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.