Chikao Nakayama

In Vitro and in Vivo Characterization of a Novel Semaphorin 3A Inhibitor, SM-216289 or Xanthofulvin

SM-216289 (xanthofulvin) isolated from the fermentation broth of a fungal strain, Penicillium sp. SPF-3059, was identified as a strong semaphorin 3A (Sema3A) inhibitor. Sema3A-induced growth cone collapse of dorsal root ganglion neurons in vitro was completely abolished in the presence of SM-216289 at levels less than 2 μm (IC50 = 0.16 μm). When dorsal root ganglion explants were co-cultured with Sema3A-producing COS7 cells in a collagen gel matrix, SM-216289 enabled neurites to grow toward the COS7 cells. SM-216289 diminished the binding of Sema3A to its receptor neuropilin-1 in vitro, suggesting a direct interference of receptor-ligand association. Moreover, our data suggest that SM-216289 interacted with Sema3A directly and blocked the binding of Sema3A to its receptor. We examined the efficacy of SM-216289 in vivo using a rat olfactory nerve axotomy model, in which strong Sema3A induction has been reported around regenerating axons. The regeneration of olfactory nerves was significantly accelerated by a local administration of SM-216289 in the lesion site, suggesting the involvement of Sema3A in neural regeneration as an inhibitory factor. SM-216289 is an excellent molecular probe to investigate the function of Sema3A, in vitro and in vivo, and may be useful for the treatment of traumatic neural injuries.

Analysis of effects and pharmacokinetics of subcutaneously administered BDNF

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family and has been shown to be a potent and effective trophic factor for motor neurons and other neurons of the peripheral and central nervous. Little is known, however, about the relationship between the efficacy and pharmacokinetics of s.c. administered BDNF. In this study, the efficacy of BDNF on motor neuron protection in sciatic or facial nerve axotomy models was examined and compared with the concommitant concentrations of BDNF in plasma. Delayed treatment (started at 1 week after surgery) of BDNF was also shown to retard choline acetyltransferase reduction in sciatic nerve axotomy models.

Enhancement of BDNF and activated-ERK immunoreactivity in spinal motor neurons after peripheral administration of BDNF

Brain-derived neurotrophic factor (BDNF) shows neurotrophic effects on adult motor neurons when given systemically, But it is unknown whether systemically administered BDNF is transported to central cell bodies to affect them directly. Here we used immunohistochemistry to investigate the transport of peripherally injected BDNF to spinal motor neurons and the subsequent activation of a signaling pathway. We first injected BDNF into the flexor digitorum brevis (FDB) and analyzed the motor nucleus that projects to the FDB for BDNF immunoreactivity (BDNF-ir) and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 immunoreactivity (pERK1/2-ir). Both immunoreactivities were observed in the motor neuron cell bodies. Next, BDNF was injected subcutaneously (s.c.) into rats with a unilaterally axotomized sciatic nerve. pERK1/2-ir was detected in motor neurons of the lesioned side. BDNF-ir and pERK1/2-ir were also observed on the unlesioned side when a high dose of BDNF was injected. Therefore, we examined BDNF-ir and pERK1/2-ir after injecting BDNF s.c. into normal rats. Both immunoreactivities were observed in motor nuclei on both sides. Finally, we examined pERK1/2-ir after a lower dose of BDNF was injected, which prevents the decrease in choline acetyl transferase that occurs in the motor neuron upon axotomy. Spinal motor nuclei contained a few cell bodies with pERK1/2-ir. These findings represent the first direct evidence that subcutaneously injected BDNF is transported to motor neurons and that it activates a signaling pathway in the spinal cord and exhibits neurotrophic effects in vivo.

Synthetic Nucleosides and Nucleotides. XX1. Synthesis of Various 1-β-D-Xylofuranosyl-5-Alkyluracils and Related Nucleosides

Abstract Treatment of D-xylose (1) with 0.5% methanolic hydrogen chloride under controlled conditions followed by benzoylation and acetolysis afforded crystalline 1-O-acetyl-2, 3, 5-tri-O-benzoyl-α-D-xylofuranose (4) in good yield. Coupling of 4 with 2, 4-bis-trimethylsilyl derivatives of 5-alkyluracils (methyl, ethyl, propyl and butyl) (5a-5d), 5-fluorouracil (5e) and uracil (5f) in acetonitrile in the presence of stannic chloride gave 1-(2,3,5-tri-O-benzoyl-β-D-xylofuranosyl)-nucleosides (6a-6f). Saponification of 6 with sodium methoxide afforded 1-β-D-xylofuranosyl-5-substituted uracils (7a-7f). Condensation of 4 with free adenine in similar fashion and deblocking gave carcinostatic 9-β-D-xylofuranosyladenine (7g).

Riluzole slows the progression of neuromuscular dysfunction in the wobbler mouse motor neuron disease

In the wobbler mouse motor neuron disease (MND), we firstly evaluated the effect of riluzole, the only approved drug for amyotrophic lateral sclerosis, and compared it with that of brain-derived neurotrophic factor (BDNF). Wobbler mice received either daily subcutaneous treatment with BDNF (5, 20, and 40 mg/kg) or oral riluzole in drinking water (100 and 200 microg/ml), beginning immediately after the clinical onset of MND. We examined motor functions, such as grip strength and rota-rod walking performance, weekly, and the amplitude of the compound muscle action potential (CMAP) in the forelimb biceps at the end of treatment. BDNF treatment slowed the disease progression maximally at a dose of 20 mg/kg, consistent to the previous evidence. Only high-dose riluzole treatment increased grip strength at weeks 1 (P=0.0023) and 2 (P=0.021), time before falling in the rota-rod test throughout all 4 weeks of treatment (P=0.0022 to 0.0282), and CMAP amplitude (P=0.0069) at the end of treatment, compared with the vehicle. Furthermore, the riluzole treatment increased the number of the cervical cord anterior horn neurons that were immunoreactive for SMI-32, a specific motor neuron marker, by the end of treatment (P=0.0063), although it did not affect the vacuolar degeneration on the SMI-32-positive neurons. This study demonstrated that riluzole was comparable to BDNF in slowing the progression of neuromuscular dysfunction in the wobbler mouse MND, which may provide a useful model for examining the mechanisms of selective motor neuron degeneration.

Brain-derived neurotrophic factor shows a protective effect and improves recovery of the ERG b-wave response in light-damage

We investigated the neuroprotective effects of brain‐derived neurotrophic factor (BDNF) and its influence on the functional recovery of the retina following light‐induced retinal damage by electroretinogram (ERG). Rats were exposed to constant fluorescent light for 2, 5, 7, or 14 days, then returned to a cyclic light environment for 14 days. The result indicated that BDNF had few effects on the a‐wave amplitude, but there was a statistically significant difference in the b‐wave amplitudes between BDNF‐treated and control eyes from day 0–14 of the recovery period following 2 days of light exposure (p < 0.05). Our findings suggest that BDNF not only protects the retinal neuronal function but also enhances the recovery from retinal light damage.

In Vitro Antiherpesviral Activity of 5-Alkyl Derivatives of 1-β-d-Arabinofuranosyluracil

Several 5-alkyl derivatives of 1-β-d-arabinofuranosyluracil (araU) were tested for antiherpesviral activity and inhibitory action on cell growth in human embryonic lung fibroblasts. 1-β-d-Arabinofuranosylcytosine, 9-β-d-arabinofuranosyladenine, and 5-iododeoxyuridine (IUdR) were included as reference materials. Among the 5-alkyl derivatives of araU, arabinosylthymine was the most active, followed by 5-ethyl- and 5-propyl-araU. 5-Ethyl-araU was as active as IUdR and more active than 9-β-d-arabinofuranosyladenine against herpes simplex virus (HSV) type 1 and did not inhibit cell growth at a concentration as high as 1,000 μg/ml. 5-Butyl- and 5-methoxymethyl-araU, as well as araU, exhibited relatively low activity. The araU derivatives tested were as active against HSV WT-34, an isolate from a patient with keratitis, as against HSV type 1. Against an IUdR-resistant isolate, HSV WT-20, arabinosylthymine was less inhibitory than IUdR. Deoxyribonucleic acid synthesis in HSV type 1-infected cells was markedly inhibited by arabinosylthymine, IUdR, and 5-ethyl-araU, whereas cellular deoxyribonucleic acid synthesis in uninfected cells was significantly inhibited by IUdR but not by arabinosylthymine or 5-ethyl-araU.

Ciliary neurotrophic factor protects rat retina cells in vitro and in vivo via PI3 kinase

Purpose. Neurotrophic factors and neurotrophins are well-known to have neuroprotective efficacy against retinal injury. The aim of this experiment is to investigate the signal transduction pathway of ciliary neurotrophic factor (CNTF) on the upregulation of viability of retinal primary culture and retinal protection against constant light damage in vivo. CNTF is known to enhance the viability of retinal culture and provide protection under constant light exposure conditions, but little is known about how the signal transduction pathways of CNTF affect retina function. Methods. Primary retinal cultures were prepared from 7-day-old Wistar rats. Brain-derived neurotrophic factor (BDNF) (0.1, 1, 10 ng/ml), CNTF (0.1, 1, 10 ng/ml), PD98059 (10, 100, 1000 nM), or LY294002 (10, 100, 1000 nM) was added to these cultures at the time of cell preparation. After 3 days, the percentage of cells surviving was assessed using alamarBlue. For the in vivo experiment, inhibitors for the MAPKK (PD98059, 10 µg/eye) or PI3K (LY294002, 10 µg/eye) pathways were injected into the vitreous together with CNTF (1 µg/eye) 2 days before constant light exposure. Electroretinogram (ERG) analysis was performed to investigate which pathway was used by CNTF. Results. CNTF at 1, 10, or 100 ng/ml enhanced cell viability in retinal cultures. The cell-survival activity of CNTF was blocked by 10 ng/ml LY294002 (Dunnets test, p < 0.05). In vivo, the neuroprotective activity of CNTF in constant-light conditions was attenuated by 10 µg/eye LY294002 (Dunnets test, p < 0.05). Conclusions. These data suggest that CNTF promotes cell survival via the PI3K signaling pathway in vitro and in vivo.

Methionine-free brain-derived neurotrophic factor in wobbler mouse motor neuron disease: dose-related effects and comparison with the methionyl form

We compared the clinical and pharmacodynamic effects of N-terminal methionine brain-derived neurotrophic factor (met-BDNF) and endogenous met-free BDNF in wobbler mouse motor neuron disease (MND). Met- or met-free BDNF at 5 or 20 mg/kg was subcutaneously injected daily, six times/week for 4 weeks. At 20 mg/kg, grip strength (P<0.05, met-free BDNF; P<0.01, met-BDNF) and running speed (P<0.01 for both groups) improved compared to vehicle. At 5 mg/kg, the beneficial effect was more modest. Plasma BDNF levels after the final injection were dose-dependent and did not differ between BDNF groups. Endogenous met-free BDNF exerts effects similar to met-BDNF in wobbler MND.

Construction and Characterization of Ciliary Neurotrophic Factor (CNTF) Antagonists: Microenvironmental Difference in the CNTF Receptor Between Rat and Chicken Cells for Recognizing the D1 Cap Region

Abstract: Antagonistic mutants of ciliary neurotrophic factor (CNTF) were constructed and their properties characterized. K155A and K155W mutants lost cell survival promoting activity for chicken dorsal root ganglion (DRG) neurons and inhibited the activity of the wild type. However, they retained slight agonistic activity for the survival of rat DRG neurons, indicating there is a difference between chicken and rat cells for receptor recognition around the D1 cap region including K155 residue. The chicken receptor recognizes the D1 cap region more strictly than does the rat receptor. The substitution of F152, which locates at the top of the D1 cap region, was combined with the K155A mutation. A combination of the two mutations gave an antagonistic feature to not only chicken but also rat cells. Both F152S/K155A and F152D/K155A mutants lacked cell survival promoting activity and had an antagonistic effect on rat DRG neurons. The three‐dimensional structure of CNTF suggests the following. F152 and K155 bind to the receptor with hydrophobic and electrostatic interactions, respectively. F152 locates close to L156 with a van der Waals contact, and K155 contacts with Q42 through a hydrogen bond. Both interactions play indispensable roles in maintaining the structure around the D1 cap region of CNTF.