Yoshihiko Nakatani

Effect of rabbit anti-asialo-GM1 (GA1) polyclonal antibodies on neuromuscular transmission and acetylcholine-induced action potentials: Neurophysiological and immunohistochemical studies

We produced anti–asialo-GM1 (GA1) polyclonal antibodies by sensitizing New Zealand rabbits with GA1 and investigated the epitopes and pathogenic role of anti-GA1 antibodies that appeared in serum. The serum blocked neuromuscular transmission, but not acetylcholine (ACh)-induced potentials, in muscle–spinal cord cocultured cells. The effect was complement independent. The antibodies inhibited voltage-gated Ca2+ channel (VGCC). The epitopes recognized by the antibodies were located in the outer membrane of Schwann cells and motor axons of Wistar rat ventral roots and on motor axons extended from spinal cord to muscle cells in muscle–spinal cocultured cells. The ACh-induced potential was not reduced by the addition of sera, suggesting the blockade is presynaptic. Thus, anti-GA1 antibodies may block neuromuscular transmission by suppressing VGCC on axonal terminals of motor nerves.

IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells.

We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

Corticosterone suppresses the proliferation of BV2 microglia cells via glucocorticoid, but not mineralocorticoid receptor

AIMS Corticosterone (CORT), which is often referred to as the stress hormone, is a well-known regulator of peripheral immune responses and also shows anti-inflammatory properties in the brain. Microglia play a key role in immune response and inflammation in the brain. However, it is still unclear how CORT affects microglia. In this study, we focused on the effects of CORT on the proliferation and survival of microglia using mouse microglia cell line BV2. MAIN METHODS We used WST-8 and LDH (lactate dehydrogenase) assays to check the effects of CORT for the proliferation and survival in BV2 microglia cells. We also analyzed the expression pattern of proteins which related to CORT signal cascades using western blotting analysis. KEY FINDINGS Under treatment with 0.1, 1 and 10μM CORT for 24h, the BV2 proliferation rate decreased to 83, 77 and 70% of that in the control. Moreover, this inhibition was blocked by treatment with mifepristone, a glucocorticoid receptor (GR) antagonist, but not by spironolactone, a mineralocorticoid receptor (MR) antagonist. Moreover, an LDH assay showed that CORT was dose-dependently cytotoxic toward BV2 microglia cells and this cytotoxicity was partially abolished by treatment with mifepristone. In addition, treatment with CORT resulted in the translocation of GR, but not MR, from the cytosol to the nucleus. SIGNIFICANCE Our findings suggested that CORT suppresses the proliferation of BV2 microglia cells accompanied with a cytotoxic effect that is induced by the formation of a CORT-GR complex.

Cav2.1 Voltage-dependent Ca2+ Channel Current is Inhibited by Serum from Select Patients with Guillain-Barré Syndrome

To investigate the pathophysiological mechanisms of immune-mediated peripheral neuropathies, we studied the effects of sera from patients with Guillain-Barré syndrome (GBS) on the Cav2.1 voltage-dependent calcium channel (VDCC) current in Purkinje cells. Using the whole-cell recording technique, Cav2.1 VDCC current was measured in cerebellar Purkinje cells in the presence of serum from GBS patients with acute motor axonal neuropathy (AMAN) or acute inflammatory demyelinating polyneuropathy (AIDP). The AMAN patient sera significantly inhibited the Cav2.1 VDCC current compared with healthy volunteer sera, and this inhibition was fully reversible by washing out the AMAN serum. Similarly, IgG purified from AMAN sera also inhibited the Cav2.1 VDCC current. However, the activation and inactivation kinetics of the Cav2.1 VDCC currents were not affected by serum from an AMAN patient. Moreover, the VDCC current of Purkinje cells was also inhibited by IgG anti-GM1 monoclonal antibody (anti-GM1 mAb). In an immunocytochemical study using double fluorescence staining, Purkinje cells were stained by monoclonal IgG anti-GM1 mAb. In contrast, AIDP patient and healthy volunteer sera did not affect the Cav2.1 VDCC current. These results suggest that in some case of GBS, particularly of AMAN patients with IgG anti-GM1 mAb, muscle weakness may be induced by dysfunction of Cav2.1 VDCC functioning at the motor nerve terminals.

Expression and localization of Kv1 potassium channels in rat dorsal and ventral spinal roots

We investigated the expression and localization of Kv1 channels in dorsal spinal roots (DRs) and ventral spinal roots (VRs) in rats. Among Kv1.1-1.6 tested by RT-PCR, mRNAs of Kv1.1, 1.2, and 1.5 were moderately expressed, those of Kv1.3 and Kv1.6 were weakly expressed, and that of Kv1.4 was hardly expressed at all in both DRs and VRs, whereas all six mRNAs were detected in spinal cord. Western blotting revealed that the major immunoreactive proteins were Kv1.1 and Kv1.2 in both DRs and VRs. Quantitative analysis indicated that levels of Kv1.1 and Kv1.2 protein were significantly higher in DRs than VRs. Immunohistochemical examination showed that Kv1.1 and Kv1.2 were colocalized in juxtaparanodal regions of axons in both DRs and VRs. Finally, immunoprecipitation experiments revealed that Kv1.1 and Kv1.2 were coassembled. These findings indicate that Kv1 subtypes in DRs and VRs are somewhat different from those in spinal cord, and that the numbers of Kv1.1 and Kv1.2 channels are higher in DRs than VRs.

Neuroprotective effect of yokukansan against cytotoxicity induced by corticosterone on mouse hippocampal neurons.

Yokukansan, a traditional Japanese herbal medicine, has been used for the management of neurodegenerative disorders and for the treatment of neurosis, insomnia, and behavioral and psychological symptoms of dementia. Recently, several studies have shown that yokukansan has a neuroprotective effect. The aim of this study was to examine the neuroprotective effect of yokukansan on hippocampal neurons from embryonic mouse brain against the effects of corticosterone, which is considered to be a stress hormone and to be cytotoxic toward neurons. The cell survival rates were measured by the WST-8 assay and LDH assay. Twenty-four hours after treatment with corticosterone, cell numbers were significantly decreased compared with the control or treatment with vehicle in a dose-dependent manner. When cells were treated with 30 μM corticosterone, the decrease in the number of cells was significantly recovered by treatment with yokukansan (100-1,000 μg/ml) in a dose-dependent manner. However, yokukansan did not suppress the decrease in cell numbers that was induced by treatment with 100 μM corticosterone. In the LDH assay, treatment with yokukansan at a high concentration (500-1,000 μg/ml) suppressed the LDH concentration induced by treatment with both 30 μM and 100 μM corticosterone compared to treatment with corticosterone alone, respectively. These results suggest that yokukansan protects against the cytotoxic effect of a low concentration of corticosterone on hippocampal neurons.

Ca2+ Channel Currents Inhibited by Serum from Select Patients with Guillain-Barré Syndrome

We performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a coculture of rat muscle and spinal cord by exposure to serum, as well as purified IgG, from patients with the acute motor axonal neuropathy (AMAN) variant of Guillain-Barré syndrome (GBS). However, exposure to serum from two patients with the acute inflammatory demyelinating polyneuropathy (AIDP) form of GBS had no effect. Using a whole-cell recording technique, we then investigated the effects of serum and purified IgG from patients with GBS on voltage-dependent calcium channel (VDCC) currents in nerve growth factor-differentiated PC12 cells. Serum from patients with GBS (AMAN) inhibited VDCC currents in PC12 cells, which was fully reversible by washing with the bath solution. Similarly, purified IgG from the serum of two patients with GBS (AMAN) also inhibited VDCC currents in PC12 cells. In contrast, sera from patients with AIDP and healthy volunteers did not affect VDCC currents in PC12 cells. These results suggest that muscle weakness in some patients with GBS might be induced by inhibition of Ca2+ channel currents within motor nerve terminals.

Topology and patch-clamp analysis of the sodium channel in relationship to the anti-lipid a antibody in campylobacteriosis

An infecting strain VLA2/18 of Campylobacter jejuni was obtained from an individual with campylobacteriosis and used to prepare chicken sera by experimental infection to investigate the role of serum anti‐ganglioside antibodies in Guillain‐Barré syndrome. Both sera of the patient and chicken contained anti‐ganglioside antibodies and anti‐Lipid A (anti‐Kdo2‐Lipid A) antibodies directed against the lipid A portion of the bacterial lipooligosaccharide. The anti‐Kdo2‐Lipid A activities inhibited voltage‐gated Na (Nav) channel of NSC‐34 cells in culture. We hypothesized that anti‐Kdo2‐Lipid A antibody acts on the functional inhibition of Nav1.4. To test this possibility, a rabbit peptide antibody (anti‐Nav1.4 pAb) against a 19‐mer peptide (KELKDNHILNHVGLTDGPR) on the α subunit of Nav1.4 was produced. Anti‐Nav1.4 pAb was cross‐reactive to Kdo2‐Lipid A. Anti‐Kdo2‐lipid A antibody activity in the chicken serum was tested for the Na+ current inhibition in NSC‐34 cells in combination with μ‐Conotoxin and tetrodotoxin. Contrary to our expectations, the anti‐Kdo2‐Lipid A antibody activity was extended to Nav channels other than Nav1.4. By overlapping structural analysis, it was found that there might be multiple peptide epitopes containing certain dipeptides showing a structural similarity with v‐Lipid A. Thus, our study suggests the possibility that there are multiple epitopic peptides on the extracellular domains of Nav1.1 to 1.9, and some of them may represent target sites for anti‐Kdo2‐Lipid A antibody, to induce neurophysiological changes in GBS by disrupting the normal function of the Nav channels.

Effects of IgM Anti-Galnac-GD1a Monoclonal Antibody on NeuromuscularTransmission and Calcium Channel Binding in the Rat Neuromuscular Junction

Guillain-Barre syndrome with antibodies against ganglioside N-acetylgalactosaminyl GD1a (GalNAc-GD1a) is characterized by a rapid onset of predominantly distal pure motor neuropathy. However, the pathogenic role of anti- GalNAc-GD1a antibody and calcium channels in neuromuscular junctions (NMJs) remains unclear. We investigated the effects of IgM anti-GalNAc-GD1a monoclonal antibody (IgM anti-GalNAc-GD1a mAb) on spontaneous muscle action potentials (SMAP) in a rat spinal cord–muscle co-culture system and the localization of IgM anti-GalNAc- GD1a mAb and calcium channel binding in the rat hemi-diaphragm. Immunohistochemistry of the rat hemidiaphragm showed that IgM anti-GalNAc-GD1a mAb binding overlapped with anti-neurofilament 200 antibody and α- bungarotoxin staining, demonstrating that IgM anti-GalNAc-GD1a mAb was localized at the motor nerve terminal. Moreover, IgM anti-GalNAc-GD1a mAb binding overlapped with anti-Cav2.1 antibody in the nerve terminal. We suggest that the inhibitory effect of IgM anti-GalNAc-GD1a mAb on SMAP is related to the GalNAc-GD1a epitope on P/Q-type calcium channels in motor nerve terminals at NMJs.

Neurophysiological and immunohistochemical studies of IgG anti-GM1 monoclonal antibody on neuromuscular transmission: effects in rat neuromuscular junctions

Guillain–Barré syndrome, which is a variant of acute inflammatory neuropathy, is associated with anti-GM1 antibodies and causes ataxia. We investigated the effects of IgG anti-GM1 monoclonal antibody (IgG anti-GM1 mAb) on spontaneous muscle action potentials in a rat spinal cord–muscle co-culture system and the localization of IgG anti-GM1 mAb binding in the rat hemi-diaphragm. The frequency of spontaneous muscle action potentials in innervated muscle cells was acutely inhibited by IgG anti-GM1 mAb. When cultures were pretreated with GM2 synthase antisense oligodeoxynucleotide, IgG anti-GM1 mAb failed to inhibit spontaneous muscle action potentials, demonstrating the importance of the GM1 epitope in the action of IgG anti-GM1 mAb. Immunohistochemistry of rat hemi-diaphragm showed that IgG anti-GM1 mAb binding overlapped with neurofilament 200 (NF200) antibodies staining, but not α-bungarotoxin (α-BuTx) staining, demonstrating that IgG anti-GM1 mAb was localized at the presynaptic nerve terminal. IgG anti-GM1 mAb binding overlapped with syntaxin antibody and S-100 antibody in the nerve terminal. After collagenase treatment, IgG anti-GM1 mAb and NF200 antibodies did not show staining, but α-BuTx selectively stained the hemi-diaphragm. IgG anti-GM1 mAb binds to the presynaptic nerve terminal of neuromuscular junctions. Therefore, we suggest that the inhibitory effect of IgG anti-GM1 mAb on spontaneous muscle action potentials is related to the GM1 epitope in presynaptic motor nerve terminals at the NMJs.