The outgrowth response of the axons of developing and regenerating rat retinal ganglion cells in vitro to neurotrophin treatment

BDNF and NT-4 (but not NT-3 or CNTF) significantly enhanced the outgrowth of early embryonic and adult regenerating RGC axons when provided with a supportive substrate in vitro. BDNF and NT-4 treatment transiently increased RGC axon outgrowth from E15 rat retinas but not from retinas at older embryonic ages. The transient effect of BDNF and NT-4 and the inability of the neurotrophins to promote outgrowth from older embryonic retinal explants suggests a time frame of neurotrophin action and that other chemical factors (target-derived or otherwise) may be necessary for the continued maintenance of developing RGC axons. BDNF and NT-4 also enhanced the outgrowth of regenerating axons from adult retinal explants, but appeared to have a more subtle effect on axon outgrowth, in that the growth-promoting effects of BDNF and NT-4 appeared continuous throughout the incubation period. The suppression of RGC axon outgrowth from embryonic and adult retinae cultured in trkB-IgG-containing medium suggests that the response of developing and regenerating axons, to BDNF and NT-4 are likely to occur through trkB signalling.     

Extracellular-Regulated Kinases and Phosphatidylinositol 3-Kinase Are Involved in Brain-Derived Neurotrophic Factor-Mediated Survival and neuritogenesis of the Neuroblastoma Cell Line SH-SY5Y

Retinoic acid (RA) induces the differentiation of many cell lines, including those derived from neuroblastoma. RA treatment of SH-SY5Y cells induces the appearance of functional Trk B and Trk C receptors. Acute stimulation of RA-predifferentiated SH-SY5Y cells with brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), or neurotrophin 4/5 (NT-4/5), but not nerve growth factor (NGF), induces Trk autophosphorylation, followed by phosphorylation of Akt and the extracellular signal-regulated kinases (ERKs) 1 and 2. In addition, BDNF, NT-3, or NT-4/5, but not NGF, promotes cell survival and neurite outgrowth in serum-free medium. The mitogen-activated protein kinase and ERK kinase (MEK) inhibitor PD98059 blocks BDNF-induced neurite outgrowth and growth-associated protein-43 expression but has no effects on cell survival. On the other hand, the phosphatidylinositol 3-kinase inhibitor LY249002 reverses the survival response elicited by BDNF, leading to a cell death with morphological features of apoptosis.     

Neurite Outgrowth Effect of 4-O-methylhonokiol by Induction of Neurotrophic Factors Through ERK Activation

Compounds isolated from Magnolia officinalis such as magnolol, honokiol and obovatol exhibit several pharmacological effects on CNS including depressant, anxiolytic and anticonvulsant effects, as well as neuroprotective effects against chemical and heat damages. Recently, honokiol was found to have a neurotrophic effect in fetal rat cortical neurons. In the present study, we show that 4-O-methylhonokiol, a novel compound from Magnolia officinalis, promotes neurite outgrowth in a concentration-dependent manner in rat embryonic neuronal cells. In parallel with the neurite outgrowth activity, the expression of neurite outgrowth marker proteins is also increased by treatment with 4-O-methylhonokiol. We also found that 4-O-methylhonokiol promotes the release of NGF and BDNF into cell culture medium. In addition, lower concentration of 4-O-methylhonokiol (1 and 2 μM) further enhanced neurite outgrowth and expression of neurite outgrowth marker proteins in the presence of NGF (50 ng/ml) or BDNF (10 ng/ml). Subsequently, we found that 4-O-methylhonokiol activates ERK in a concentration-dependent manner. However, the neurite outgrowth activity and the NGF and BDNF release induced by 4-O-methylhonokiol are suppressed by an ERK-specific inhibitor. These results suggest that 4-O-methylhonokiol has the ability to induce neurite outgrowth via the increase of neurotrophic factor levels through ERK activation.     

BDNF and NT4/5 promote survival and neurite outgrowth of pontocerebellar mossy fiber neurons

The neurotrophins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT3), and NT4/5 are all found in the developing cerebellum. Granule cells, the major target neurons of mossy fibers, express BDNF during mossy fiber synaptogenesis. To determine whether neurotrophins contribute to the development of cerebellar afferent axons, we characterized the effects of neurotrophins on the growth of mossy fiber neurons from mice and rats in vitro. For a mossy fiber source, we used the basilar pontine nuclei (BPN), the major source of cerebellar mossy fibers in mammals. BDNF and NT4/5 increased BPN neuron survival, neurite outgrowth, growth cone size, and elongation rate, while neither NT3 nor NGF increased survival or outgrowth. In addition, BDNF and NT4/5 reduced the size of neurite bundles. Consistent with these effects, in situ hybridization on cultured basilar pontine neurons revealed the presence of mRNA encoding the TrkB receptor which binds both BDNF and NT4/5 with high affinity. We detected little or no message encoding the TrkC receptor which preferentially binds NT3. BDNF and NT4/5 also increased TrkB mRNA levels in BPN neurons. In addition to previously established functions as an autocrine/paracrine trophic factor for granule cells, the present results indicate that cerebellar BDNF may also act as a target‐derived trophic factor for basilar pontine mossy fibers. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 254–269, 1999     

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.     

Differential effects of the trophic factors BDNF, NT-4, GDNF, and IGF-I on the isthmo-optic nucleus in chick embryos

The isthmo-optic nucleus (ION) of chick embryos is a model system for the study of retrograde trophic signaling in developing CNS neurons. The role of brain-derived neurotrophic factor (BDNF) is well established in this system. Recent work has implicated neurotrophin-4 (NT-4), glial cell line-derived neurotrophic factor (GDNF), and insulin-like growth factor I (IGF-I) as additional trophic factors for ION neurons. Here it was examined in vitro and in vivo whether these factors are target-derived trophic factors for the ION in 13- to 16-day-old chick embryos. Unlike BDNF, neither GDNF, NT-4, nor IGF-I increased the survival of ION neurons in dissociated cultures identified by retrograde labeling with the fluorescent tracer DiI. BDNF and IGF-I promoted neurite outgrowth from ION explants, whereas GDNF and NT-4 had no effect. Injections of NT-4, but not GDNF, in the retina decreased the survival of ION neurons and accelerated cell death in the ION. NT-4-like immunoreactivity was present in the retina and the ION. Exogenous, radiolabeled NT-4, but not GDNF or IGF-I, was retrogradely transported from the retina to the ION. NT-4 transport was significantly reduced by coinjection of excess cold nerve growth factor (NGF), indicating that the majority of NT-4 bound to p75 neurotrophin receptors during axonal transport. Binding of NT-4 to chick p75 receptors was confirmed in L-cells, which express chick p75 receptors. These data indicate that GDNF has no direct trophic effects on ION neurons. IGF-I may be an afferent trophic factor for the ION, and NT-4 may act as an antagonist to BDNF, either by competing with BDNF for p75 and/or trkB binding or by signaling cell death via p75.     

Trophic responsiveness of purified postnatal and adult rat retinal ganglion cells

Central neurons lose the ability for axonal re-growth during development and typically do not regenerate their axons following axotomy once they become mature unless given a growth-permissive environment i.e. peripheral nerve graft. In the present study, the growth responsiveness of purified retinal ganglion cells (RGCs) at different ages to neurotrophic factors and Schwann cell (SC)-secreted factors were examined directly. The purity of adult RGCs was 97% as assessed by retrograde labelling with 4,6-diamidino-2-phenylindole. The stability of cultures were demonstrated by long-term survival (30 days) in medium contained brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and forskolin (F) (BCF). RGCs from postnatal (P) (P0, P4, P8, P21) and adult (P90) rats showed decreasing levels of survival and neuritogenesis when grown in BCF. In contrast, the opposite was observed in SC-conditioned medium (CM)-treated P0-P8 RGCs which were increasingly responsive. SCCM induced maximal neurite outgrowth in P8 RGCs via the activation of extracellular regulated kinase 1/2 (Erk1/2). Inhibition of mitogen-activated protein kinase-Erk1/2 signaling using an Erk1/2-specific inhibitor (UO126) abolished SCCM-induced Erk1/2 phosphorylation and neuritogenesis completely. Although both SCCM and BCF failed to sustain the same levels of growth in P21 or P90 cultures as observed in P8 cultures, SCCM promoted higher survival and neuritogenesis than BCF-treated adult RGCs. This study is the first report of adult rat RGC purification and demonstrates that mature RGCs need multiple factors for survival and neurite outgrowth.     

BDNF and NT4/5 promote survival and neurite outgrowth of pontocerebellar mossy fiber neurons.

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and NT4/5 are all found in the developing cerebellum. Granule cells, the major target neurons of mossy fibers, express BDNF during mossy fiber synaptogenesis. To determine whether neurotrophins contribute to the development of cerebellar afferent axons, we characterized the effects of neurotrophins on the growth of mossy fiber neurons from mice and rats in vitro. For a mossy fiber source, we used the basilar pontine nuclei (BPN), the major source of cerebellar mossy fibers in mammals. BDNF and NT4/5 increased BPN neuron survival, neurite outgrowth, growth cone size, and elongation rate, while neither NT3 nor NGF increased survival or outgrowth. In addition, BDNF and NT4/5 reduced the size of neurite bundles. Consistent with these effects, in situ hybridization on cultured basilar pontine neurons revealed the presence of mRNA encoding the TrkB receptor which binds both BDNF and NT4/5 with high affinity. We detected little or no message encoding the TrkC receptor which preferentially binds NT3. BDNF and NT4/5 also increased TrkB mRNA levels in BPN neurons. In addition to previously established functions as an autocrine/paracrine trophic factor for granule cells, the present results indicate that cerebellar BDNF may also act as a target-derived trophic factor for basilar pontine mossy fibers.     

Nerve fiber formation and catecholamine content in adult rat adrenal medullary transplants after treatment with NGF, NT-3, NT-4/5, bFGF, CNTF, and GDNF

Adrenal chromaffin cells have been characterized by the ability to change the phenotype in response to neurotrophic factor stimulation. The adrenal gland expresses numerous trophic factors endogenously, but there is still a lack of knowledge as to how the adrenal medullary cells respond to these factors. Accordingly, we evaluated nerve fiber outgrowth and cell morphology, and measured catecholamine content in adult rat adrenal medullary tissue transplanted to the anterior chamber of the eye after exposure to neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), or glial cell line-derived neurotrophic factor (GDNF) compared with the effects after exposure to recombinant human nerve growth factor (rhNGF). The results show that rhNGF was the most potent factor in inducing neurite outgrowth from the grafted chromaffin cells. CNTF was also a powerful inducer of nerve fiber formation, while NT-4/5, GDNF, and bFGF were less potent. NT-3 did not produce neurite outgrowth above that seen in vehicle-treated eyes. Combining two neurotrophins, rhNGF and NT-3, reduced nerve fiber formation. Tyrosine hydroxylase (TH) immunohistochemistry revealed good cell survival in all grafts, and no morphological differences were detected with the different treatments. The adrenaline: noradrenaline: dopamine ratio was approximately 49%: 49%: 2%, independent of treatment, and the catecholamine content was equal irrespective of treatment. In conclusion, all neurotrophic factors used, except for NT-3, promoted neurite outgrowth from adult rat chromaffin transplants. Differences in outgrowth induced by the various trophic factors did not, however, change the catecholamine content in grafts when analyzed together with the graft-derived nerve plexus.     

Fibroblast growth factor receptor signaling in Xenopus retinal axon extension

Fibroblast growth factor receptors (FGFRs) and N-cadherin both regulate axon extension in developing Xenopus retinal ganglion cells (RGCs). Cultured cerebellar neurons have been shown to require FGFR activity for N-cadherin–stimulated neurite outgrowth, raising the possibility that N-cadherin is a FGFR ligand. To investigate this possibility in the developing visual system, retinal neurons were transfected with a dominant-negative FGFR (XFD) and plated on purified N-cadherin substrates. XFD-expressing neurons extended markedly shorter processes than control GFP-expressing neurons, implicating a role for FGFRs in N-cadherin–stimulated neurite outgrowth. To examine whether N-cadherin and FGFRs share the same pathway or use distinct second messenger pathways, specific inhibitors of implicated signaling molecules were added to neurons stimulated by N-cadherin, basic fibroblast growth factor (bFGF), or brain-derived nerve factor (BDNF) (which stimulates RGC outgrowth by a FGFR-independent mechanism). Diacylglycerol (DAG) lipase and Ca²⁺/calmodulin kinase II inhibitors both significantly reduced outgrowth stimulated by N-cadherin or bFGF but not by BDNF. Furthermore, we show that inhibiting DAG lipase activity in RGC axons extending in vivo toward the optic tectum reversibly slows axon extension without collapsing their growth cones. Thus, a common second-messenger signaling pathway mediating both N-cadherin– and bFGF-stimulated neurite extension is consistent with a model in which N-cadherin directly modulates the FGFR or a model whereby both FGFR and N-cadherin regulate the same second-messenger system. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 633–641, 1998     

Adenoviral vector-directed expression of neurotrophin-3 in rat dorsal root ganglion explants results in a robust neurite outgrowth response

The neurotrophins are a family of proteins that promote neuronal survival and neurite outgrowth during development and can also enhance the regeneration of injured adult neurons. The local and continuous delivery of these proteins at the site of injury is problematic, since this requires repeated intraparenchymal injections or the use of invasive canula-micropump devices. In the present study we report the generation and characterization of an adenoviral vector for a member of the neurotrophins, neurotrophin-3 (Ad-NT-3). Using Ad-NT-3, we examined the expression and biological activity of NT-3 in dorsal root ganglia (DRG) explant cultures. Gene transfer with Ad-NT-3 results in the synthesis of genuine NT-3 and in a dosage-dependent neurite outgrowth response in DRG explants. Transduction of DRG explants with a viral vector dosage of 5 × 10⁵ to 5 × 10⁶ plaque-forming units induced the formation of a dense halo of neurites comparable to outgrowth observed following the addition of 100 ng/mL exogenous NT-3. In addition, a single infection with Ad-NT-3 produced biologically active NT-3 for at least 20 days in culture, as evidenced by continued neurite extension. This indicates that adenoviral vector-mediated expression of NT-3 results in high-level production of biologically active NT-3 and could therefore be used as a strategy to promote the regeneration of injured peripheral and central nerve projections. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 172–184, 1997     

Distinct roles for Sema3A,Sema3F,and an unidentified trophic factor in controlling the advance of geniculate axons to gustatory lingual epithelium

Geniculate ganglion axons arrive in the lingual mesenchyme on embryonic day 13 (E13), 3–4 days before penetrating fungiform papilla epithelium (E17). This latency may result from chemorepulsion by epithelial Sema3A (Dillon et al. (2004) Journal of Comparative Neurology470, 13–24), or Sema3F, which we report is also expressed in this epithelium. Sema3A and Sema3F repelled or suppressed geniculate neurite outgrowth, respectively, and these effects were stage and neurotrophic factor dependent. BDNF-stimulated outgrowth is repelled by Sema3A until E17, but insensitive to Sema3F from E16. NT-4-stimulated neurite outgrowth is sensitive to Sema3A and Sema3F through E18, but NT-4 has not been detected in E15–18 tongue. E15–18 tongue explants did not exhibit net chemorepulsion of geniculate neurites, but the ability of tongue explants to support geniculate neurite outgrowth fluctuates: E12–13 (Rochlin et al. (2000), Journal of Comparative Neurology, 422, 579–593) and E17–18 explants promote and may attract geniculate neurites, but stages corresponding to intralingual arborization do not. The E18 trophic and tropic effects were evident even in the presence of BDNF or NT-4, suggesting that some other factor is responsible. Intrinsic neurite outgrowth capability (without exogenous neurotrophic factors) fluctuated similarly: ganglia deteriorated at E15, but exhibited moderate outgrowth at E18.The chemorepulsion studies are consistent with a role for Sema3A, not Sema3F, in restricting geniculate axons from the epithelium until E17, when axons penetrate the epithelium. The transient inability of tongue explants to promote geniculate neurite outgrowth may signify an alternative mechanism for restricting geniculate axons from the epithelium: limiting trophic factor access.     

Neuronal Survival,Morphology and Outgrowth of Spiral Ganglion Neurons Using a Defined Growth Factor Combination

Objectives

The functionality of cochlear implants (CI) depends, among others, on the number and excitability of surviving spiral ganglion neurons (SGN). The spatial separation between the SGN, located in the bony axis of the inner ear, and the CI, which is inserted in the scala tympani, results in suboptimal performance of CI patients and may be decreased by attracting the SGN neurites towards the electrode contacts. Neurotrophic factors (NTFs) can support neuronal survival and neurite outgrowth.

Methods

Since brain-derived neurotrophic factor (BDNF) is well known for its neuroprotective effect and ciliary neurotrophic factor (CNTF) increases neurite outgrowth, we evaluated if the combination of BDNF and CNTF leads to an enhanced neuronal survival with extended neurite outgrowth. Both NTFs were added in effective high concentrations (BDNF 50ng/ml, CNTF 100ng/ml), alone and in combination, to cultured dissociated SGN of neonatal rats for 48 hours.

Results

The neuronal survival and neurite outgrowth were significantly higher in SGN treated with the combination of the two NTFs compared to treatment with each factor alone. Additionally, with respect to the morphology, the combination of BDNF and CNTF leads to a significantly higher number of bipolar neurons and a decreased number of neurons without neurites in culture.

Conclusion

The combination of BDNF and CNTF shows a great potential to increase the neuronal survival and the number of bipolar neurons in vitro and to regenerate retracted nerve fibers.     

Neurotrophin-4/5, brain-derived neurotrophic factor,and neurotrophin-3 promote survival of cultured vestibular ganglion neurons and protect them against neurotoxicity of ototoxins

The ability of neurotrophin-4/5 (NT-4/5), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF) to promote survival of postnatal rat vestibular ganglion neurons (VGNs) was examined in dissociated cell cultures. Of the four neurotrophins, NT-4/5 and BDNF were equally effective but more potent than NT-3 in promoting the survival of VGNs. In contrast, NGF showed no detectable effects. As expected, TrkB-IgG (a fusion protein of extracellular domain of TrkB and Fc domain of human immunoglobulin G) specifically inhibited the survival-promoting effects by NT-4/5 or BDNF and TrkC-IgG fusion protein completely blocked that of NT-3. Immunohistochemistry with TrkB, TrkA, and p75 antisera revealed that VGNs made TrkB and p75 proteins, but not TrkA protein. Ototoxic therapeutic drugs such as cisplatin and gentamicin often induce degeneration of hair cells and ganglion neurons in both auditory and vestibular systems that leads to impairment of hearing and balance. When cisplatin and gentamicin were added to the dissociated VGN culture in which the hair cells were absent, additional cell death of VGNs was induced, suggesting that the two ototoxins may have a direct neurotoxic effect on ganglion neurons in addition to their known toxicity on hair cells. However, if the cultures were co-treated with neurotrophins, NT-4/5, BDNF, and NT-3, but not NGF, prevented or reduced the neurotoxicity of the two ototoxins. Thus, the three neurotrophins are survival factors for VGNs and are implicated in the therapeutic prevention of VGN loss caused by injury and ototoxins. © 1995 John Wiley & Sons, Inc.     

Paracrine-Mediated Neuroprotection and Neuritogenesis of Axotomised Retinal Ganglion Cells by Human Dental Pulp Stem Cells: Comparison with Human Bone Marrow and Adipose-Derived Mesenchymal Stem Cells

We have investigated and compared the neurotrophic activity of human dental pulp stem cells (hDPSC), human bone marrow-derived mesenchymal stem cells (hBMSC) and human adipose-derived stem cells (hAMSC) on axotomised adult rat retinal ganglion cells (RGC) in vitro in order to evaluate their therapeutic potential for neurodegenerative conditions of RGC. Using the transwell system, RGC survival and length/number of neurites were quantified in coculture with stem cells in the presence or absence of specific Fc-receptor inhibitors to determine the role of NGF, BDNF, NT-3, VEGF, GDNF, PDGF-AA and PDGF-AB/BB in stem cell-mediated RGC neuroprotection and neuritogenesis. Conditioned media, collected from cultured hDPSC/hBMSC/hAMSC, were assayed for the secreted growth factors detailed above using ELISA. PCR array determined the hDPSC, hBMSC and hAMSC expression of genes encoding 84 growth factors and receptors. The results demonstrated that hDPSC promoted significantly more neuroprotection and neuritogenesis of axotomised RGC than either hBMSC or hAMSC, an effect that was neutralized after the addition of specific Fc-receptor inhibitors. hDPSC secreted greater levels of various growth factors including NGF, BDNF and VEGF compared with hBMSC/hAMSC. The PCR array confirmed these findings and identified VGF as a novel potentially therapeutic hDPSC-derived neurotrophic factor (NTF) with significant RGC neuroprotective properties after coculture with axotomised RGC. In conclusion, hDPSC promoted significant multi-factorial paracrine-mediated RGC survival and neurite outgrowth and may be considered a potent and advantageous cell therapy for retinal nerve repair.     

Neurotrophic factor regulation of developing avian oculomotor neurons: differential effects of BDNF and GDNF.

Neurotrophic factors support the development of motoneurons by several possible mechanisms. Neurotrophins may act as target-derived factors or as afferent factors derived from the central nervous system (CNS) or sensory ganglia. We tested whether brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), and glial cell line-derived neurotrophic factor (GDNF) may be target-derived factors for neurons in the oculomotor (MIII) or trochlear (MIV) nucleus in chick embryos. Radio-iodinated BDNF, NT-3, NT-4, and GDNF accumulated in oculomotor neurons via retrograde axonal transport when the trophic factors were applied to the target. Systemic GDNF rescued oculomotor neurons from developmental cell death, while BDNF and NT-3 had no effect. BDNF enhanced neurite outgrowth from explants of MIII and MIV nuclei (identified by retrograde labeling in ovo with the fluorescent tracer DiI), while GDNF, NT-3, and NT-4 had no effect. The oculomotor neurons were immunoreactive for BDNF and the BDNF receptors p75(NTR) and trkB. To determine whether BDNF may be derived from its target or may act as an autocrine or paracrine factor, in situ hybridization and deprivation studies were performed. BDNF mRNA expression was detected in eye muscles, but not in CNS sources of afferent innervation to MIII, or the oculomotor complex itself. Injection of trkB fusion proteins in the eye muscle reduced BDNF immunoreactivity in the innervating motoneurons. These data indicate that BDNF trophic support for the oculomotor neurons was derived from their target.     

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues¹ and survival factors² located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons³. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons⁴. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro^5,6.Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol⁷) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival⁸.     

Activation of transient receptor potential vanilloid-1 (TRPV1) influences how retinal ganglion cell neurons respond to pressure-related stress

Our recent studies implicate the transient receptor potential vanilloid-1 (TRPV1) channel as a mediator of retinal ganglion cell (RGC) function and survival. With elevated pressure in the eye, TRPV1 increases in RGCs, supporting enhanced excitability, while Trpv1 -/- accelerates RGC degeneration in mice. Here we find TRPV1 localized in monkey and human RGCs, similar to rodents. Expression increases in RGCs exposed to acute changes in pressure. In retinal explants, contrary to our animal studies, both Trpv1 -/- and pharmacological antagonism of the channel prevented pressure-induced RGC apoptosis, as did chelation of extracellular Ca²⁺. Finally, while TRPV1 and TRPV4 co-localize in some RGC bodies and form a protein complex in the retina, expression of their mRNA is inversely related with increasing ocular pressure. We propose that TRPV1 activation by pressure-related insult in the eye initiates changes in expression that contribute to a Ca²⁺-dependent adaptive response to maintain excitatory signaling in RGCs.     

Expression and function of TRK-B and BDNF in human neuroblastomas.

There is considerable interest in the role of the TRK family of neuotrophin receptors in regulating growth and differentiation in normal and neoplastic nerve cells. A neuroblastoma is a common pediatric tumor derived from the neural crest, and the majority of favorable neuroblastomas express a high level of TRK-A mRNA. However, little is known about the expression or function of TRK-B in these tumors. TRK-B encodes a tyrosine kinase that binds to brain-derived neuotrophic factor (BDNF), as well as neurotrophin-3 (NT-3) and NT-4/5. We have studied the N-myc-amplified human neuroblastoma cell line, SMS-KCN, which expresses both TRK-B and BDNF. Exogenous BDNF induces tyrosine phosphorylation of TRK-B as well as phosphorylation of phospholipase C-gamma 1, the extracellular signal-regulated kinases 1 and 2, and phosphatidylinositol-3 kinase. BDNF also induces expression of the immediate-early genes c-FOS and NGFI-A but not NGFI-B or NGFI-C. In addition, BDNF appears to promote cell survival and neurite outgrowth. SMS-KCN cells also express TRK-A, which is phosphorylated in response to nerve growth factor. However, the downstream TRK-A signaling is apparently defective. Finally, we determined that in a series of 74 primary neuroblastomas, 36% express TRK-B mRNA, 68% express BDNF mRNA, and 31% express both. Truncated TRK-B appears to be preferentially expressed in more-differentiated tumors (ganglioneuromas and ganglioneuroblastomas), whereas full-length TRK-B is expressed almost exclusively in immature neuroblastomas with N-myc amplification. Our findings suggest that in TRK-B-expressing human neuroblastomas, BDNF promotes survival and induces neurite outgrowth in an autocrine or paracrine manner. The BDNF/TRK-B pathway may be particularly important for growth and differentiation of neuroblastomas with N-myc amplification.     

Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration

Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into two components: (1) RGC survival, and (2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased four- to five-fold at the protein level by 3-5 days after nerve injury. Subsequently, Bcl-2 levels increased 1.7-fold, accompanied by a slight reduction in caspase-3 activity 10-20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2-3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury.