Biomedical subjects

J X Comella

Publications and source records attributed to J X Comella.

At least 19 recordsLinked to original sources

PubMed2006

Reactive oxygen species and p38 mitogen-activated protein kinase activate Bax to induce mitochondrial cytochrome c release and apoptosis in response to malonate.

Malonate, an inhibitor of mitochondrial complex II, is a widely used toxin to study neurodegeneration in Huntington's disease and ischemic stroke. We have shown previously that malonate increased reactive oxygen species (ROS) production in human SH-SY5Y neuroblastoma cells, leading to oxidative stress, cytochrome c release, and apoptotic cell death. Expression of a green fluorescent protein-Bax fusion protein in SH-SY5Y neuroblastoma cells demonstrated a Bax redistribution from the cytosol to mitochondria after 12 to 24 h of malonate treatment that coincided with mitochondrial potential collapse and chromatin condensation. Inhibition of Bax translocation using furosemide, as well as Bax gene deletion, afforded significant protection against malonate-induced apoptosis. Further experiments revealed that malonate induced a prominent increase in the level of activated p38 mitogen-activated protein (MAP) kinase and that treatment with the p38 MAP kinase inhibitor SKF86002 potently blocked malonate-induced Bax translocation and apoptosis. Treatment with vitamin E diminished ROS production, reduced the activation status of p38 MAP kinase, inhibited Bax translocation, and protected against malonate-induced apoptosis. Our data suggest that malonate-induced ROS production and subsequent p38 MAP kinase activation mediates the activation of the pro-apoptotic Bax protein to induce mitochondrial membrane permeabilization and neuronal apoptosis.

Animals↗

PubMed2003

Mu-opioid receptor activation prevents apoptosis following serum withdrawal in differentiated SH-SY5Y cells and cortical neurons via phosphatidylinositol 3-kinase.

Opioid peptides and alkaloids exert their effects via G protein-coupled receptors (GPCRs). It has been shown that, in addition to trophic factors, some GPCRs are able to activate the phosphatidylinositol 3-kinase/Akt (PI 3-K/Akt) signal transduction pathway, thus leading to cell survival. The aim of this study was to test whether activation of mu-opioid receptors has protective effects on serum withdrawal-induced cell death and to study the possible implication of PI 3-K in this process. In SH-SY5Y neuroblastoma cells fully differentiated by exposure to retinoic acid for five days, the enkephalin derivative selective mu-agonist DAMGO (0.1-2 microM) and the alkaloid morphine (0.1-10 microM) promoted cell survival after serum deprivation (MTT and trypan blue exclusion assays), without inducing cell proliferation. These effects were fully reversed by naloxone, by the selective mu-antagonist beta-funaltrexamine (beta-FNA) and also by the specific PI 3-K inhibitor LY294002. The two agonists stimulated Akt phosphorylation and the effect was also abolished by beta-FNA and by LY294002. In mouse primary cortical neurons, DAMGO reduced the percentage of apoptosis after 6, 12, 24 and 48 h of serum withdrawal; as determined by Hoechst staining. This effect was blocked by beta-FNA, by pre-treatment with pertussis toxin and by LY294002. DAMGO also stimulated Akt phosphorylation via PI 3-K in this primary neuronal culture. Together, these results indicate that stimulation of the mu-opioid receptor promotes neuronal survival in a G(i/o)-linked, PI 3-K-dependent signaling cascade and suggest that Akt may be a key downstream kinase involved in this anti-apoptotic effect.

Apoptosis↗

PubMed2003

Lack of Apaf-1 expression confers resistance to cytochrome c-driven apoptosis in cardiomyocytes.

Apoptosis plays a role in cardiomyocyte death in several cardiovascular disorders. Here, we show that primary postnatal cardiomyocytes did not die upon activation of the intrinsic (cytochrome c-dependent) apoptotic pathway. Release of cytochrome c from mitochondria to the cytosol occurred, but did not activate the effector phase of apoptosis. Myocardial cells did not express apoptotic protease-activating factor-1 (Apaf-1), the allosteric activator of caspase-9 acting downstream of cytochrome c release. Forced expression of Apaf-1 restored the competence to complete the cytochrome c-induced apoptotic program and this effect was prevented by overexpression of Bcl-X(L). However, cardiomyocytes were able to enter the apoptotic program when it was initiated by activation of death receptors, as observed during serum deprivation and metabolic inhibition. Our results indicate that regulation of Apaf-1 expression may be a new regulatory mechanism developed in postmitotic cells in order to prevent irreversible commitment to die after release of cytochrome c.

Animals↗

PubMed2002

Isolation of AmphiCASP-3/7, an ancestral caspase from amphioxus (Branchiostoma floridae). Evolutionary considerations for vertebrate caspases.

Caspases are a large family of cysteine proteases that play an essential role as effectors of apoptosis in metazoans. Thirteen different caspases have been identified in vertebrates so far, and their function in apoptotic or inflammatory responses is well documented. We have taken advantage of the broadly accepted condition of amphioxus (Cephalochordata, Branchiostoma floridae) as the closest living relative to vertebrates to study the molecular evolution of caspases. Here we report for the first time the pattern of programmed cell death during development of cephalochordates. We also describe the isolation and functional characterisation of the first caspase related gene in amphioxus, which we named AmphiCASP-3/7. The amphioxus caspase is expressed throughout development, from the gastrula to larva stage. AmphiCASP-3/7 induced cell death when ectopically expressed in human HEK 293T cells, and the recombinant protein was inhibited by DEVD peptides. AmphiCASP-3/7 reflects the primitive condition of the executor vertebrates caspases -3 and -7, prior to vertebrate specific duplication. Interestingly, AmphiCASP-3/7 is functionally closer to vertebrate caspase-7, as shown by substrate specificity both in vitro and in MCF7 cells. Our phylogenetic and functional data help in drawing the evolutionary history of caspases, and illustrates an example of acquisition in vertebrates of novel functional properties after gene duplication.

Animals↗

PubMed2001

Neuronal survival induced by neurotrophins requires calmodulin.

It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, we have analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells.

Animals↗

PubMed2001

The absence of oligonucleosomal DNA fragmentation during apoptosis of IMR-5 neuroblastoma cells: disappearance of the caspase-activated DNase.

Caspase-activated DNase is responsible for the oligonucleosomal DNA degradation during apoptosis. DNA degradation is thought to be important for multicellular organisms to prevent oncogenic transformation or as a mechanism of viral defense. It has been reported that certain cells, including some neuroblastoma cell lines such as IMR-5, enter apoptosis without digesting DNA in such a way. We have analyzed the causes for the absence of DNA laddering in staurosporine-treated IMR-5 cells, and we have found that most of the molecular mechanisms controlling apoptosis are well preserved in this cell line. These include degradation of substrates for caspases, blockade of cell death by antiapoptotic genes such as Bcl-2 or Bcl-X(L), or normal levels and adequate activation of caspase-3. Moreover, these cells display normal levels of caspase-activated DNase and its inhibitory protein, inhibitor of caspase-activated DNase, and their cDNA sequences are identical to those reported previously. Nevertheless, IMR-5 cells lose caspase-activated DNase during apoptosis and recover their ability to degrade DNA when human recombinant caspase-activated DNase is overexpressed. Our results lead to the conclusion that caspase-activated DNase is processed during apoptosis of IMR-5 cells, making these cells a good model to study the relevance of this endonuclease in physiological or pathological conditions.

Apoptosis↗

PubMed2001

c-Src is required for glial cell line-derived neurotrophic factor (GDNF) family ligand-mediated neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway.

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), consisting of GDNF, neurturin, persephin, and artemin, signal via a multicomponent complex composed of Ret tyrosine kinase and the glycosyl-phosphatidylinositol (GPI)-anchored coreceptors GFRalpha1-alpha4. In previous work we have demonstrated that the localization of Ret to membrane microdomains known as lipid rafts is essential for GDNF-induced downstream signaling, differentiation, and neuronal survival. Moreover, we have found that Ret interacts with members of the Src family kinases (SFK) only when it is localized to these microdomains. In the present work we show by pharmacological and genetic approaches that Src activity was necessary to elicit optimal GDNF-mediated signaling, neurite outgrowth, and survival. In particular, p60Src, but not the other ubiquitous SFKs, Fyn and Yes, was responsible for the observed effects. Moreover, Src appeared to promote neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway because the PI-3K inhibitor LY294002 prevented GFL-mediated neuronal survival and prevented activated Src-mediated neuronal survival. In contrast, the inhibition of Src activity had no effects on NGF-mediated survival, indicating that the requirement for Src was selective for GFL-mediated neuronal survival. These data confirm the importance of protein-protein interactions between Ret and raft-associated proteins in the signaling pathways elicited by GDNF, and the data implicate Src as one of the major signaling molecules involved in GDNF-mediated bioactivity.

Animals↗

PubMed2001

Cytokines promote motoneuron survival through the Janus kinase-dependent activation of the phosphatidylinositol 3-kinase pathway.

To determine which intracellular pathways mediate the survival effects of ciliary neurotrophic factor and cardiotrophin-1 cytokines on motoneurons, we studied the activation of the Jak/STAT, the PI 3-kinase/Akt, and the ERK pathways. At shorter time points, cytokines induced the activation of STAT3 and ERK, but not PI 3-kinase. Jak3 inhibitor suppressed cytokine- and muscle extract-induced survival. In contrast, PD 98059, a MEK inhibitor, was not able to prevent cytokine-induced survival, demonstrating that ERK is not involved. Surprisingly, the PI 3-kinase inhibitor LY 294002 prevented the survival-promoting effects of cytokines. When assays of PI 3-kinase activity were performed at later stages following cytokine treatment a significant increase was observed compared to control cultures. This delayed increase of activity could be completely prevented by treatment with protein synthesis or Jak3 inhibitors. Collectively, these results demonstrate that cytokines induce motoneuron survival through a PI 3-kinase activation requiring de novo protein synthesis dependent on Jak pathway.

Animals↗

PubMed2000

PC12 cells have caveolae that contain TrkA. Caveolae-disrupting drugs inhibit nerve growth factor-induced, but not epidermal growth factor-induced, MAPK phosphorylation.

Nerve growth factor (NGF) induces survival and differentiation of the neural crest-derived PC12 cell line. Caveolae are cholesterol-enriched, caveolin-containing plasma membrane microdomains involved in vesicular transport and signal transduction. Here we demonstrate the presence of caveolae in PC12 cells and their involvement in NGF signaling. Our results showed the expression of caveolin-1 by Western blot and confocal immuno-microscopy. The presence of plasma membrane caveolae was directly shown by rapid-freeze deep-etching electron microscopy. Moreover, combined deep-etching and immunogold techniques revealed the presence of the NGF receptor TrkA in the caveolae of PC12 cells. These data together with the cofractionation of Shc, Ras, caveolin, and TrkA in the caveolae fraction supported a role for these plasma membrane microdomains in NGF signaling. To approach this hypothesis, caveolae were disrupted by treatment of PC12 cells with cholesterol binding drugs. Either filipin or cyclodextrin treatment increased basal levels of MAPK phosphorylation. In contrast, pretreatment of PC12 cells with these drugs inhibited the NGF- but not the epidermal growth factor-induced MAPK phosphorylation without affecting the TrkA autophosphorylation. Taken together, our results demonstrate the presence of caveolae in PC12 cells, which contain the high affinity NGF receptor TrkA, and the specific involvement of these cholesterol-enriched plasma membrane microdomains in the propagation of the NGF-induced signal.

Animals↗

PubMed2000

Combined use of the green and yellow fluorescent proteins and fluorescence-activated cell sorting to select populations of transiently transfected PC12 cells.

One of the more time-consuming procedures in the study of exogenously expressed proteins in cell lines is the selection of individual transfected clones. In recent years, green fluorescent protein variants with excitation/emission spectra matching the typical flow cytometer configurations have been generated and are in common use. We employed PC12 cells transfected with vectors encoding fluorescent proteins and a fluorescence selection procedure using a fluorescence-activated cell-sorter. In order to select the optimal co-electroporation and sorting conditions, we used the simultaneous detection of two variants of the green fluorescent protein, that possess separable emission peaks when excited at 488 nm. Using these variants and the adequate combination of band-pass filters, we were able to analyze and establish the conditions for identifying and sorting cells transfected with enhanced green fluorescent protein, that simultaneously express another plasmid of interest. Using this procedure, the cells sorted that express both plasmids exceeded 90%. The whole procedure did not alter the physiological responsiveness of the transfected cells to growth factors, and has been successfully applied to the constitutive activation of the mitogen-activated protein kinase pathway, resulting in the spontaneous differentiation of PC12 cells. Also, this procedure has been used with other set of expression vectors encoding proteins that protect PC12 cells from apoptosis caused by different stimuli. The method that we present here provides an easy and fast procedure to obtain a high proportion of positively transfected populations of PC12 cells.

Animals↗

PubMed2000

Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells.

A rapid and simple procedure is presented to obtain nearly pure populations of human neuron-like cells from the SH-SY5Y neuroblastoma cell line. Sequential exposure of SH-SY5Y cells to retinoic acid and brain-derived neurotrophic factor in serum-free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5-bromo-2'-deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain-derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron-specific enolase, and growth-associated protein-43 as well as neuronal polarity markers such as tau and microtubule-associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large-scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.

Apoptosis↗

PubMed2000

Nerve growth factor activation of the extracellular signal-regulated kinase pathway is modulated by Ca(2+) and calmodulin.

Nerve growth factor is a member of the neurotrophin family of trophic factors that have been reported to be essential for the survival and development of sympathetic neurons and a subset of sensory neurons. Nerve growth factor exerts its effects mainly by interaction with the specific receptor TrkA, which leads to the activation of several intracellular signaling pathways. Once activated, TrkA also allows for a rapid and moderate increase in intracellular calcium levels, which would contribute to the effects triggered by nerve growth factor in neurons. In this report, we analyzed the relationship of calcium to the activation of the Ras/extracellular signal-regulated kinase pathway in PC12 cells. We observed that calcium and calmodulin are both necessary for the acute activation of extracellular signal-regulated kinases after TrkA stimulation. We analyzed the elements of the pathway that lead to this activation, and we observed that calmodulin antagonists completely block the initial Raf-1 activation without affecting the function of upstream elements, such as Ras, Grb2, Shc, and Trk. We have broadened our study to other stimuli that activate extracellular signal-regulated kinases through tyrosine kinase receptors, and we have observed that calmodulin also modulates the activation of such kinases after epidermal growth factor receptor stimulation in PC12 cells and after TrkB stimulation in cultured chicken embryo motoneurons. Calmodulin seems to regulate the full activation of Raf-1 after Ras activation, since functional Ras is necessary for Raf-1 activation after nerve growth factor stimulation and calmodulin-Sepharose is able to precipitate Raf-1 in a calcium-dependent manner.

Animals↗

PubMed1999

Receptors of the glial cell line-derived neurotrophic factor family of neurotrophic factors signal cell survival through the phosphatidylinositol 3-kinase pathway in spinal cord motoneurons.

The members of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors (GDNF, neurturin, persephin, and artemin) are able to promote in vivo and in vitro survival of different neuronal populations, including spinal cord motoneurons. These factors signal via multicomponent receptors that consist of the Ret receptor tyrosine kinase plus a member of the GDNF family receptor alpha (GRFalpha) family of glycosylphosphatidylinositol-linked coreceptors. Activation of the receptor induces Ret phosphorylation that leads the survival-promoting effects. Ret phosphorylation causes the activation of several intracellular pathways, but the biological effects caused by the activation of each of these pathways are still unknown. In the present work, we describe the ability of the GDNF family members to promote chicken motoneuron survival in culture. We show the presence of Ret and GFRalpha-1, GFRalpha-2, and GFRalpha-4 in chicken motoneurons using in situ hybridization and reverse transcription-PCR techniques. By Western blot analysis and kinase assays, we demonstrate the ability of these factors to induce the phosphatidylinositol 3 kinase (PI 3-kinase) and the extracellular regulated kinase (ERK)-mitogen-activated protein (MAP) kinase pathways activation. To characterize the involvement of these pathways in the survival effect, we used the PI 3-kinase inhibitor LY 294002 and the MAP kinase and ERK kinase (MEK) inhibitor PD 98059. We demonstrate that LY 294002, but not PD 98059, prevents GDNF-, neurturin-, and persephin-induced motoneuron survival, suggesting that PI 3-kinase intracellular pathway is responsible in mediating the neurotrophic effect.

Animals↗

PubMed1999

Calcium influx activates extracellular-regulated kinase/mitogen-activated protein kinase pathway through a calmodulin-sensitive mechanism in PC12 cells.

Evidence suggests that membrane depolarization is able to promote neuronal survival through a sustained, although moderate, increase in the intracellular calcium. We have used the PC12 cell line to study the possible intracellular pathways that can be activated by calcium influx. Previously, we observed that membrane depolarization-induced calcium influx was able to activate the extracellular-regulated kinase (ERK)/mitogen-activated protein kinase pathway and most of this activation was calmodulin-dependent. We demonstrated that a part of the ERK activation is due to the phosphorylation of the epidermal growth factor receptor. Here, we show that both the epidermal growth factor receptor phosphorylation and the Shc-Grb2-Ras activation are not calmodulin-modulated. Moreover, dominant negative mutant Ha-ras (Asn-17) prevents the activation on ERKs by membrane depolarization, suggesting that Ras and calmodulin are both necessaries to activate ERKs by membrane depolarization. We failed to observe any significant induction and/or modulation of the A-Raf, B-Raf or c-Raf-1 kinase activities, thus suggesting the existence of a MEK kinase different from the classical Raf kinases that directly or indirectly can be modulated by Ca2+/calmodulin.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Binding patterns of lectins with GalNAc specificity in the mouse dorsal root ganglia and spinal cord.

To localize membrane glycoconjugates in neurons of the mouse spinal cord and dorsal root ganglia (DRG), cryostat sections of newborn (P0), 7 day-old (P7), P14, P21 and P31 animals were stained with ten FITC-conjugated plant lectins, the majority of them recognizing N-acetyl-D-galactosamine (GalNAc) terminal sugar residues. In the dorsal root ganglia of P0 animals, the different lectins showed distinct patterns of labeling in either cells of the nervous system, including neurons, or other structures such as nerves or blood vessels. Moreover, some of these lectins showed important changes in their pattern of labeling during postnatal development. This was especially relevant for lectins that label a subpopulation of small-sized cells that have been previously identified as the nociceptive cells of the DRG. Enzymatic digestion of sections with neuraminidase removes sialic acid from the carbohydrate chains of glycoconjugates thus exposing novel sugar residues. When this treatment was applied to DRG sections from postnatal animals the pattern of lectin staining was either changed or eliminated and heterogeneous subsets of glycoconjugates normally masked by this sugar were exposed. In the spinal cord of PO animals, none of the lectins labeled cells in the central gray matter. However, after the enzymatic digestion of sections with neuraminidase, spinal cord motoneurons and some other cells were labeled by two of the lectins suggesting that GalNAc residues present in these cells are normally masked by terminal sialic acid. Altogether, these results show important changes in the temporal and spatial expression of glycoconjugates that may be relevant for the postnatal development of the CNS and PNS of mice.

Acetylgalactosamine↗

PubMed1999

Activation of phosphatidylinositol 3-kinase, but not extracellular-regulated kinases, is necessary to mediate brain-derived neurotrophic factor-induced motoneuron survival.

Chick embryo spinal cord motoneurons develop a trophic response to some neurotrophins when they are maintained in culture in the presence of muscle extract. Thus, after 2 days in culture, brain-derived neurotrophic factor (BDNF) promotes motoneuron survival. In the present study we have analyzed the intracellular pathways that may be involved in the BDNF-induced motoneuron survival. We have observed that BDNF activated the extracellular-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and the phosphatidylinositol (PI) 3-kinase pathways. To examine the contribution of these pathways to the survival effect triggered by BDNF, we used PD 98059, a specific inhibitor of MAP kinase kinase, and LY 294002, a selective inhibitor of PI 3-kinase. PD 98059, at doses that significantly reduced the phosphorylation of ERKs, did not show any prominent effect on neuronal survival. However, LY 294002 at doses that inhibited the phosphorylation of Akt, a down-stream element of the PI 3-kinase, completely abolished the motoneuron survival effects of BDNF. Moreover, cell death triggered by LY 294002 treatment exhibited features similar to those observed after muscle extract deprivation. Our results suggest that the PI 3-kinase pathway plays an important role in the survival effect triggered by BDNF on motoneurons, whereas activation of the ERK MAP kinase pathway is not relevant.

Animals↗

PubMed1999

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.

Brain-Derived Neurotrophic Factor↗

PubMed1998

Development of survival responsiveness to brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4/5, but not to nerve growth factor, in cultured motoneurons from chick embryo spinal cord.

During embryonic development, most neuronal populations undergo a process usually referred to as naturally occurring neuronal death. For motoneurons (MTNs) of the lumbar spinal cord of chick embryos, this process takes place in a well defined period of time, between embryonic days 6 and 10 (E6-E10). Neurotrophins (NTs) are the best characterized family of neurotrophic factors and exert their effects through activation of their specific Trk receptors. In vitro and in vivo studies have demonstrated that rodent motoneurons survive in response to BDNF, NT3, and NT4/5. In contrast, the trophic dependencies of chicken motoneurons have been difficult to elucidate, and various apparently conflicting reports have been published. In the present study, we describe how freshly isolated motoneurons from E5.5 chick embryos did not respond to any neurotrophin in vitro. Yet, because motoneurons were maintained alive in culture in the presence of muscle extract, they developed a delayed specific survival response to BDNF, NT3, and NT4/5 that is clearly dose-dependent, reaching saturation at doses of 100 pg/ml. This trophic response correlated with increasing expression of the corresponding functional receptors TrkB and TrkC. Moreover, TrkB receptor is able to become autophosphorylated and to activate classical intracellular signaling pathways such as the extracellular signal-regulated protein kinase when it is stimulated with its cognate ligand BDNF. Therefore, our results reconcile the reported differences between in vivo and in vitro studies on the ability of chicken MTNs to respond to some members of the neurotrophin family of trophic factors.

Animals↗