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.

The lipid kinase PIK3C3 (also called Vps34) regulates both the endosomal and autophagic pathways. However, the effect of inactivating PIK3C3 on neuronal endosomal versus autophagic processes in vivo has not been studied. We generated mice in which Pik3c3 was conditionally deleted in differentiated sensory neurons. Within a few days after Pik3c3 deletion, mutant largediameter myelinated neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated more slowly. These differential degenerative phenotypes are unlikely caused by a disruption in the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct phenotype in all sensory neurons (i.e., formation of very large intracellular inclusion bodies and slow degeneration over a period of several months). More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. Analyses of Pik3c3/Atg7 double mutant neurons revealed that this unconventional initiation pathway still depends on ATG7. Our studies represent in vivo characterization of PIK3C3 functions in mammals and provide insights into the complexity of neuronal endo-lysosomal and autophagic pathways. P IK3C3 (also known as Vps34) is a class III phosphatidylinositol-3-kinase that specifically catalyzes the formation of phosphatidylinositol-3-phosphate (PI3P) (1). Studies in invertebrate organisms as well as in nonneuronal cells showed that PIK3C3/Vps34 regulates multiple aspects of both the endocytic/ endosomal and autophagic pathways (2-5). In yeast, there are two distinct Vps34 complexes: complex I (Vps34, Vps15, Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Atg6, and Vps38) functions in the vacuolar proteinsorting process (6). In mammals, homologs of Vps15 and Atg6 are p150 and Beclin1, respectively (7), and evidence exists for Beclin1-independent functions of PIK3C3 in the endocytic pathways (8). A mammalian homolog of complex I (PIK3C3, p150, Beclin1, and Atg14L) activates autophagy, and a homolog of complex II (PIK3C3, p150, Beclin1, and UVRAG/Vps38) regulates trafficking at late endosomes (7). Interestingly, Bif-1 and Rubicon can interact with complex II to promote autophagy (9, 10). The chemical inhibitor of PIK3C3 (3-MA or wortmanin) has been frequently used as an inhibitor for autophagy in numerous studies, including those studying autophagy in neurons (11)(12)(13)(14). However, because of the lack of genetic studies on Pik3c3 in mammals, it is not clear how inactivating PIK3C3 in neurons in vivo differentially affects endosomal versus autophagic processes.Neurons are highly susceptible to disruptions in both endocytic and autophagic pathways. Genetic mutations in ubiquitously expressed proteins regulating the endocytic (15,16) Lowe (23,...

Recent studies have shown that delayed transplantation of neural stem/progenitor cells (NSPCs) into the injured spinal cord can promote functional recovery in adult rats. Preclinical studies using nonhuman primates, however, are necessary before NSPCs can be used in clinical trials to treat human patients with spinal cord injury (SCI). Cervical contusion SCIs were induced in 10 adult common marmosets using a stereotaxic device. Nine days after injury, in vitro-expanded human NSPCs were transplanted into the spinal cord of five randomly selected animals, and the other sham-operated control animals received culture medium alone. Motor functions were evaluated through measurements of bar grip power and spontaneous motor activity, and temporal changes in the intramedullary signals were monitored by magnetic resonance imaging. Eight weeks after transplantation, all animals were sacrificed. Histologic analysis revealed that the grafted human NSPCs survived and differentiated into neurons, astrocytes, and oligodendrocytes, and that the cavities were smaller than those in sham-operated control animals. The bar grip power and the spontaneous motor activity of the transplanted animals were significantly higher than those of sham-operated control animals. These findings show that NSPC transplantation was effective for SCI in primates and suggest that human NSPC transplantation could be a feasible treatment for human SCI.

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Axonal damage is a major morphological alteration in the CNS of patients with multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the underlying mechanism for the axonal damage associated with MS/EAE and its contribution to the clinical symptoms remain unclear. The expression of a fusion protein, named "Wallerian degeneration slow" (Wld s ), can protect axons from degeneration, likely through a ␤-nicotinamide adenine dinucleotide (NAD)-dependent mechanism. In this study, we find that, when induced with EAE, Wld s mice showed a modest attenuation of behavioral deficits and axon loss, suggesting that EAE-associated axon damage may occur by a mechanism similar to Wallerian degeneration. Furthermore, nicotinamide (NAm), an NAD biosynthesis precursor, profoundly prevents the degeneration of demyelinated axons and improves the behavioral deficits in EAE models. Finally, we demonstrate that delayed NAm treatment is also beneficial to EAE models, pointing to the therapeutic potential of NAm as a protective agent for EAE and perhaps MS patients.

A major obstacle for successful axon regeneration in the adult central nervous system (CNS) arises from inhibitory molecules in CNS myelin, which signal through a common receptor complex on neurons consisting of the ligand-binding Nogo-66 receptor (NgR) and two transmembrane coreceptors, p75 and LINGO-1. However, p75 expression is only detectable in subpopulations of mature neurons, raising the question of how these inhibitory signals are transduced in neurons lacking p75. In this study, we demonstrate that TROY (also known as TAJ), a TNF receptor family member selectively expressed in the adult nervous system, can form a functional receptor complex with NgR and LINGO-1 to mediate cellular responses to myelin inhibitors. Also, both overexpressing a dominant-negative TROY or presence of a soluble TROY protein can efficiently block neuronal response to myelin inhibitors. Our results implicate TROY in mediating myelin inhibition, offering new insights into the molecular mechanisms of regeneration failure in the adult nervous system.

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Neuronal Nogo-66 receptor 1 (NgR1) has been proposed to function as an obligatory coreceptor for the myelin-derived ligands Nogo-A, oligodendrocyte myelin glycoprotein (OMgp), and myelin-associated glycoprotein (MAG) to mediate neurite outgrowth inhibition by these ligands. To examine the contribution of neuronal NgR1 to outgrowth inhibition, we used two different strategies, genetic ablation of NgR1 through the germline and transient short hairpin RNA interference (shRNAi)-mediated knock-down. To monitor growth inhibition, two different paradigms were used, chronic presentation of substrate-bound inhibitor to measure neurite extension and acute application of soluble inhibitor to assay growth cone collapse. We find that regardless of the NgR1 genotype, membrane-bound MAG strongly inhibits neurite outgrowth of primary cerebellar, sensory, and cortical neurons. Similarly, substrate-bound OMgp strongly inhibits neurite outgrowth of NgR1 wild-type and mutant sensory neurons. Consistent with these results, shRNAi-mediated knock-down of neuronal NgR1 does not result in a substantial release of L-MAG (large MAG) inhibition. When applied acutely, however, MAG-Fc and OMgp-Fc induce a modest degree of growth cone collapse that is significantly attenuated in NgR1-null neurons compared with wild-type controls. Based on our findings and previous studies with Nogo-66, we propose that neuronal NgR1 has a circumscribed role in regulating cytoskeletal dynamics after acute exposure to soluble MAG, OMgp, or Nogo-66, but is not required for these ligands to mediate their growth-inhibitory properties in chronic outgrowth experiments. Our results thus provide unexpected evidence that the growth conecollapsing activities and substrate growth-inhibitory activities of inhibitory ligands can be dissociated. We also conclude that chronic axon growth inhibition by myelin is mediated by NgR1-independent mechanisms.