Tadashi Miyatake

Neurophysiological and immunohistochemical studies on Guillain-Barré syndrome with IgG anti-GalNAc-GD1a antibodies—effects on neuromuscular transmission

We investigated the epitopes and functional role of IgG anti-GalNAc-GD1a antibodies appearing in serum from a patient with Guillain-Barre syndrome (GBS) and IgG anti-GalNAc-GD1a antibody that was produced by immunization of a rabbit with GalNAc-GD1a. Both sera blocked neuromuscular transmission in muscle-spinal cord co-culture cells. The acetylcholine-induced potential did not reduce by adding sera, suggesting that the blockade is presynaptic. The effect was complement-independent and purified IgG from serum of the patient or the rabbit had the same effects. The epitope with both anti-GalNAc-GD1a antibodies was observed in the soma of large neurons in the anterior horns of the adult rat spinal cord and their motor axons of rat ventral roots. Both anti-GalNAc GD1a antibodies reacted strongly with the motor nerve terminals in rats. The anti-GalNAc-GD1a antibodies may block neuromuscular transmission by attacking on presynaptic motor axon, probably affecting the ion channels in the presynaptic motor axon.

Biochemical and pathological study of endogenous 1-benzyl-1,2,3,4-tetrahydroisoquinoline-induced parkinsonism in the mouse.

We administered 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BnTIQ; 80 mg/kg, i.p.), an endogenous neurotoxin known to cause bradykinesia, the Parkinsons disease-like symptom, in order to obtain biochemical and pathological evidence of behavioral abnormalities. Immunohistochemical analysis demonstrated that 1-BnTIQ did not decrease the number of tyrosine hydroxylase-positive cells in the substantia nigra. Biochemical analysis demonstrated significantly increased striatal dopamine (DA) content, while DA metabolites in the striatum remained at control levels. We concluded that the 1-BnTIQ-induced bradykinesia has a different mechanism of action than that underlying the MPTP-induced depletion of striatal DA neurons.

Stereoselective effect of (R)- and (S)-1-methyl-1,2,3,4-tetrahydroisoquinolines on a mouse model of Parkinson’s disease

We carried out behavioral, pathological, and biochemical studies in order to determine whether the stereo-structure of 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ) affects the onset of Parkinsons disease-like symptoms, which are induced by 1,2,3,4-tetrahydroisoquinoline (TIQ) in mice. Pretreatment with (R)-1-MeTIQ or its racemate (RS)-1-MeTIQ prevented the TIQ-induced bradykinesia. Pretreatment with a combination of L-DOPA and carbidopa significantly prevented subsequent TIQ-induced bradykinesia. Furthermore, the pathological study demonstrated that either (R)-1-MeTIQ or its racemate protected against TIQ-induced loss of tyrosine hydroxylase-positive cells of the substantia nigra pars compacta. (R)-1-MeTIQ and its racemate also prevented the TIQ-induced reduction in the levels of dopamine and its metabolites in the striatum. Serotonin and its metabolite were not affected by repeated administration of (RS)-1-MeTIQ or its derivatives. On the other hand, (S)-1-MeTIQ induced moderate but significant bradykinesia, whereas (R)-1-MeTIQ did not induce this behavioral abnormality at all. In addition, (S)-enantiomer prevented the onset of TIQ-induced bradykinesia, though to a lesser extent than did either (R)-enantiomer or its racemate. However, (S)-enantiomer did not prevent the loss of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. We concluded that (R)-1-MeTIQ, and not (S)-enantiomer, plays a crucial role in protection against TIQ-induced parkinsonism, a fact which suggests that enantiomeric biochemical events such as 1-MeTIQ biosynthesis may participate in the pathogenesis of Parkinsons disease.

Recent studies on the roles of antiglycosphingolipids in the pathogenesis of neurological disorders

Evidence is mounting to suggest a causal role of humoral immunity arising from antiglycosphingolipid (GSL) antibodies in a variety of neurological disorders. These disorders include the demyelinating and axonal forms of Guillain‐Barre syndrome, multifocal motor neuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, and IgM paraproteinemia. Many claims have been made regarding other neurological disorders, which should be carefully scrutinized for their validity, based on several criteria proposed in this review. These criteria include 1) characterization of the causative antigens and immunoglobulins, 2) correlation of the pathological lesions and clinical manifestation of the antigens, 3) establishment of animal models using pure GSLs as the antigens, 4) immunopathogenic mechanisms of the neurodenerative process, 5) mechanisms for the malfunctioning of blood–nerve barrier and the ensuing leakage of circulating antibodies into peripheral nerve parenchyma, and 6) the roles of anti‐GSL antibodies that may cause humorally mediated nerve dysfunction and injury as well as interference with ion channel function at the node of Ranvier, where carbohydrate epitopes are located. Finally, the origin of the anti‐GSL antibodies is discussed in light of the recent circumstantial evidence pointing to a molecular mimicry mechanism with infectious agents. With a better understanding of the immunopathogenic mechanisms, it will then be possible to devise rational and effective diagnostic and therapeutic strategies for the treatment of these neurological disorders. J. Neurosci. Res. 65:363–370, 2001.

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.

Characterization of muscarinic receptor subtypes in the rostral ventrolateral medulla and effects on morphine-induced antinociception in rats

The present study investigated the role of muscarinic receptor subtypes in the nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha of the rat rostral ventrolateral medulla in morphine-induced antinociception. The antinociceptive effects of morphine were evoked by systemic administration or microinjection into the nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha. Administration of morphine produced antinociception for hot plate and tail immersion responses to noxious heat stimuli. These effects were antagonized by prior exposure to naloxone and inhibited by mecamylamine pretreatment. Morphine-induced antinociception was significantly inhibited by atropine in a dose-dependent manner. Muscarinic toxin-1 and pirenzepine inhibited morphine-induced antinociception for both the hot plate and tail immersion tests. At a dose of 5 nmol/site, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) also inhibited morphine-induced antinociception, although low doses of this drug did not significantly affect hot plate test response latency when morphine was systemically administered. These results suggest that the antinociceptive effects induced by morphine in part involve the muscarinic M(1) and M(3) receptors of the rat nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha.

GalNAc-GD1a is localized specifically in ventral spinal roots, but not in dorsal spinal roots

We investigated the localization of GalNAc-GD1a biochemically in the human and bovine peripheral nervous system (PNS). The high-performance thin-layer chromatography (HPTLC)-overlay method with rabbit IgG polyclonal antibody against GalNAc-GD1a (anti-GalNAc-GD1a antibody) revealed expression of GalNAc-GD1a in the ventral spinal nerve roots (VRs) but not in the dorsal spinal nerve roots (DRs) of both species. The amount of GalNAc-GD1a in the human and bovine VRs was 2.22 +/- 0.35 microg/g wet tissue and 7.71 +/- 0.49 microg/g wet tissue, respectively. These results suggest that IgG anti-GalNAc-GD1a antibody may be involved in disturbance of peripheral motor nerves and in the pathogenesis of pure motor neuropathy.

GM2 Ganglioside Regulates the Function of Ciliary Neurotrophic Factor Receptor in Murine Immortalized Motor Neuron-Like Cells (NSC-34)

We previously reported that ciliary neurotrophic factor (CNTF) increased the serum-free cell survival of immortalized motor neuron-like cells (NSC-34), and addition of the exogenous ganglioside GalNAcβ4(Neu5Acα3)Galβ4GlcCer (GM2) facilitated cell survival together with CNTF. Moreover β 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity increased in NSC-34 cells cultured with CNTF. We now have examined whether CNTF-induced cell survival is associated with the collaboration between GM2 and the CNTF receptor (CNTF-R). Despite the presence of CNTF (50 ng/ml), anti-CNTF-R antibody caused cell death and prevented the up-regulation of GM2 synthase expression. The addition of GM2 (1 to 20 μM) abrogated the anti-CNTF-R antibody effect which shortened cell survival and blocked GM2 synthase activation. Use of [125I]CNTF showed the specificity of CNTF binding in NSC-34 cells in situ. GM2 produced a 5-fold increase in the CNTF binding affinity per cell but did not change the binding site number. The study by metabolic labeling with [1−14C]N-acetyl-D-galactosamine ([14C]GalNAc) showed that biosynthesized GM2 was involved in the immunoprecipitation of CNTF-R. These findings indicate that up-regulated GM2 synthesis induces functional conversion of CNTF-R to the activated state, in which it has affinity for CNTF. We conclude that GM2 is a bio-regulating molecule of CNTF-R in motor neurons.

Modulation of noradrenergic and serotonergic transmission by noxious stimuli and intrathecal morphine differs in the dorsal raphe nucleus of anesthetized rat: in vivo voltammetric studies

We examined the effects of cutaneous noxious heat as well as the intrathecal administration of morphine on the oxidation current of peaks 1 and 2 in the dorsal raphe nucleus (DRN) of anesthetized rats. Differential normal pulse voltammetry with carbon fiber electrodes identified distinct oxidation currents at +120 mV (peak 1: catechol signals) and +280 mV (peak 2: 5-hydroxyindole signals). The catechol signal was significantly increased by 22.9 +/- 4.2% after applying cutaneous noxious heat at 52 degrees C. The 5-hydroxyindole signal was decreased by 39.8 +/- 4.3 and by 25.2 +/- 4.7% after stimulation with cutaneous noxious heat at 52 and 45 degrees C, respectively. A low dose of morphine (2.5 microg) potentiated the increase in the catechol signal and the decrease in the 5-hydroxyindole signal induced by noxious heat, and a high dose (10.0 microg) attenuated both. The effects of morphine at low (2.5 microg) and high doses (10.0 microg) were antagonized by naloxone (0.5 mg/kg, i.p.). These results indicate that noxious heat stimulation increased the catechol signal and decreased the 5-hydroxyindole signal in the DRN. The intrathecal administration of morphine affects the noxious stimulation-induced activity of noradrenergic and serotonergic neurotransmission in the DRN.

β-Phenylethylamine modulates acetylcholine release in the rat striatum: involvement of a dopamine D2 receptor mechanism

We examined the effects of beta-phenylethylamine on striatal acetylcholine release in freely moving rats using in vivo microdialysis. beta-Phenylethylamine at 12.5 mg/kg, i.p. did not affect acetylcholine release in the striatum, whereas 25 and 50 mg/kg, i.p. immediately induced an increase in acetylcholine release in the striatum at 15-45 min. This increase following intraperitoneal administration of beta-phenylethylamine (25 mg/kg) was not affected by locally applied SCH-23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine, 10 microM), a dopamine D(1) receptor antagonist, nor by raclopride (10 microM), a dopamine D(2) receptor antagonist. The increased release of acetylcholine induced by beta-phenylethylamine was suppressed by local infusion of tetrodotoxin (1 microM). In contrast, the extracellular acetylcholine level in the striatum was significantly decreased by local application of beta-phenylethylamine (10 and 100 microM) in the striatum via a microdialysis probe. The decrease was completely blocked by local co-application of raclopride (10 microM). The beta-phenylethylamine-induced decrease in striatal acetylcholine release was not affected by co-perfusion with SCH-23390 (10 microM). These results indicate that systemic administration of beta-phenylethylamine increases acetylcholine release, whereas locally applied beta-phenylethylamine decreases striatal acetylcholine release in freely moving rats. Furthermore, the dopaminergic system, through the dopamine D(2) receptor, is involved in the locally applied beta-phenylethylamine-induced decrease in acetylcholine in the striatum.