Kaoru Kikuchi

A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell–mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.

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.

Cloning and Characterization of a Novel Class VI Semaphorin, Semaphorin Y ☆

Semaphorins comprise a large family of proteins implicated in axonal guidance. We cloned a novel transmembrane semaphorin, semaphorin Y (Sema Y), which has a class VI sema domain. Sema Y shows growth cone collapsing activity on DRG neurons in vitro, and the target regions of the DRG neurons express sema Y mRNA during development. Sema Y may be a stop signal for these neurons in their target areas. Interestingly, sema Y mRNA was also detected in other neurons and their targets. Two isoforms of Sema Y derived from alternative splicing were identified and their expression was found to be regulated in a tissue- and age-dependent manner. Distribution of sema Y mRNA suggests that Sema Y might also be important during maintenance of axonal connections and/or differentiation and migration of cells. Sequence comparison among class VI semaphorins revealed two short conserved sequence stretches in their cytoplasmic domains, suggesting interaction of these semaphorins with a common intracellular component(s).

Molecular cloning of a novel member of semaphorin family genes, semaphorin Z.

Semaphorins/collapsins (semaphorins) comprise a large family of proteins implicated in axonal guidance during development. We cloned a novel member (semaZ) of the semaphorin gene family from a rat brain cDNA library. Sema Z was thought to be an integral membrane glycoprotein of 887 amino acids including a sema domain composed of 532 amino acids. The amino acid sequence of Sema Z showed 28-35% identity with other semaphorins in its sema domain, including 15 conserved cysteine residues. The cytoplasmic domain of Sema Z was found to be rich in prolines. Our phylogenetic analysis based on the amino acid sequence of the sema domains and the location of conserved N-glycosylation sites suggested that the sema domain of Sema Z belongs to a new class, class VI. We detected the semaZ mRNA in the first branchial arch of embryonic day 11 (E11) rat embryo, and subsequently in the myotomes and the dorsal root ganglia in developing somites from E11.5 through E13.5, but not in the brain. However, at E15, 18, 21 and P0, semaZ was highly expressed in the brain. Sema Z might play a role in both peripheral and central nervous system development.

Rewiring of regenerated axons by combining treadmill training with semaphorin3A inhibition

BackgroundRats exhibit extremely limited motor function recovery after total transection of the spinal cord (SCT). We previously reported that SM-216289, a semaphorin3A inhibitor, enhanced axon regeneration and motor function recovery in SCT adult rats. However, these effects were limited because most regenerated axons likely do not connect to the right targets. Thus, rebuilding the appropriate connections for regenerated axons may enhance recovery. In this study, we combined semaphorin3A inhibitor treatment with extensive treadmill training to determine whether combined treatment would further enhance the “rewiring” of regenerated axons. In this study, which aimed for clinical applicability, we administered a newly developed, potent semaphorin3A inhibitor, SM-345431 (Vinaxanthone), using a novel drug delivery system that enables continuous drug delivery over the period of the experiment.ResultsTreatment with SM-345431 using this delivery system enhanced axon regeneration and produced significant, but limited, hindlimb motor function recovery. Although extensive treadmill training combined with SM-345431 administration did not further improve axon regeneration, hindlimb motor performance was restored, as evidenced by the significant improvement in the execution of plantar steps on a treadmill. In contrast, control SCT rats could not execute plantar steps at any point during the experimental period. Further analyses suggested that this strategy reinforced the wiring of central pattern generators in lumbar spinal circuits, which, in turn, led to enhanced motor function recovery (especially in extensor muscles).ConclusionsThis study highlights the importance of combining treatments that promote axon regeneration with specific and appropriate rehabilitations that promote rewiring for the treatment of spinal cord injury.

V170 Area Plays an Essential Role in the Biological Activity of Human Ciliary Neurotrophic Factor

Abstract: The structure‐function relationships of human ciliary neurotrophic factor (CNTF) were analyzed by mutagenic means. Amino acid substitutions at helix D caused marked changes in the biological activity of CNTF, suggesting that the residues at helix D of CNTF participate in receptor recognition. In particular, both the cell survival‐promoting activity and receptor binding ability of V170 mutant CNTF proteins correlated well with the hydrophobicity of amino acids at position 170. The reduction of hydrophobicity at position 170 resulted in a loss of biological activity, indicating that the hydrophobicity of V170 is essential for the receptor binding and cell survival‐promoting activity. Substitutions of R171 or D175 evoked very little folding ability and negated the biological activity of CNTF. As R171 and D175 interact electrostatically with each other and with E75 and R72, respectively, these interactions would be indispensable for stabilizing the whole CNTF protein and for maintaining the structure of the receptor binding epitope.

Translational research into species differences of endocrine toxicity via steroidogenesis inhibition by SMP-028--for human safety in clinical study.

SMP-028 is a drug candidate developed for the treatment of asthma. In a 13-week repeated dose toxicity study of SMP-028 in rats and monkeys, differences of endocrine toxicological events between rats and monkeys were observed. In rats, these toxicological events mainly consisted of pathological changes in the adrenal, testis, ovary, and the other endocrine-related organs. On the other hand, in monkeys, no toxicological events were observed. The goal of this study is to try to understand the reason why only rats, but not monkeys, showed toxicological events following treatment with SMP-028 and to eventually predict the possible toxicological effect of this compound on human endocrine organs. Our results show that SMP-028 inhibits neutral cholesterol esterase more strongly than other steroidogenic enzymes in rats. Although SMP-028 also inhibits monkeys and human neutral cholesterol esterase, this inhibition is much weaker than that of rat neutral cholesterol esterase. These results indicate (1) that the difference in endocrine toxicological events between rats and monkeys is mainly due to inhibition of steroidogenesis by SMP-028 in rats, not in monkeys, and (2) that SMP-028 may not affect steroidogenesis in humans and therefore might cause no endocrine toxicological events in clinical studies.