Biomedical subjects

E M Shooter

Publications and source records attributed to E M Shooter.

At least 19 recordsLinked to original sources

PubMed2004

Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 are sorted to dense-core vesicles and released via the regulated pathway in primary rat cortical neurons.

Neurotrophins (NTs) play an important role in the modulation of synaptic transmission and in morphological changes in synaptic structures. Although there is agreement that brain-derived neurotrophic factor (BDNF) is sorted to large dense-core vesicles (LDCVs) and released via the regulated secretory pathway, there has been some dispute regarding the mode of secretion of nerve growth factor (NGF) and neurotrophin-3 (NT-3), two structurally related members of the NT family. In this study, we examined the subcellular localization and release characteristics of NGF, BDNF, and NT-3 in adenovirus-infected primary cortical neurons. We found that all members of the NT family colocalized with markers for the endoplasmic reticulum and Golgi within cell bodies and in a punctate manner with a marker for LDCVs within processes. Moreover, their release was triggered by depolarization, indicating that NGF, BDNF, and NT-3 are released via the regulated secretory pathway. When neurons were coinfected with two separate adenoviruses coding for NGF or BDNF, both NTs showed almost complete vesicular colocalization within single cells, suggesting that different NTs might be packaged into shared vesicles. We also examined whether the two splice variants of NGF, the short and long precursors, differ in their release characteristics. We found that neurons infected with viruses coding for either splice variant released NGF in a regulated way. Overall, our study supports the notion that all members of the NT family undergo activity-dependent regulated release from neurons, enabling them to act as "synaptotrophins" on electrically active neurons.

Animals↗

PubMed2002

BDNF overexpression increases dendrite complexity in hippocampal dentate gyrus.

There is increasing evidence that brain-derived neurotrophic factor (BDNF) modulates synaptic and morphological plasticity in the developing and mature nervous system. Plasticity may be modulated partially by BDNF's effects on dendritic structure. Utilizing transgenic mice where BDNF overexpression was controlled by the beta-actin promoter, we evaluated the effects of long-term overexpression of BDNF on the dendritic structure of granule cells in the hippocampal dentate gyrus. BDNF transgenic mice provided the opportunity to investigate the effects of modestly increased BDNF levels on dendrite structure in the complex in vivo environment. While the elevated BDNF levels were insufficient to change levels of TrkB receptor isoforms or downstream TrkB signaling, they did increase dendrite complexity of dentate granule cells. These cells showed an increased number of first order dendrites, of total dendritic length and of total number of branch points. These results suggest that dendrite structure of granule cells is tightly regulated and is sensitive to modest increases in levels of BDNF. This is the first study to evaluate the effects of BDNF overexpression on dendrite morphology in the intact hippocampus and extends previous in vitro observations that BDNF influences synaptic plasticity by increasing complexity of dendritic arbors.

Animals↗

PubMed2001

Neurotrophins are key mediators of the myelination program in the peripheral nervous system.

Although knowledge of the functions of neurotrophins has advanced rapidly in recent years, studies concerning the involvement of neurotrophins in glial-neuronal interactions rarely extend further than their roles in supporting the survival and differentiation of neuronal cells. In this study endogenous brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) were identified in Schwann cell/dorsal root ganglia neuronal cocultures and shown to modulate the myelination program of the peripheral nervous system. The differential expression of BDNF and NT3 were examined and compared with the expression profiles of myelin proteins in the cocultures throughout the myelination process. BDNF levels correlated with active myelin formation, whereas NT3 expression was initially high and then down regulated throughout the proliferation and premyelination periods. Addition of exogenous BDNF enhanced myelination, whereas the removal of the endogenous BDNF by using the BDNF receptor TrkB-Fc fusion protein inhibited the formation of mature myelin internodes. Interestingly, exogenous NT3 significantly inhibited myelination, whereas the removal of the endogenous NT3 by using the NT3 receptor TrkC-Fc fusion protein resulted in an enhancement similar to that obtained with the addition of BDNF. In addition, in vivo studies were performed during the development of the mouse sciatic nerve. Subcutaneous injections of BDNF resulted in an enhancement of myelin formation in the sciatic nerve, whereas the removal of the endogenous BDNF dramatically inhibited myelination. Injections of NT3 inhibited myelin formation, and the removal of the endogenous NT3 enhanced myelination. These results demonstrate that BDNF and NT3 possess different modulatory roles in the myelination program of the peripheral nervous system and that their mechanisms of action are specific and highly regulated.

Animals↗

PubMed2001

Binding of nerve growth factor to its p75 receptor in stressed cells induces selective IkappaB-beta degradation and NF-kappaB nuclear translocation.

Nerve growth factor (NGF) regulates the activity of the transcription factor NF-kappaB (nuclear factor-kappaB) through its low affinity receptor, p75. In the present study we found that NGF binding to p75 induces nuclear translocation of p65 and increases NF-kappaB binding activity in a cell line overexpressing p75, but only after the cells have been subjected to a previous stress. Under physiological conditions, in the absence of stress, NGF is unable to alter p65 nuclear levels. Tumor necrosis factor-alpha (TNF-alpha) induces a down-regulation of IkappaB-alpha, -beta and -epsilon both in physiological and in stress, i.e. serum-free, conditions. In contrast, NGF only induces the specific degradation of IkappaB-beta after serum withdrawal, without affecting IkappaB-alpha or -epsilon either in the presence or in the absence of stress. IkappaB-beta consists of several isoforms, whose relative abundance is regulated by serum withdrawal. NGF does not target all the IkappaB-beta isoforms with the same potency, being more effective in reducing the levels of the isoforms up-regulated by serum withdrawal. TRAF-6 is expressed at the same level under both physiological and stress conditions. These results indicate that NGF is able to induce NF-kappaB nuclear translocation by a mechanism that involves specific IkappaB-beta degradation only after the cells have been subjected to a severe stress.

Animals↗

PubMed2001

Early days of the nerve growth factor proteins.

Adult male mouse submaxillary glands served as the preferred starting material for the isolation of the nerve growth factor (NGF) proteins in most of the isolation studies done. Two types of NGF proteins were isolated from extracts of the gland, a high-molecular-weight 7S NGF complex and a low-molecular-weight protein variously called NGF, betaNGF, or 2.5S NGF. The latter, which mediated all known biological functions of NGF, were closely related forms of a basic NGF dimer in which the N and C termini of two monomers (chains) were modified by proteolytic enzymes to different extents with no effect on biological activity. The betaNGF dimer showed a novel protein structure in which the two chains interacted non-covalently over a wide surface. Correspondingly, the betaNGF dimer was found to be unusually stable and the form through which NGFs actions were mediated at physiological concentrations. The betaNGF dimer was one of three subunits in 7S NGF; the other two were the gamma subunit, an arginine esteropeptidase or kallikrein, and the alpha subunit, an inactive kallikrein. Two zinc ions were also present in the complex and contributed greatly to its stability. There was much debate about whether 7S NGF was a specific protein complex of interacting subunits and, if so, what functions it might play in the biology of NGF. Observations of the inhibition of the enzyme activity of the gamma subunit and of the biological activity of betaNGF in 7S NGF were important in determining that 7S NGF was a naturally occurring complex and the sole source of NGF in the gland extract or in saliva. Specific interactions between the active site of the gamma subunit and the C-terminal arginine residues of the NGF chains, confirmed in the three-dimensional structure of 7S NGF, suggested a role for the gamma subunit in pro-NGF processing during the assembly of 7S NGF. In spite of the detailed knowledge of 7S NGF structure, no information on the role of this complex in the neurobiology of NGF has emerged. With the exception of the submaxillary gland of an African rodent, no other source of NGF has been convincingly shown to synthesize the alpha and gamma subunits, and they may well be irrelevant to NGFs actions.

Animals↗

PubMed2000

Palmitoylation of the p75 neurotrophin receptor has no effect on its interaction with TrkA or on TrkA-mediated down-regulation of cell adhesion molecules.

The short- and long-term effects of nerve growth factor (NGF) were studied on fibroblast cell lines stably expressing both TrkA and either wild-type p75 or a mutant that lacks the palmitoylation site of p75. The lack of palmitoylation had no effect on the ability of p75 to enhance the short-term NGF-induced tyrosine phosphorylation of TrkA over a wide range of NGF concentrations. Long-term treatment of the cell lines with NGF led to loss of cell adhesion to the culture dishes that increased with increasing concentrations of NGF and increased expression of TrkA. Treatment of the cell lines with mutant NGFs that bound selectively to TrkA or p75 alone revealed that cell detachment was mediated solely through TrkA. Increased cell detachment correlated with a decrease in the expression levels of fibronectin and cadherin, cell surface molecules involved in cell adhesion. The loss of cell adhesion with the cell line expressing the palmitoylation-deficient p75 were identical to those expressing wild type, as was anticipated from the lack of involvement of p75 in this process.

Animals↗

PubMed2000

p75 reduces TrkB tyrosine autophosphorylation in response to brain-derived neurotrophic factor and neurotrophin 4/5.

Neurotrophins mediate their signals through two different receptors: the family of receptor tyrosine kinases, Trks, and the low affinity pan-neurotrophin receptor p75. Trk receptors show more restricted ligand specificity, whereas all neurotrophins are able to bind to p75. One important function of p75 is the enhancement of nerve growth factor signaling via TrkA by increasing TrkA tyrosine autophosphorylation. Here, we have examined the importance of p75 on TrkB- and TrkC-mediated neurotrophin signaling in an MG87 fibroblast cell line stably transfected with either p75 and TrkB or p75 and TrkC, as well as in PC12 cells stably transfected with TrkB. In contrast to TrkA signaling, p75 had a negative effect on TrkB tyrosine autophosphorylation in response to its cognate neurotrophins, brain-derived neurotrophic factor and neurotrophin 4/5. On the other hand, p75 had no effect on TrkB or TrkC activation in neurotrophin 3 treatment. p75 did not effect extracellular signal-regulated kinase 2 tyrosine phosphorylation in response to brain-derived neurotrophic factor, neurotrophin 3, or neurotrophin 4/5. These results suggest that the observed reduction in TrkB tyrosine autophosphorylation caused by p75 does not influence Ras/mitogen-activated protein kinase signaling pathway in neurotrophin treatments.

Animals↗

PubMed2000

Combined assessment of tau and neuronal thread protein in Alzheimer's disease CSF.

OBJECTIVE: Comparative study of CSF levels of tau and AD7C-neuronal thread protein (NTP) in patients with AD and control subjects. BACKGROUND: AD is characterized by neurofibrillary tangles composed of the abnormally hyperphosphorylated microtubule-associated protein tau. AD7C-NTP is a proposed AD marker expressed at early stages of neurofibrillary degeneration. METHODS: Enzyme-linked immunosorbent assays specific for tau and AD7C-NTP. CSF samples were obtained from 35 demented patients (25 with antemortem clinical diagnosis of probable AD, 5 with neuropathologic diagnosis of definite AD, 5 with Lewy body pathology), 29 nondemented patients with PD, and 16 elderly healthy control subjects. Receiver operating characteristics (ROC) and multivariate discriminant analysis for AD versus controls. Correlational analysis of CSF tau and AD7C-NTP and of each marker with Mini-Mental State Examination (MMSE) scores was performed. RESULTS: Levels of both tau and AD7C-NTP were significantly elevated in the AD patients compared with control subjects. ROC analysis showed that CSF tau distinguished between patients with AD and nondemented control subjects with 63% sensitivity and 89% specificity, AD7C-NTP with 70% sensitivity and 87% specificity. Combined evaluation of both markers with discriminant analysis raised the specificity to 93% at a 63% sensitivity level. Both markers positively correlated with each other within the AD group, but not among control subjects. CSF levels of AD7C-NTP, but not of tau, showed a small but significant inverse correlation (r = -0.43) with MMSE scores of AD patients. CONCLUSIONS: CSF levels of tau and AD7C-NTP may be useful biomarkers for AD.

Aged↗

PubMed2000

Translational control of ribosomal protein L4 mRNA is required for rapid neurite regeneration.

Under some circumstances neurons can be primed to rapidly regenerate injured neuritic processes independent of new gene expression. Such transcription-independent neurite extension occurs in adult rat sensory neurons cultured after sciatic nerve crush and in NGF-differentiated PC12 cells whose neurites have been mechanically sheared. In the PC12 cells, neurite regeneration occurs by means of translational control of mRNAs which were transcribed prior to neurite injury. The survival of such translationally regulated mRNAs is relatively short in the differentiated PC12 cells (< or =10 h). By subtractive hybridization, we have isolated a short-lived mRNA from differentiated PC12 cells. This mRNA, which encodes the ribosomal protein L4, is translationally regulated during neurite regeneration in PC12 cells. Antisense oligonucleotides to L4 mRNA inhibit neurite regeneration from the differentiated PC12 cells as well as axonal elongation from conditioned sensory neurons, indicating that ongoing translation of L4 mRNA is needed for these forms of rapid transcription-independent neurite growth. Taken together, these data point to the importance of translational regulation of existing neuronal mRNAs in the regenerative responses to neuronal injury. Although there are other examples of neuronal translational control, there are no other known neuronal proteins whose levels are regulated predominantly by translational rather than transcriptional control.

Animals↗

PubMed2000

Role of the peripheral myelin protein 22 N-linked glycan in oligomer stability.

Peripheral myelin protein 22 (PMP22) is a 22-kDa glycoprotein containing a single N-linked carbohydrate moiety. This posttranslational modification is conserved in PMP22 across species and within members of the PMP22 gene family; however, the function of the oligosaccharide is not known. To study the role of the PMP22 carbohydrate, site-directed mutagenesis was used to alter the glycosylation consensus sequence and produce a glycosylation-deficient mutant protein. This modified PMP22 was expressed in primary Schwann cells (SCs), and the effect of the N-glycan on the turnover rate, oligomerization, and intracellular trafficking of PMP22 was determined. Our data show a slight decrease in turnover rate from a half-life of approximately 70 min for the wild-type (wt) protein to 100 min for the glycosylation mutant. Although the presence of glycosylation-deficient PMP22 oligomers could be detected in SCs, we observed a decrease in oligomer stability compared with the wt oligomers. Both wt and mutant proteins showed similar localization in the endoplasmic reticulum and Golgi compartments and were transported to the SC surface. These results suggest that the N-glycan of PMP22 facilitates, in part, the stability of the PMP22 oligomer; however, the implications of PMP22 oligomerization remain unknown.

Amino Acid Substitution↗

PubMed1999

Preparation of nuclear extracts from myelinating Schwann cells.

Myelination of peripheral nerve fibres is performed by Schwann cells and is associated with the coordinate upregulation of lipid synthesis and multiple genes encoding myelin-specific proteins. Both the decision to enter into a myelinating phenotype and subsequently, the quantity of myelin that each Schwann cell elaborates appear to be controlled by axonal signals. Understanding of the relevant signaling pathways and the downstream transcription factors and cis elements that confer myelin gene expression is notably limited. In large part, this has resulted directly from a lack of methods for obtaining nuclear extracts from myelinating Schwann cells thus precluding the application of numerous powerful molecular techniques. In this report, we describe a method that overcomes this limitation for the myelinating Schwann cells in the sciatic nerves of the mouse. During the evolution of the method, its effectiveness was monitored using an oligonucleotide containing the binding site for KROX-20, a transcription factor known to be present in myelinating Schwann cells. Following technical development, the optimized protocol has proven to be entirely reliable and thus novel experimental strategies now can be applied to the investigation of the molecular mechanisms controlling gene expression in peripheral nerves.

Animals↗

PubMed1999

Transport of Trembler-J mutant peripheral myelin protein 22 is blocked in the intermediate compartment and affects the transport of the wild-type protein by direct interaction.

Peripheral myelin protein 22 (PMP22) is an integral membrane protein that is essential for the normal formation and maintenance of peripheral myelin. Duplications, deletions, or mutations in the PMP22 gene account for a set of dominantly inherited peripheral neuropathies. The heterozygous Trembler-J (TrJ) genotype in mice is similar genetically to a Charcot-Marie-Tooth disease type 1A pedigree in humans, whereas the homozygous TrJ condition leads to the most severe form of PMP22-associated neuropathies. To characterize the consequences of the TrJ mutation, we labeled wild-type (wt-) and TrJ-PMP22 in the third loop of the protein with different epitope tags and expressed them separately or together in COS7 cells and primary Schwann cells. Here we show that the transport of the mutant TrJ-PMP22 is interrupted in the intermediate compartment, preventing its insertion into the plasma membrane and affecting the morphology of the endoplasmic reticulum. In addition, TrJ-PMP22 forms a heterodimer with the wt-PMP22. This interaction causes a fraction of the wt-PMP22 to be retained with TrJ-PMP22 in the intermediate compartment of COS7 and Schwann cells. The relative stability of a wt-mutant PMP22 heterodimer as compared with the wt-wt PMP22 homodimer may determine whether a particular mutation is semidominant or dominant. The neuropathy itself appears to result both from decreased trafficking of wt-PMP22 to the plasma membrane and from a toxic gain of function via the accumulation of wt- and TrJ-PMP22 in the intermediate compartment.

Animals↗

PubMed1999

Temporal expression pattern of peripheral myelin protein 22 during in vivo and in vitro myelination.

Peripheral myelin protein 22 (PMP22) was initially described as a minor component of peripheral myelin. Mutations affecting the PMP22 gene cause demyelinating neuropathies, supporting a role for the protein in PNS myelination. Furthermore, PMP22 carries the L2/HNK-1 carbohydrate epitope suggesting an adhesion/recognition function. Despite advances in characterizing the PMP22 gene, the specific role(s) of the protein in myelin remains unknown. In this study we determined the temporal expression pattern of PMP22 in comparison to galactocerebroside (GalC) and myelin associated glycoprotein (MAG), early constituents of PNS myelin, and to protein zero (P0) and myelin basic protein (MBP), late components of myelin. In sciatic nerve lysates, PMP22 was detected at postnatal day 3, after MAG, but before MBP expression. The same results were obtained in cocultures of dorsal root ganglion neurons and Schwann cells (SCs). Low levels of PMP22 were found in early, anti-MAG and anti-GalC immunoreactive, myelinating cocultures. However, PMP22 could only be detected in the SC plasma membrane after basal lamina formation. In long-term myelinating cocultures PMP22 levels continued to increase and the protein was found in anti-P0 and anti-MBP immunoreactive myelin segments. Furthermore, PMP22, MBP, and P0 protein levels were greatly enhanced by progesterone treatment of the cocultures. The highest levels of PMP22 expression were associated with late stages of myelination; however the presence of the protein in nonmyelinating SCs and in SCs commencing myelination supports multiple roles for PMP22 in peripheral nerve biology.

Animals↗

PubMed1999

PMP22 accumulation in aggresomes: implications for CMT1A pathology.

Peripheral myelin protein 22 (PMP22) is a 22-kDa glycoprotein mainly expressed by Schwann cells (SCs). Duplication or deletion of the PMP22 gene locus is associated with heritable peripheral neuropathies suggesting that the correct level of PMP22 protein is essential for SC functioning. Previously we reported that in SCs the majority (80%) of newly synthesized PMP22 is rapidly degraded, possibly due to inefficient folding. Here we show that inhibition of the proteasome pathway results in a marked accumulation of PMP22 in the perinuclear cytoplasm. Double immunolabeling with an anti-ubiquitin antibody and various organelle markers indicates that the accumulated PMP22 is found in unique intracellular inclusions, called aggresomes. Moreover, overexpression of PMP22 in SCs can induce perinuclear accumulation of the protein. Together, these studies suggest that the proteasome pathway is critical for the regulation of PMP22 protein levels and raise the possibility that aggresomes may be involved in the pathogenesis of PMP22-associated peripheral neuropathies.

Animals↗

PubMed1998

Neurotrophins induce release of neurotrophins by the regulated secretory pathway.

Recent studies have established that neurotrophin synthesis and secretion are regulated by activity and that these factors are involved in activity-dependent processes in the nervous system. Neurotrophins also are known to induce increases in intracellular calcium, a trigger for regulated secretion. This finding raises the possibility that neurotrophins themselves may stimulate regulated secretion of neurotrophins. To address this question, we studied the release of neurotrophins from transfected PC12 cells, a widely used model for neuronal secretion and neurotrophin signal transduction. We found that neurotrophins induced the regulated secretion of brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and neurotrophin-4/5. The effect of brain-derived neurotrophic factor on release of NT-3 could be abolished by REX, a p75 blocking antibody, but not by K252a, an inhibitor of neurotrophin tyrosine kinase receptor (Trk) signaling. The nerve growth factor effect on release of NT-3 could be blocked only by simultaneous application of REX and K252a, suggesting that they are mediated by TrkA as well as p75. Our data show that neurotrophins are able to induce the regulated secretion of neurotrophins and suggest a signal-transducing role for both TrkA and p75 in this process. The neurotrophin-induced release of neurotrophins may be relevant for activity-dependent processes such as synaptic plasticity and memory formation.

Animals↗

PubMed1998

Duration and magnitude of nerve growth factor signaling depend on the ratio of p75LNTR to TrkA.

The role of the low affinity neurotrophin receptor p75LNTR in neurotrophin signal transduction remains open. Recent reports show that this receptor generates intracellular signals independent of Trk activity, and others imply that it collaborates with Trk(s) to enhance cellular responses to low neurotrophin concentrations. We have used the Cytosensor microphysiometer as a direct marker of intracellular metabolic activity to address the physiologic role of p75LNTR in nerve growth factor (NGF) signal transduction. NGF treatment of PC12 or TrkA-transfected Chinese hamster ovary (CHO) cells results in a rapid, transient increase in the extracellular acidification rate as measured by the Cytosensor; in both cell types, p75LNTR enhances this response. p75LNTR affects both the magnitude of and the duration of the extracellular acidification response to NGF. Moreover, it is not merely the presence of p75LNTR, but also the ratio of p75LNTR:TrkA which determines cellular responsiveness to NGF. In transiently transfected CHO cells, a 5:1 ratio of p75LNTR:trkA cDNAs produced the greatest change in NGF-induced acid secretion. Pretreatment of PC12 cells with anti-p75LNTR antibodies decreased the responsiveness to NGF. However, long-term NGF exposure to PC12 cells in which p75LNTR expression was decreased to approximately 10% of wild-type levels showed a longer duration of acid secretion compared to wild-type PC12 cells. Together, these data suggest that p75LNTR may play a dual role in modulating NGF signal transduction by enhancing and extending cellular responses to short-term ligand exposures while attenuating the metabolic response to long-term ligand exposures. With regard to potential Trk-independent p75LNTR signal transduction mechanisms, we detected no change in extracellular acidification response in 75LNTR-transfected CHO cells, PCNA-15 fibroblasts, or Schwann cells, all of which express large amounts of p75LNTR and no Trk. Thus, p75LNTR cannot produce any signal detected by microphysiometry in the absence of TrkA.

Animals↗

PubMed1998

Subcellular localization of epitope-tagged neurotrophins in neuroendocrine cells.

A growing body of evidence suggests that neurotrophins (NTs) play a critical role in synaptic plasticity and other activity dependent processes in the CNS. Release of these growth factors by neurons and neuroendocrine cells was recently shown to occur via the regulated secretory pathway, representing a possible mechanism for preferentially supplying NTs locally to active synapses. However, the identity and characteristics of the intracellular storage compartment for NTs undergoing stimulus-coupled secretion remains controversial. As a step towards addressing these issues we have investigated the subcellular localization of epitope-tagged nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) in neuroendocrine cells. Placement of the myc-epitope tag at the neurotrophin carboxy terminus did not affect essential properties of the NTs such as their ability to induce Trk tyrosine phosphorylation or their sorting into the regulated secretory pathway in PC12 and AtT-20 neuroendocrine cells. Epitope-tagged NTs colocalize with dense core vesicle (DCV)-markers at the light microscopic level in both cell lines investigated. Furthermore, at an EM level immunoreactivity (IR) for myc-tagged NGF was found over dense core granules (DCGs) in PC12 cells. These data provide evidence that NTs can be stored in DCVs in neuronal model cell lines and, potentially, in neurons as well.

Animals↗

PubMed1998

Phosphatidylcholine-specific phospholipase inhibitor D609 differentially affects MAP kinases and immediate-early genes in PC12 cells.

The effects of tricyclodecan-9-yl xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipases, on PC12 cells were investigated. D609 repressed nerve growth factor (NGF)-mediated induction of c-fos mRNA with an IC50 approximately 50 microg/ml. Interestingly, maximal c-fos-suppressing doses of D609 did not affect activity of extracellular signal-regulated kinases. Surprisingly, D609 enhanced the extracellular acidification rate of PC12 cells, even in the absence of NGF. D609 alone induced c-jun mRNA with the same potency as it repressed the NGF-induced expression of c-fos. Like NGF, D609 alone induced c-jun even in the presence of dominant-negative Ras. Immediate-early induction of c-jun mRNA by NGF and D609 was abrogated by pretreatment with the kinase inhibitor olomoucine. Jun kinase, which is inhibited by olomoucine, was found to be activated by D609. Thus, D609 might induce c-jun in PC12 cells as a consequence of Jun kinase activation through a Ras-independent pathway. Under the same conditions, D609 repressed NGF-mediated induction of c-fos mRNA.

Animals↗