Yasufumi Sato

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.

Cartilage Acidic Protein–1B (LOTUS), an Endogenous Nogo Receptor Antagonist for Axon Tract Formation

A molecule that functions in normal olfactory tract development could provide clues to failed neuronal regeneration in adults. Neural circuitry formation depends on the molecular control of axonal projection during development. By screening with fluorophore-assisted light inactivation in the developing mouse brain, we identified cartilage acidic protein–1B as a key molecule for lateral olfactory tract (LOT) formation and named it LOT usher substance (LOTUS). We further identified Nogo receptor–1 (NgR1) as a LOTUS-binding protein. NgR1 is a receptor of myelin-derived axon growth inhibitors, such as Nogo, which prevent neural regeneration in the adult. LOTUS suppressed Nogo-NgR1 binding and Nogo-induced growth cone collapse. A defasciculated LOT was present in lotus-deficient mice but not in mice lacking both lotus- and ngr1. These findings suggest that endogenous antagonism of NgR1 by LOTUS is crucial for normal LOT formation.

Mosaic development of the olfactory cortex with Pax6-dependent and -independent components

The olfactory cortex is the target area of olfactory bulb axons and is suggested to be derived from neuroepithelial progenitors of various ventricular domains during development. In the present study, we examined the development of the olfactory cortex, using the newly developed monoclonal antibody (mAb) 9-4c, which recognizes reticulon 1-A and -B. The mAb labeled neuroepithelial progenitors at the pallio-subpallial boundary (PSB) and their putative descendants in the deep layers of the olfactory cortex. In the Pax6 mutant embryo, labeling at the PSB was specifically lacking, and the number of immunopositive cells in the olfactory cortex was markedly reduced. In contrast, the guidepost neurons of olfactory bulb axons, lot cells, developed relatively normally in the superficial layer of the olfactory cortex in the mutant embryo. These guidepost neurons have been recently shown to originate in the pallium and eventually guide the initial projection of olfactory bulb axons. The olfactory bulb projection in the Pax6 mutant embryo also suggested the dualistic nature of the olfactory cortex development; the initial projection of olfactory bulb axons developed relatively normally, whereas the final projection of their collateral branches was severely defective.

Chronotopic organization of olfactory bulb axons in the lateral olfactory tract

The arrangement of axons in a tract can have a specific effect on the organization of functional neuronal circuits. Here we describe olfactory bulb axons chronologically arranged in the lateral olfactory tract. Newly differentiated projection neurons over the whole olfactory bulb are similarly marked with transient expression of c‐kit protein. Their axons are assembled together and project into the ventral superficial part of the tract, displacing the older axons. This special assembly of the axons explains the nontopographic relationships between the olfactory bulb and the lateral olfactory tract axons that have been described in previous studies and could possibly influence the subsequent selection of the olfactory target areas by these axons. J. Comp. Neurol. 475:247–260, 2004.

Induction of axon growth arrest without growth cone collapse through the N-terminal region of four-transmembrane glycoprotein M6a.

During development, axons elongate vigorously, carefully controlling their speed, to connect with their targets. In general, rapid axon growth is correlated with active growth cones driven by dynamic actin filaments. For example, when the actin‐driven tip is collapsed by repulsive guidance molecules, axon growth is severely impaired. In this study, we report that axon growth can be suppressed, without destroying the actin‐based structure or motility of the growth cones, when antibodies bind to the four‐transmembrane glycoprotein M6a concentrated on the growth cone edge. Surprisingly, M6a‐deficient axons grow actively but are not growth suppressed by the antibodies, arguing for an inductive action of the antibody. The binding of antibodies clusters and displaces M6a protein from the growth cone edge membrane, suggesting that the spatial rearrangement of this protein might underlie the unique growth cone behavior triggered by the antibodies. Molecular dissection of M6a suggested involvement for the N‐terminal intracellular domain in this antibody‐induced growth cone arrest.

Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones

Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry‐induced increase of phosphorylation of eukaryotic elongation factor‐2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2‐mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore‐assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin‐dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)‐evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP‐induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth.

Actin-Independent Behavior and Membrane Deformation Exhibited by the Four-Transmembrane Protein M6a

M6a is a four-transmembrane protein that is abundantly expressed in the nervous system. Previous studies have shown that over-expression of this protein induces various cellular protrusions, such as neurites, filopodia, and dendritic spines. In this detailed characterization of M6a-induced structures, we found their varied and peculiar characteristics. Notably, the M6a-induced protrusions were mostly devoid of actin filaments or microtubules and exhibited free random vibrating motion. Moreover, when an antibody bound to M6a, the membrane-wrapped protrusions were suddenly disrupted, leading to perturbation of the surrounding membrane dynamics involving phosphoinositide signaling. During single-molecule analysis, M6a exhibited cytoskeleton-independent movement and became selectively entrapped along the cell perimeter in an actin-independent manner. These observations highlight the unusual characteristics of M6a, which may have a significant yet unappreciated role in biological systems.

The carboxyl-terminal region of Crtac1B/LOTUS acts as a functional domain in endogenous antagonism to Nogo receptor-1.

Myelin-derived axon growth inhibitors, such as Nogo, bind to Nogo receptor-1 (NgR1) and thereby limit the action of axonal regeneration after injury in the adult central nervous system. Recently, we have found that cartilage acidic protein-1B (Crtac1B)/lateral olfactory tract usher substance (LOTUS) binds to NgR1 and functions as an endogenous NgR1 antagonist. To examine the functional domain of LOTUS in the antagonism to NgR1, analysis using the deletion mutants of LOTUS was performed and revealed that the carboxyl-terminal region (UA/EC domain) of LOTUS bound to NgR1. The UA/EC fragment of LOTUS overexpressed together with NgR1 in COS7 cells abolished the binding of Nogo66 to NgR1. Overexpression of the UA/EC fragment in cultured chick dorsal root ganglion neurons suppressed Nogo66-induced growth cone collapse. These findings suggest that the UA/EC region is a functional domain of LOTUS serving for an antagonistic action to NgR1.

Development of the amygdalohypothalamic projection in the mouse embryonic forebrain

The amygdalohypothalamic projection, a major component of the stria terminalis, is involved in the conduction of emotional and olfactory information integrated in the amygdala to the hypothalamus to elicit emotional reactions. Despite the extensive studies on functional aspects of the amygdaloid complex, developmental mechanisms of the amygdala and related structures are still poorly understood. To investigate the development of the amygdalohypothalamic projection in the mouse embryonic brain, carbocyanine dye was applied to the amygdala to label the growing axons anterogradely and to the hypothalamus to label the amygdaloid neurons retrogradely. The initial outgrowth of the stria terminalis was found to be as early as E11.5. The pathway crossed in a saddle over the internal capsule, another prominent connection in the developing forebrain of the mammalian embryo. Bipolar immature neurons were distributed along the stria terminalis at the telencephalo-diencephalic boundary, and the internal capsule was also surrounded by these cells. These cells expressed immunoreactivities to calretinin and the lot-1 antigen which has been shown to be involved in guidance of the developing lateral olfactory tract. Ultrastructural analysis revealed an adherens-like junction between the stria terminalis and the apposed cells, implying contact-mediated guidance. These results suggest that, in the development of the stria terminalis, the axonal outgrowth is guided by a mechanism similar to that of the developing lateral olfactory tract, a major amygdalopetal connection.

Physiological roles of LOTUS, an endogenous Nogo receptor antagonist, in lateral olfactory tract formation

In the rodent primary somatosensory cortex, layer IV cells are concentrated around barrel walls, forming cell-sparse barrel hollows and septa that delineate individual barrels. During early postnatal stage, thalamocortical axons (TCAs) from individual thalamic barrelloids are almost entirely confined to a single barrel cluster, followed by arrangement of cortical layer IV neurons into barrel hollows and septa. Addition to this, unidirectional dendrite formation of barrel neurons toward barrel hollows occurs around P7 for efficient synapse formation with TCAs. To elucidate the molecular mechanism of barrel development, we searched for genes expressed in the barrel cortex, by using Allen Brain Atlas and tested their temporal and spatial expression during early postnatal stage using in situ hybridization (ISH). As a result, we found several genes, whose expression is restricted in the barrel or septa. Among these genes, we focused on Btbd3, BTB/POZ domain containing 3, whose expression is restricted in the barrel cells. We first revealed that Btbd3 expression start around P4, when TCAs exhibit more axon branches in cortical layer IV. Next, we revealed that its barrel-like expression is disrupted in the cortex with the abnormal TCA innervation. Furthermore, we tested its function in barrel development with employing in utero electroporation and its shRNA construct. Although no major structural difference was obtained in Btbd3 knockdown barrel cortex, dendrite orientation of barrel cells are disrupted. Our results indicate that Btbd3 function to regulate dendrite patterning in TCA-dependent fashion.