ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA

Background

Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.

Methodology/Principal Findings

Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR^−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR^−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR^−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.

Conclusions

proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.     

Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine

Purpose

RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.

Methods

661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology.

Results

Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested.

Conclusions

661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.     

Neurotrophic effects of BDNF and CNTF,alone and in combination,on postnatal day 5 rat acoustic ganglion neurons

The neuronal survival promoting ability of brain derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), individually and in combination, was evaluated in dissociated cell cultures of postnatal day 5 (P5) rat acoustic ganglia. The neuritogenic promoting effect of these same neurotrophic factors was examined in organotypic explants of P5 rat acoustic ganglia. The results showed that BDNF was maximally effective at a concentration of 10 ng/mL in promoting both survival and neuritogenesis of these postnatal auditory neurons in vitro. CNTF was maximally effective at a concentration of 0.01 ng/mL at promoting both survival and neuritogenesis in the acoustic ganglion cultures. BDNF had its strongest effect on neuronal survival while CNTF was most effective in stimulating neurite outgrowth. These two neurotrophic factors, when added together at their respective maximally effective concentrations, behave in an additive manner for promoting both survival and neuritic outgrowth by the auditory neurons. © 1996 John Wiley & Sons, Inc.     

Isoform-specific effects of ApoE on neurite outgrowth in Olfactory Epithelium culture

Background

The apolipoprotein E4 (apoE4) genotype is a major risk factor for developing late-onset Alzheimer’s disease (AD). Inheritance of apoE4 is also associated with impairments in olfactory function in early stages of AD. In this project we examined the effects of the three common isoforms of human apoE (apoE2, apoE3, and apoE4) on neuronal differentiation and neurite outgrowth in explant cultures of mouse olfactory epithelium (OE).

Results

The OE cultures derived from apoE-deficient/knockout (KO) mice have significantly fewer neurons with shorter neurite outgrowth than cultures from wild-type (WT) mice. Treatment of the apoE KO culture with either purified human apoE2 or with human apoE3 significantly increased neurite outgrowth. In contrast, treatment with apoE4 did not have an effect on neurite outgrowth. The differential effects of human apoE isoforms on neurite outgrowth were abolished by blocking the low-density lipoprotein receptor-related protein (LRP) with lactoferrin and receptor-associated protein (RAP).

Conclusion

ApoE2 and apoE3 stimulate neurite outgrowth in OE cultures by interacting with the lipoprotein receptor, LRP. ApoE4, the isoform associated with AD, failed to promote neurite outgrowth, suggesting a potential mechanism whereby apoE4 may lead to olfactory dysfunction in AD patients.     

Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury

Background

The blood brain barrier (BBB) and truncated trkB receptor on astrocytes prevent the penetration of brain derived neurotrophic factor (BDNF) applied into the peripheral (PNS) and central nervous system (CNS) thus restrict its application in the treatment of nervous diseases. As BDNF is anterogradely transported by axons, we propose that peripherally derived and/or applied BDNF may act on the regeneration of central axons of ascending sensory neurons.

Methodology/Principal Findings

The present study aimed to test the hypothesis by using conditioning lesion of the sciatic nerve as a model to increase the expression of endogenous BDNF in sensory neurons and by injecting exogenous BDNF into the peripheral nerve or tissues. Here we showed that most of regenerating sensory neurons expressed BDNF and p-CREB but not p75NTR. Conditioning-lesion induced regeneration of ascending sensory neuron and the increase in the number of p-Erk positive and GAP-43 positive neurons was blocked by the injection of the BDNF antiserum in the periphery. Enhanced neurite outgrowth of dorsal root ganglia (DRG) neurons in vitro by conditioning lesion was also inhibited by the neutralization with the BDNF antiserum. The delivery of exogenous BDNF into the sciatic nerve or the footpad significantly increased the number of regenerating DRG neurons and regenerating sensory axons in the injured spinal cord. In a contusion injury model, an injection of BDNF into the footpad promoted recovery of motor functions.

Conclusions/Significance

Our data suggest that endogenous BDNF in DRG and spinal cord is required for the enhanced regeneration of ascending sensory neurons after conditioning lesion of sciatic nerve and peripherally applied BDNF may have therapeutic effects on the spinal cord injury.     

Reciprocal Regulation of Axonal Filopodia and Outgrowth during Neuromuscular Junction Development

Background

The assembly of the vertebrate neuromuscular junction (NMJ) is initiated when nerve and muscle first contact each other by filopodial processes which are thought to enable close interactions between the synaptic partners and facilitate synaptogenesis. We recently reported that embryonic Xenopus spinal neurons preferentially extended filopodia towards cocultured muscle cells and that basic fibroblast growth factor (bFGF) produced by muscle activated neuronal FGF receptor 1 (FGFR1) to induce filopodia and favor synaptogenesis. Intriguingly, in an earlier study we found that neurotrophins (NTs), a different set of target-derived factors that act through Trk receptor tyrosine kinases, promoted neuronal growth but hindered presynaptic differentiation and NMJ formation. Thus, here we investigated how bFGF- and NT-signals in neurons jointly elicit presynaptic changes during the earliest stages of NMJ development.

Methodology/Principal Findings

Whereas forced expression of wild-type TrkB in neurons reduced filopodial extension and triggered axonal outgrowth, expression of a mutant TrkB lacking the intracellular kinase domain enhanced filopodial growth and slowed axonal advance. Neurons overexpressing wild-type FGFR1 also displayed more filopodia than control neurons, in accord with our previous findings, and, notably, this elevation in filopodial density was suppressed when neurons were chronically treated from the beginning of the culture period with BDNF, the NT that specifically activates TrkB. Conversely, inhibition by BDNF of NMJ formation in nerve-muscle cocultures was partly reversed by the overexpression of bFGF in muscle.

Conclusions

Our results suggest that the balance between neuronal FGFR1- and TrkB-dependent filopodial assembly and axonal outgrowth regulates the establishment of incipient NMJs.     

Cell Surface Sialylation and Fucosylation Are Regulated by L1 via Phospholipase Cγ and Cooperate to Modulate Neurite Outgrowth,Cell Survival and Migration

Background

Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown.

Methodology/Principal Findings

Using a panel of carbohydrate surface markers, we have shown that cell surface sialylation and fucosylation were downregulated in L1^−/y neurons versus L1^+/y neurons. Consistently, mRNA levels of sialyltransferase ST6Gal1, and fucosyltransferase FUT9 were significantly reduced in L1^−/y neurons. Moreover, treatment of L1^+/y neurons with L1 antibodies, triggering signal transduction downstream of L1, led to an increase in cell surface sialylation and fucosylation compared to rat IgG-treated cells. ShRNAs for both ST6Gal1 and FUT9 blocked L1 antibody-mediated enhancement of neurite outgrowth, cell survival and migration. A phospholipase Cγ (PLCγ) inhibitor and shRNA, as well as an Erk inhibitor, reduced ST6Gal1 and FUT9 mRNA levels and inhibited effects of L1 on neurite outgrowth and cell survival.

Conclusions

Neuronal surface sialylation and fucosylation are regulated via PLCγ by L1, modulating neurite outgrowth, cell survival and migration.     

Calpain Inhibitor PD150606 Attenuates Glutamate Induced Spiral Ganglion Neuron Apoptosis through Apoptosis Inducing Factor Pathway In Vitro

Objective

This research aimed to investigate whether glutamate induced spiral ganglion neurons (SGNs) apoptosis through apoptosis inducing factor (AIF) pathway. And verify whether PD150606, a calpain inhibitor could prevent apoptosis by inhibiting cleaving and releasing AIF in mitochondrion.

Methods

SGNs of postnatal days 0-3 were harvested and cultured in dishes. 20 mM Glu, the caspase inhibitor Z-VAD-FMK and calpain inhibitor PD150606 were added into cultured dishes separately. We used optical microscope and immunofluoresence staining to observe cell morphology and AIF distribution, RT-PCR and Westernblot to analyse AIF and calpain expression in SGNs. TUNEL assay was used to test cell apoptosis.

Results

Cell morphology and nuclear translocation of AIF were altered in SGNs by 20 mM Glu treated in vitro. The axon of SGN shortened, more apoptosis SGN were observed and the expression of AIF and calpain were up-regulated in Glu-treated group than the normal one (P<0.05). The same experiments were conducted in 20 mM+PD150606 treated group and 20 mM+Z-VAD-FMK group. Obviously AIF were located from cytoplasm to the nuclear and the expressions of AIF and calpain were down-regulated by PD150606 (P<0.05). Positive cells in TUNEL staining decreased after PD150606 treating. However, Z-VAD-FMK had no influence on AIF, calpain expression or cell apoptosis.

Conclusion

The AIF-related apoptosis pathway is involved in the process of Glu-induced SGN injury. Furthermore, the inhibition of calpain can prevent AIF from releasing the nuclear or inducing SGN apoptosis.     

Essential role of ERK activation in neurite outgrowth induced by α-lipoic acid

Background

Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth.

Methods

To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for β-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth.

Results

LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth.

Conclusion

Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism.     

Role of Brain-Derived Neurotrophic Factor in Beneficial Effects of Repetitive Transcranial Magnetic Stimulation for Upper Limb Hemiparesis after Stroke

Background

Repetitive transcranial magnetic stimulation (rTMS) can improve upper limb hemiparesis after stroke but the mechanism underlying its efficacy remains elusive. rTMS seems to alter brain-derived neurotrophic factor (BDNF) and such effect is influenced by BDNF gene polymorphism.

Objectives

To investigate the molecular effects of rTMS on serum levels of BDNF, its precursor proBDNF and matrix metalloproteinase-9 (MMP-9) in poststroke patients with upper limb hemiparesis.

Methods

Poststroke patients with upper limb hemiparesis were studied. Sixty-two patients underwent rehabilitation plus rTMS combination therapy and 33 patients underwent rehabilitation monotherapy without rTMS for 14 days at our hospital. One Hz rTMS was applied over the motor representation of the first dorsal interosseous muscle on the non-lesional hemisphere. Fugl-Meyer Assessment and Wolf Motor Function (WMFT) were used to evaluate motor function on the affected upper limb before and after intervention. Blood samples were collected for analysis of BDNF polymorphism and measurement of BDNF, proBDNF and MMP-9 levels.

Results

Two-week combination therapy increased BDNF and MMP-9 serum levels, but not serum proBDNF. Serum BDNF and MMP-9 levels did not correlate with motor function improvement, though baseline serum proBDNF levels correlated negatively and significantly with improvement in WMFT (ρ = -0.422, p = 0.002). The outcome of rTMS therapy was not altered by BDNF gene polymorphism.

Conclusions

The combination therapy of rehabilitation plus low-frequency rTMS seems to improve motor function in the affected limb, by activating BDNF processing. BDNF and its precursor proBDNF could be potentially suitable biomarkers for poststroke motor recovery.     

Two distinct filopodia populations at the growth cone allow to sense nanotopographical extracellular matrix cues to guide neurite outgrowth

Background

The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth.

Methodology/Principal Findings

We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth.

Conclusions/Significance

We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.     

Phosphorylation of the zebrafish M6Ab at serine 263 contributes to filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos

Background

Mammalian M6A, a member of the proteolipid protein (PLP/DM20) family expressed in neurons, was first isolated by expression cloning with a monoclonal antibody. Overexpression of M6A was shown to induce filopodium formation in neuronal cells; however, the underlying mechanism of is largely unknown. Possibly due to gene duplication, there are two M6A paralogs, M6Aa and M6Ab, in the zebrafish genome. In the present study, we used the zebrafish as a model system to investigate the role of zebrafish M6Ab in filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos.

Methodology/Principal Findings

We demonstrated that zebrafish M6Ab promoted extensive filopodium formation in NGF-treated PC12 cells, which is similar to the function of mammalian M6A. Phosphorylation at serine 263 of zebrafish M6Ab contributed to this induction. Transfection of the S263A mutant protein greatly reduced filopodium formation in PC12 cells. In zebrafish embryos, only S263D could induce neurite outgrowth.

Conclusions/Significance

Our results reveal that the phosphorylation status of zebrafish M6Ab at serine 263 is critical for its role in regulating filopodium formation and neurite outgrowth.     

An SK3 channel/nWASP/Abi-1 complex is involved in early neurogenesis

Background

The stabilization or regulated reorganization of the actin cytoskeleton is essential for cellular structure and function. Recently, we could show that the activation of the SK3-channel that represents the predominant SK-channel in neural stem cells, leads to a rapid local outgrowth of long filopodial processes. This observation indicates that the rearrangement of the actin based cytoskeleton via membrane bound SK3-channels might selectively be controlled in defined micro compartments of the cell.

Principal Findings

We found two important proteins for cytoskeletal rearrangement, the Abelson interacting protein 1, Abi-1 and the neural Wiskott Aldrich Syndrome Protein, nWASP, to be in complex with SK3- channels in neural stem cells (NSCs). Moreover, this interaction is also found in spines and postsynaptic compartments of developing primary hippocampal neurons and regulates neurite outgrowth during early phases of differentiation. Overexpression of the proteins or pharmacological activation of SK3 channels induces obvious structural changes in NSCs and hippocampal neurons. In both neuronal cell systems SK3 channels and nWASP act synergistic by strongly inducing filopodial outgrowth while Abi-1 behaves antagonistic to its interaction partners.

Conclusions

Our results give good evidence for a functional interplay of a trimeric complex that transforms incoming signals via SK3-channel activation into the local rearrangement of the cytoskeleton in early steps of neuronal differentiation involving nWASP and Abi-1 actin binding proteins.     

Explore the Features of Brain-Derived Neurotrophic Factor in Mood Disorders

Objectives

Brain-derived neurotrophic factor (BDNF) plays important roles in neuronal survival and differentiation; however, the effects of BDNF on mood disorders remain unclear. We investigated BDNF from the perspective of various aspects of systems biology, including its molecular evolution, genomic studies, protein functions, and pathway analysis.

Methods

We conducted analyses examining sequences, multiple alignments, phylogenetic trees and positive selection across 12 species and several human populations. We summarized the results of previous genomic and functional studies of pro-BDNF and mature-BDNF (m-BDNF) found in a literature review. We identified proteins that interact with BDNF and performed pathway-based analysis using large genome-wide association (GWA) datasets obtained for mood disorders.

Results

BDNF is encoded by a highly conserved gene. The chordate BDNF genes exhibit an average of 75% identity with the human gene, while vertebrate orthologues are 85.9%-100% identical to human BDNF. No signs of recent positive selection were found. Associations between BDNF and mood disorders were not significant in most of the genomic studies (e.g., linkage, association, gene expression, GWA), while relationships between serum/plasma BDNF level and mood disorders were consistently reported. Pro-BDNF is important in the response to stress; the literature review suggests the necessity of studying both pro- and m-BDNF with regard to mood disorders. In addition to conventional pathway analysis, we further considered proteins that interact with BDNF (I-Genes) and identified several biological pathways involved with BDNF or I-Genes to be significantly associated with mood disorders.

Conclusions

Systematically examining the features and biological pathways of BDNF may provide opportunities to deepen our understanding of the mechanisms underlying mood disorders.     

Developmental changes in the protective effect of exogenous brain-derived neurotrophic factor and neurotrophin-3 against ototoxic drugs in cultured rat vestibular ganglion neurons

We examine developmental changes in the responsiveness of rat vestibular ganglion neurons (VGNs) to two neurotrophic factors (NTFs), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and investigate the protective effects of these NTFs against ototoxic drugs during postnatal development in dissociated cultures. VGNs were obtained from rats on postnatal days (P) 1, 3, 7 and 14. BDNF facilitated neuronal survival as well as neurite sprouting of VGNs obtained from younger rats (P1 and P3), whereas these effects were not observed in older rats (P7 and P14). BDNF was also effective in facilitating neurite extension in VGNs at each of the postnatal ages. NT-3 also facilitated neuronal survival and neurite extension of VGNs from younger rats but these effects were significantly smaller than those of BDNF (p?<?0.05). The protective effects of BDNF and NT-3 against ototoxic drugs, gentamicin and cisplatin, were also age-dependent: they were effective for neuronal survival, neurite sprouting and neurite extension in VGNs from younger rats, whereas these effects tended to disappear in VGNs from older rats. Analysis of the changes in the expression of the receptors of NTFs revealed that expression of TrkB and TrkC proteins and their mRNA did not change during the developmental period, whereas expression of p75^NTR protein was down-regulated together with that of p75^NTR mRNA during the developmental period. Developmental changes in the responsiveness to exogenous NTFs in VGNs, which is not caused by the changes of their receptors but probably caused by changes in the intracellular signaling pathways, should be taken into consideration in the prevention of neuronal degeneration caused by ototoxic drugs.     

Magnetic Beads Enhance Adhesion of NIH 3T3 Fibroblasts: A Proof-of-Principle In Vitro Study for Implant-Mediated Long-Term Drug Delivery to the Inner Ear

Introduction

Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI) electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB) onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated.

Materials and Methods

Murine NIH 3T3 fibroblasts—genetically modified to produce BDNF—were labelled with MB.

Results

Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release.

Discussion

Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest.     

Overexpression of Bcl-2 or Bcl-xL prevents spiral ganglion neuron death and inhibits neurite growth

Spiral ganglion neurons (SGNs) provide afferent innervation to the cochlea and rely on contact with hair cells (HCs) for their survival. Following deafferentation due to hair cell loss, SGNs gradually die. In a rat culture model, we explored the ability of prosurvival members of the Bcl-2 family of proteins to support the survival and neurite outgrowth of SGNs. We found that overexpression of either Bcl-2 or Bcl-xL significantly increases SGN survival in the absence of neurotrophic factors, establishing that the Bcl-2 pathway is sufficient for SGN cell survival and that SGN deprived of trophic support die by an apoptotic mechanism. However, in contrast to observations in central neurons and PC12 cells where Bcl-2 appears to promote neurite growth, both Bcl-2 and Bcl-xL overexpression dramatically inhibit neurite outgrowth in SGNs. This inhibition of neurite growth by Bcl-2 occurs in nearly all SGNs even in the presence of multiple neurotrophic factors implying that Bcl-2 directly inhibits neurite growth rather than simply rescuing a subpopulation of neurons incapable of extending neurites without additional stimuli. Thus, although overexpression of prosurvival members of the Bcl-2 family prevents SGN loss following trophic factor deprivation, the inhibition of neurite growth by these molecules may limit their efficacy for support of auditory nerve maintenance or regeneration following hair cell loss.     

Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60-70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a "central-target" (CNS) derived neurotrophic factor--BDNF, while they are differentially responsive to "peripheral-target"-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.     

Prevention of thrombin-induced motoneuron degeneration with different neurotrophic factors in highly enriched cultures

Previous reports have shown that neuronal and glial cells express functionally active thrombin receptors. The thrombin receptor (PAR-1), a member of a growing family of protease activated receptors (PARs), requires cleavage of the extracellular amino-terminus domain by thrombin to induce signal transduction. Studies from our laboratory have shown that PAR-1 activation following the addition of thrombin or a synthetic thrombin receptor activating peptide (TRAP) induces motoneuron cell death both in vitro and in vivo. In addition to increasing motoneuron cell death, PAR- 1 activation leads to decreases in the mean neurite length and side branching in highly enriched motoneuron cultures. It has been suggested that motoneuron survival depends on access to sufficient target-derived neurotrophic factors through axonal branching and synaptic contacts. However, whether the thrombininduced effects on motoneurons can be prevented by neurotrophic factors is still unknown. Using highly enriched avian motoneuron cultures, we show here that alone, soluble chick skeletal muscle extracts (CMX), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF) significantly increased motoneuron survival compared to controls, whereas nerve growth factor (NGF) did not have a significant effect on motoneuron survival. Furthermore, cotreatment with muscle-derived agents (i.e., CMX, BDNF, GDNF) significantly prevented the death of motoneurons induced by alpha-thrombin. Yet, non-muscle-derived agents (CNTF and NGF) had little or no significant effect in reversing thrombin-induced motoneuron death. CMX and CNTF significantly increased the mean length of neurites, whereas NGF, BDNF, and GDNF failed to enhance neurite outgrowth compared to controls. Furthermore, CMX and CNTF significantly prevented thrombin-induced inhibition of neurite outgrowth, whereas BDNF and GDNF only partially reversed thrombin-induced inhibition of neurite outgrowth. These findings show differential effects of neurotrophic factors on thrombin-induced motoneuron degeneration and suggest specific overlaps between the trophic and stress pathways activated by some neurotrophic agents and thrombin, respectively.