Robo in the rat brain.

Signal Transduction of Ras-like GTPase Activation in the Nervous System / 대한해부학회지

Synaptic plasticity has been thought to be a mechanism of synaptic maturation, learning and memory. In this study, the possible involvement of Rac, RhoA, proline-rich tyrosine kinase 2 (PYK2) and focal adhesion kinase (FAK) in the synaptic plasticity was investigated using PC12 cells and rat brains. The followings are the results. 1. Depolarization induced extracellular signal-regulated kinase (ERK) activation but did not activate Rac and RhoA in PC12 cells. 2. ERK activation and c-fos expression were observed after electroconvulsive shock (ECS) but the activity of Rac and RhoA was not changed following ECS. 3. PYK2 not FAK activation was observed after ECS. 4. The activity of PYK2 was increased with postnatal development but that of FAK was decreased with ages. 5. The expression of Rac and PYK2 was clearly observed in the postsynaptic density but that of RhoA and FAK was not. These findings indicate that PYK2 seems to play an important role in activity-dependent synaptic plasticity in vivo brain.