Survival of neural stem cell grafts in the lesioned spinal cord is enhanced by a combination of treadmill locomotor training via insulin-like growth factor-1 signaling.

Combining cell transplantation with activity-based rehabilitation is a promising therapeutic approach for spinal cord repair. The present study was designed to investigate potential interactions between the transplantation (TP) of neural stem cells (NSCs) obtained at embryonic day 14 and treadmill training (TMT) in promoting locomotor recovery and structural repair in rat contusive injury model. Combination of TMT with NSC TP at 1 week after injury synergistically improved locomotor function. We report here that combining TMT increased the survival of grafted NSCs by >3-fold and >5-fold at 3 and 9 weeks after injury, respectively. The number of surviving NSCs was significantly correlated with the extent of locomotor recovery. NSCs grafted into the injured spinal cord were under cellular stresses induced by reactive nitrogen or oxygen species, which were markedly attenuated by TMT. TMT increased the concentration of insulin-like growth factor-1 (IGF-1) in the CSF. Intrathecal infusion of neutralizing IGF-1 antibodies, but not antibodies against either BDNF or Neurotrophin-3 (NT-3), abolished the enhanced survival of NSC grafts by TMT. The combination of TP and TMT also resulted in tissue sparing, increased myelination, and restoration of serotonergic fiber innervation to the lumbar spinal cord to a larger extent than that induced by either TP or TMT alone. Therefore, we have discovered unanticipated beneficial effects of TMT in modulating the survival of grafted NSCs via IGF-1. Our study identifies a novel neurobiological basis for complementing NSC-based spinal cord repair with activity-based neurorehabilitative approaches.

Peptides other than the neurotrophins that can be cleaved from proneurotrophins: a neglected story.

The members of the family of neurotrophic factors known as neurotrophins, NGF, BDNF, NT-3 and NT4/5 are known to be cleaved intracellularly from immature precursors, the proneurotrophins. NGF and the other neurotrophins regulate neurite outgrowth and neuronal survival during development via binding to Trk receptor tyrosine kinases and the p75 neurotrophin receptor. Surprisingly, the proneurotrophins were shown to be also biologically active ligands. ProNGF and proBDNF induce neuronal apoptosis via binding to a complex of p75 and sortilin. Therefore, life and death seems to be a delicate interplay between 'cleavage' or 'not cleavage' of the proneurotrophins. However, there is a third aspect to this story. In general, peptide-hormone precursors are known to give rise to several biologically active peptides from one precursor molecule. The paradox with the proneurotrophins is that although they have several additional potential cleavage sites that would necessarily give rise to other peptides besides the neurotrophins and thus new members in the neurotrophin family, this aspect has been largely neglected. This article aims to review evidence for biologically active peptides other than the NGF and NT-3 that can be generated from the proNGF and proNT-3.

Rat retinal microglial cells under normal conditions, after optic nerve section, and after optic nerve section and intravitreal injection of trophic factors or macrophage inhibitory factor.

Retinal microglial cells may have a role in both degeneration and neuroprotection of retinal ganglion cells (RGC) after optic nerve (ON) section. We have used NDPase enzymohistochemistry to label adult rat retinal microglial cells and have studied these cells under normal conditions, after left ON section, and after left ON section and eye puncture or intravitreal injection of different substances: vehicle, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT3), or macrophage inhibitory factor (MIF). Resident microglial cells are present in four layers in the adult rat retina: the nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), and outer plexiform layer (OPL). Left ON section induces microglial activation in the ipsilateral and contralateral retina as manifested by stronger staining intensity in both retinas and increased microglial cell densities in the NFL, IPL, and GCL of the ipsilateral retina. Left ON section followed by left eye puncture or intravitreal injection increases microglial cell density in both retinas and induces changes in the microglial cells of the ipsilateral retina that vary depending on the substance injected: BDNF injections delay microglial activation, possibly through retinal ganglion cell neuroprotection, whereas NT3 partially inhibits microglial activation in the NFL; MIF injections have no clear effects on microglial activation. In conclusion, retinal microglial cells become activated after an ON section and react more intensely when the eye is also punctured or injected, and this response may be altered by using neurotrophic factors, although the effects of MIF are less clear.

NT3 inhibits FGF2-induced neural progenitor cell proliferation via the PI3K/GSK3 pathway.

Neurotrophin 3 (NT3), a member of the neurotrophin family, antagonizes the proliferative effect of fibroblast growth factor 2 (FGF2) on cortical precursors. However, the mechanism by which NT3 inhibits FGF2-induced neural progenitor (NP) cell proliferation is unclear. Here, using an FGF2-dependent rat neurosphere culture system, we found that NT3 inhibits both FGF2-induced neurosphere growth and bromodeoxyuridine (BrdU) incorporation in a dose-dependent manner. U0126, a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, both inhibited FGF2-induced BrdU incorporation, suggesting that the extracellular signal-regulated kinase1/2 (ERK1/2) and PI3K pathways are required for FGF2-induced NP cell proliferation. NT3 significantly inhibited FGF2-induced phosphorylation of Akt and glycogen synthase kinase 3beta (GSK3beta), a downstream kinase of Akt, whereas phosphorylation of ERK1/2 was unaffected. The inhibitory effect of NT3 on FGF2-induced NP cell proliferation was abolished by LY294002, and treatment with SB216763, a specific GSK3 inhibitor, antagonized the NT3 effect, rescuing both neurosphere growth and BrdU incorporation. Moreover, experiments with anti-NT3 antibody revealed that endogenous NT3 also plays a role in inhibiting FGF2-induced NP cell proliferation, and that anti-NT3 antibody enhanced phospho-Akt and phospho-GSK3beta levels in the presence of FGF2. These findings indicate that FGF2-induced NP cell proliferation is inhibited by NT3 via the PI3K/GSK3 pathway.

Neutralization of neutrophin-3 in the ventral tegmental area or nucleus accumbens differentially modulates cocaine-induced behavioral plasticity in rats.

These experiments were designed to assess the influence of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) in the mesoaccumbens dopamine system on the initiation of behavioral sensitization to cocaine. A neutralizing antibody for NT-3, BDNF or their vehicle was administered into the ventral tegmental area (VTA) or nucleus accumbens prior to each of four daily injections of 15 mg/kg cocaine. Behavioral sensitization was operationally defined as a significant increase in the behavioral response to cocaine relative to the first daily injection. Results indicated that the NT-3 antibody had differential effects when administered into the VTA or nucleus accumbens. Intra-VTA microinjection of anti-NT-3 resulted in enhanced sensitization to repeated cocaine injections in that the cocaine-induced behavioral response in the anti-NT-3 group was significantly greater than the vehicle group following the second and third daily injections of cocaine. Administration of anti-NT-3 into the nucleus accumbens increased the behavioral response to cocaine over all 4 days of cocaine administration, with no sensitization of this behavioral response. In contrast, pretreatment with anti-BDNF into the VTA or nucleus accumbens had no influence on the initiation of behavioral sensitization to cocaine. Taken together, these data indicate that neutralization of NT-3 in the VTA enhances cocaine-induced behavioral sensitization, while administration of the NT-3 antibody into the nucleus accumbens increases the hyperactive behavioral response induced by cocaine but impairs the further development of behavioral sensitization.

Development of brain-derived neurotrophic factor and neurotrophin-3 immunoreactivity in the lower auditory brainstem of the postnatal gerbil.

The localization of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the gerbil auditory brainstem was studied during normal postnatal development. The principal objective of this paper was to compare the developmental distribution of BDNF and NT-3 proteins to the known developmental distribution of their cognate, high-affinity tyrosine kinase receptors. BDNF and NT-3 proteins were localized using standard immunohistochemistry. No specific immunoreactivity for BDNF or NT-3 was detected on the day of birth (P0) in any auditory structure, although fibers comprising the spinal tract of the Vth cranial nerve were well labelled with antibodies against BDNF. Diffuse immunoreactivity for both BDNF and NT-3 was first detected at P3 in the cochlear nucleus and in several second order auditory nuclei in the superior olivary complex. This diffuse immunoreactivity became clustered and restricted to neuronal cell bodies by P10. Immunoreactivity for both BDNF and NT-3 transiently disappeared in the lateral and medial superior olivary nuclei at P10. However, neurons in the medial nucleus of the trapezoid body remained immunopositive for both BDNF and NT-3. Fibers in the trapezoid body were labelled with BDNF immunoreactivity by P12. Between P12 and P15, the distribution of BDNF and NT-3 immunoreactivity in the cochlear nucleus and superior olivary complex became comparable to adult (P140) immunolabel. These results show that the normal developmental distribution of the neurotrophins BDNF and NT-3 in the lower auditory brainstem occurs during the first two postnatal weeks in parallel with the developmental expression of their cognate receptors, trkB and trkC.

A role for oligodendrocyte-derived IGF-1 in trophic support of cortical neurons.

Neurons and glia interact in the development of mammalian central nervous systems and in the maintenance of stable myelinated axons. Recent evidence suggests a role for oligodendrocytes in providing trophic support for neurons during development and in the mature nervous system. This work prompted us to study oligodendrocyte influences on neuronal survival and death in vitro. Rat embryonic cortical neurons were co-cultured with purified oligodendrocytes at different developmental stages and separately with oligodendrocyte-conditioned medium. Neuronal survival was measured by immunocytochemistry and 3H-GABA uptake. Neurons show a marked increase in survival when co-cultured directly with oligodendrocyte precursors (OPCs) and differentiated oligodendrocytes. Neurons cultured in the presence of OPCs separated by a permeable membrane and those cultured in medium conditioned by oligodendrocytes also show a significant increase in survival. Medium conditioned by differentiated oligodendrocytes provides a greater survival effect than medium conditioned by OPCs. Neutralising antibodies to IGF-1, but not to other candidate trophic factors, block the soluble survival effect of oligodendrocytes. Cells of the oligodendrocyte lineage produce IGF-1 and recombinant IGF-1 promotes neuronal survival under identical conditions. This study provides evidence that OPCs and differentiated oligodendrocytes support neuronal survival by both contact-mediated and soluble mechanisms and that IGF-1 significantly contributes to this effect.

Neurotrophin secretion from cultured microglia.

Because microglia have been suggested to produce neurotrophins, we tested this ability in vitro. Rat primary microglia were found to constitutively secrete a limited amount of brain-derived neurotrophic factor (BDNF), but nerve growth factor (NGF) and neurotrophin-3 (NT-3) were undetectable in the conditioned medium. Stimulation of the cells with lipopolysaccharide (LPS) increased BDNF secretion, and induced NGF secretion. As a first step to examine this regulation system, the association of protein kinase C (PKC) was pharmacologically analyzed. A PKC activator, phorbol-12-myristate-13-acetate, enhanced the secretion of BDNF. Pre-treatment of microglia with a PKC inhibitor, bisindolylmaleimide, suppressed LPS-stimulated BDNF secretion as well as the constitutive one. These results suggest that the PKC signaling cascade is closely associated with BDNF secretion. Among PKC isoforms, PKCalpha probably plays a role in BDNF secretion, based on the results of experiments using a specific PKC activator, 1-oleoyl-2-acetyl-sn-glycerol, and a specific PKC inhibitor, Gö 6976, and by immunoblotting. Taken together, these findings suggest that the secretion of BDNF from microglia is regulated through PKCalpha-associated signal transduction mechanism.

Protective role of neurotrophins in experimental inflammation of the rat gut.

BACKGROUND & AIMS: Sensory neuropeptides modulate the mucosal response to inflammation in experimental colitis. Because nerve growth factor (NGF) regulates the expression of neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) and is implicated as a link between the nervous system and the immune system in the inflammatory process, we investigated the functional role of NGF and neurotrophin-3 during experimental colitis. METHODS: Immunoneutralizing antibodies specific for NGF and neurotrophin (NT)-3 were used to block their endogenous activity. Mild trinitrobenzene sulfonic acid (TNBS) colitis was induced, and damage scores were assessed after 1 week. Neuropeptide content in the colon and NT messenger RNA (mRNA) expression were determined. RESULTS: The pretreatment with anti-NGF or anti-NT-3 caused a significant 2-3-fold increase in the severity of the experimental inflammation as assessed by a macroscopic damage score, histologic ulceration score, and myeloperoxidase activity in the tissue. CGRP, but not substance P, contents in the colon were significantly reduced by NGF immunoneutralization. NGF mRNA was slightly up-regulated after NGF immunoneutralization, but NT-3 mRNA was unchanged by NT-3 immunoneutralization. CGRP mRNA was not significantly changed after 1 week of colitis by NGF or NT-3 immunoneutralization, whereas beta-preprotachykinin mRNA was up-regulated after immunoneutralization. CONCLUSIONS: These findings suggest a regulatory role for NGF and NT-3 in experimental inflammation of the gut. This effect may be partly caused by the reduction of mucosal CGRP content caused by the NGF blockade.

Neurotrophin-3 controls proliferation of granular precursors as well as survival of mature granule neurons in the developing rat cerebellum.

Levels of neurotrophin-3 markedly decrease in the rat cerebellum after the first 10 days of life, suggesting an importance during early development. To further examine the effect of neurotrophin-3 on the developing cerebellum, we injected a monoclonal antibody against neurotrophin-3 into the lateral ventricle of 7.5-day-old rats. The resultant depletion of neurotrophin-3 caused a significant decrease in cerebellar wet weights noted at 7 and 23 days thereafter. Other changes noted 48 h after injection of monoclonal antibodies against neurotrophin-3 included reduced incorporation of bromode-oxyuridine into granule neurons in the external germinal layer, an elevated density of atrophic neurons that had just migrated under the Purkinje cell layer, and an increased number of apoptotic neurons in the internal granule cell layer. These changes were limited to the central lobe. The concentration of neurotrophin-3 protein in the posterior region, including the central lobe, was about four- and threefold higher than that in the anterior region of the cerebellum of 9.5- and 30-day-old rats, respectively. Immunocytochemical examination showed higher amounts of neurotrophin-3 protein in the central lobe than in the anterior lobe. Our results provide evidence that neurotrophin-3 regulates the proliferation of granule precursors and supports the survival of mature granule neurons in restricted lobules, suggesting an involvement in limited regions at a specific stage in development of the rat cerebellum.