Emeritus Faculty, Acad Council, Neurobiology
The biochemistry and molecular genetics of growth and differentiation of nerve cells. The structure, biosynthesis and mechanism of action of nerve growth factor and other neurotrophins. Gene regulation in target organs and glial cells during nerve regeneration. The role of apolipoproteins and of the myelin protein PMP-22 during nerve degeneration and regeneration and in peripheral neuropathies. Research involves the techniques of recombinant DNA and gene transfection, of cell biology and of protein chemistry.
The heterozygous Trembler-J (TrJ/+) mouse, containing a point mutation in the peripheral myelin protein 22 (Pmp22) gene, is characterized by severe hypomyelination and is a representative model of Charcot-Marie-Tooth 1A (CMT1A) disease/Dejerine-Sottas syndrome (DSS). Given that the neurotrophin-3 (NT3)-TrkC signaling pathway is inhibitory to myelination during development, we investigated the role of the NT3-TrkC pathway in myelination and manipulated this pathway to improve myelin formation in the CMT1A/DSS mouse model. Injection of NT3 to the TrJ/+ mice decreased the myelin protein P(0) level in the sciatic nerves. Suppressing the NT3-TrkC pathway with TrkC-Fc, an NT3 scavenger, enhanced myelination in vitro and in vivo in the TrJ/+ mouse. Furthermore, we found that full-length TrkC was expressed in adult TrJ/+ mouse sciatic nerves but was not detected in the wild-type adults, suggesting that the full-length TrkC is a potential target of treatment to enhance myelination in the TrJ/+ mouse.
View details for DOI 10.1002/jnr.21388
View details for Web of Science ID 000250275400007
View details for PubMedID 17628499
Endogenous neurotrophins positively and negatively regulate migration of premyelinating Schwann cells before the initiation of myelination. Neurotrophin-3 (NT3) acting through the TrkC receptor tyrosine kinase stimulates Schwann cell migration via the Rho GTPases Rac1 and Cdc42. We previously demonstrated that TrkC directly phosphorylates and activates Dbs, the guanine-nucleotide exchange factor (GEF) for Cdc42, to partially mediate Schwann cell migration. Here, we identify T lymphoma invasion and metastasis (Tiam) 1 as the Rac1-specific guanine-nucleotide exchange factor involved in NT3-induced Schwann cell migration. Furthermore, the interaction between the small GTPase Ras and Tiam1 plays an essential role in the activation of Rac1. Taken together, these results suggest that NT3 activation of TrkC stimulates Schwann cell migration through two parallel signaling units, Ras/Tiam1/Rac1 and Dbs/Cdc42, and that Schwann cell migration is uniquely regulated in the case of Ras and Rac1, by two different types of small GTPases.
View details for DOI 10.1073/pnas.0507125102
View details for Web of Science ID 000232603600073
View details for PubMedID 16203995
During the development of the peripheral nervous system, Schwann cells, the myelin-forming glia, migrate along axons before initiating myelination. We previously demonstrated that endogenous neurotrophin-3 (NT3) acting through the TrkC tyrosine kinase receptor enhances migration of premyelinating Schwann cells. This signaling pathway is mediated by the c-Jun N-terminal kinase (JNK) cascade regulated by the Rho GTPases Rac1 and Cdc42. However, missing is the link between TrkC and the GTPases. Here, we show that a guanine-nucleotide exchange factor (GEF), Dbl's big sister (Dbs), couples with TrkC to activate Cdc42 in Schwann cells. Furthermore, TrkC directly phosphorylates Dbs, thereby inducing the Cdc42-GEF activity. Taken together, activation of TrkC triggers Schwann cell migration by regulating Dbs upon direct tyrosine phosphorylation, providing a mechanism whereby a membrane receptor tyrosine kinase can induce the activation of Rho GTPase-GEFs.
View details for DOI 10.1073/pnas.0501160102
View details for Web of Science ID 000228195800050
View details for PubMedID 15758069
The Trembler-J (TrJ) mouse, containing a point mutation in the peripheral myelin protein 22 gene, is characterized by severe hypomyelination and is a representative model of Charcot-Marie-Tooth 1A disease/Dejerine-Sottas Syndrome. Previous studies have shown that protein kinase inhibitor K252a enhances wild-type Schwann cell myelination in culture. We used a dorsal root ganglion (DRG) explant culture system from the heterozygous TrJ/+ mouse to investigate if myelination could be enhanced by K252a. The TrJ/+ DRG explant cultures replicated some important features of the TrJ/+ mouse, showing reduced myelin protein accumulation, thinner myelin sheaths, and shortened myelin internodes. K252a increased myelin protein accumulation and myelin sheath thickness but did not substantially increase myelin internode length. Furthermore, the TrJ/+ DRG explant culture and sciatic nerves continued to respond to K252a during the stage when myelination is complete in the wild type. A general tyrosine kinase inhibitor, genistein, but not inhibitors of serine/threonine protein kinase inhibitors, had a similar effect to K252a. K252a is therefore able to partially overcome hypomyelination by enhancing mutant Schwann cell myelin formation in the TrJ/+ mouse.
View details for DOI 10.1002/jnr.20357
View details for Web of Science ID 000226634800006
View details for PubMedID 15605381
A characteristic feature of mouse models of the peripheral neuropathies caused by dominant mutations in peripheral myelin protein 22 (pmp22) is the appearance, in Schwann cells, of pmp22 aggregates. Using a set of dominant and recessive pmp22 mutations that cause human disease of varying degrees of severity, we compared their potential for aggregation and trafficking patterns with those of wild-type pmp22. The potential for aggregation was assessed by determining the size distribution of the various pmp22 mutant proteins under conditions where wild-type pmp22 showed little or no aggregation. All disease-causing dominant mutations showed significant aggregation and failed to traffic to the cell surface. Although the position of the dominant mutation in the pmp22 molecule determined both its potential for aggregation and how far it trafficked in the cell, there was no correlation between aggregation and the severity of the disease. On the other hand, recessive mutations were uniquely distinguished from dominant mutations by both the low potential for aggregation and their trafficking to the cell surface. In the course of these studies, it was also noted that the potential for aggregation and the trafficking of mutant pmp22s is influenced by the nature and/or location of the epitope tag.
View details for DOI 10.1016/j.nbd.2004.07.010
View details for Web of Science ID 000224757500018
View details for PubMedID 15474367
The neurotrophin brain-derived neurotrophic factor (BDNF) is an endogenous regulator of the myelination process during development in the peripheral nervous system. Enhancement of myelin formation by BDNF is mediated by the neurotrophin receptor p75(NTR). Although this neurotrophin is a positive modulator of myelination during early development, the final effects of BDNF on myelin sheaths after active myelination is completed are largely unknown. Using BDNF transgenic mice, we examined the long-term effects of BDNF on myelination of the peripheral nervous system in vivo. Elevation of BDNF levels in the transgenic mice produced an increase in both the rate and extent of the myelination process. BDNF enhanced and accelerated myelin formation during early development and this increase in myelin content and thickness was maintained in adulthood. Besides enhanced myelination, BDNF also influenced axon caliber size but to a lesser extent. This lagging increase in axon caliber compared to myelin suggests that the axon size is not the only determinant of myelin thickness.
View details for DOI 10.1002/jnr.20181
View details for Web of Science ID 000223735300005
View details for PubMedID 15352212
Axons dictate whether or not they will become myelinated in both the central and peripheral nervous systems by providing signals that direct the development of myelinating glia. Here we identify the neurotrophin nerve growth factor (NGF) as a potent regulator of the axonal signals that control myelination of TrkA-expressing dorsal root ganglion neurons (DRGs). Unexpectedly, these NGF-regulated axonal signals have opposite effects on peripheral and central myelination, promoting myelination by Schwann cells but reducing myelination by oligodendrocytes. These findings indicate a novel role for growth factors in regulating the receptivity of axons to myelination and reveal that different axonal signals control central and peripheral myelination.
View details for Web of Science ID 000222905400008
View details for PubMedID 15260955
Neurotrophins are recognized widely as essential factors in the developing nervous system. Previously, we demonstrated that neurotrophin 3 activation of TrkC inhibits Schwann cell myelination and enhances the migration of primary Schwann cells through the signaling pathway regulated by the Rho GTPases Rac1 and Cdc42. Here, we show that neurotrophins activate divergent signaling pathways to promote or inhibit Schwann cell migration. Endogenous brain-derived neurotrophic factor acting through p75(NTR) inhibits Schwann cell migration dramatically by Src kinase-dependent activation of the guanine-nucleotide exchange factor Vav2 and RhoA. Together, these results suggest that neurotrophins and their receptors differentially regulate Schwann cell migration through the signaling pathways that depend on Rho GTPases.
View details for Web of Science ID 000222037000051
View details for PubMedID 15161978
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.
View details for DOI 10.1002/jnr.20048
View details for Web of Science ID 000220028400010
View details for PubMedID 14994343
During development and nerve injury, complex interactions between glial cells and neurons are essential for establishing proper nerve function. Neurotrophins play multiple roles in the developing nervous system, including cell survival, growth, and differentiation. Here we show that migration of Schwann cells, isolated from sciatic nerves, is significantly enhanced by neurotrophin 3, but not by nerve growth factor or brain-derived neurotrophic factor. The neurotrophin-3-induced cell migration was also observed in Schwann cells isolated from sciatic nerves of p75NTR-/- mice, indicating that neurotrophin 3 enhances cell migration through TrkC. This effect was blocked by K252a, an inhibitor of the Trk receptor family. Additionally, the neurotrophin-3-induced cell migration depended on Rho GTPases (Rac1 and Cdc42) and c-Jun N-terminal kinase. We obtained the same results with Cos-7 cells expressing TrkC. Taken together, these results suggest that neurotrophin 3 activation of TrkC induces Schwann cell migration through the c-Jun N-terminal kinase signaling pathway.
View details for Web of Science ID 000186803800119
View details for PubMedID 14614136
Schwann cells in developing and regenerating peripheral nerves express elevated levels of the neurotrophin receptor p75NTR. Neurotrophins are key mediators of peripheral nervous system myelination. Our results show that myelin formation is inhibited in the absence of functional p75NTR and enhanced by blocking TrkC activity. Moreover, the enhancement of myelin formation by endogenous brain-derived neurotrophic factor is mediated by the p75NTR receptor, whereas TrkC receptors are responsible for neurotrophin-3 inhibition. Thus p75NTR and TrkC receptors have opposite effects on myelination.
View details for Web of Science ID 000179080400054
View details for PubMedID 12424382
Alterations in peripheral myelin protein 22 (PMP22) gene expression are associated with demyelinating peripheral neuropathies. Overexpression of wild type (wt) PMP22 or inhibition of proteasomal degradation lead to the formation of aggresomes, intracellular ubiquitinated PMP22 aggregates. Aggresome formation has now been observed with two mutant PMP22s, the Tr- and TrJ-PMP22 when the proteasome is inhibited. The formation of these aggresomes required intact microtubules and involved the recruitment of chaperones, including Hsp40, Hsp70, and alphaB-crystallin. Spontaneously formed ubiquitinated PMP22 aggregates were also observed in Schwann cells of homozygous TrJ mice. Significant upregulation of both the ubiquitin-proteasomal and lysosomal pathways occurred in affected nerves suggesting that two pathways of PMP22 degradation are present. Thus, the presence of aggresomes appears to be a common finding in neuropathy models of PMP22 overexpression and of some point mutations known to cause neuropathy in mice and humans.
View details for DOI 10.1006/nbdi.2002.0500
View details for Web of Science ID 000177049700004
View details for PubMedID 12127149
Mutations in the gene encoding the peripheral myelin protein 22 (PMP22), a tetraspan protein in compact peripheral myelin, are one of the causes of inherited demyelinating peripheral neuropathy. Most PMP22 mutations alter the trafficking of the PMP22 protein in Schwann cells, and this different trafficking has been proposed as the underlying mechanism of the disease. To explore this problem further, we compared the aggregation of wild-type Pmp22 with those of the two Pmp22 mutations found in Trembler (Tr) and Trembler J (TrJ) mice. All three Pmp22s can be crosslinked readily as homodimers in transfected cells. Wild-type Pmp22 also forms heterodimers with Tr and TrJ Pmp22, and these heterodimers traffic with their respective mutant Pmp22 homodimers. All three Pmp22s form complexes larger than dimers with Tr Pmp22 especially prone to aggregate into high molecular weight complexes. Despite the differences in aggregation of Tr and TrJ Pmp22, these two mutant Pmp22s sequester the same amount of wild-type Pmp22 in heterodimers and heterooligomers. Thus, the differences in the phenotypes of Tr and TrJ mice may depend more on the ability of the mutant protein to aggregate than on the dominant-negative effect of the mutant Pmp22 on wild-type Pmp22 trafficking.
View details for Web of Science ID 000173233300086
View details for PubMedID 11752407
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.
View details for Web of Science ID 000178439300025
View details for PubMedID 12220579
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.
View details for Web of Science ID 000172576900085
View details for PubMedID 11717413
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.
View details for Web of Science ID 000171634300018
View details for PubMedID 11677267
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.
View details for Web of Science ID 000170109100020
View details for PubMedID 11283322
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.
View details for Web of Science ID 000089702300007
View details for PubMedID 11020215
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.
View details for Web of Science ID 000089263000014
View details for PubMedID 10987826
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.
View details for Web of Science ID 000088683300024
View details for PubMedID 10825163
Comparative study of CSF levels of tau and AD7C-neuronal thread protein (NTP) in patients with AD and control subjects.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.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.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.CSF levels of tau and AD7C-NTP may be useful biomarkers for AD.
View details for Web of Science ID 000086460900021
View details for PubMedID 10751266
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.
View details for Web of Science ID 000083135900013
View details for PubMedID 10527811
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.
View details for Web of Science ID 000078961400014
View details for PubMedID 10066256
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.
View details for Web of Science ID 000078435400005
View details for PubMedID 10028918
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.
View details for Web of Science ID 000075246600099
View details for PubMedID 9689129
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.
View details for Web of Science ID 000073956400004
View details for PubMedID 9692675
Previous studies have indicated that certain members of the cyclin-dependent kinase/mitogen-activated protein kinase superfamily are involved in apoptosis of neuronal cells. Here, we have examined programmed cell death induced by withdrawal of neurotrophic support from CNS (rat retinal) and PNS (chick sympathetic, sensory, and ciliary) neurons. All four neuron types were equally rescued by the purine analogues olomoucine and roscovitine. Olomoucine inhibits multiple cyclin-dependent and mitogen-activated protein kinases with similar potency. Roscovitine is a more selective cyclin-dependent kinase inhibitor; but, so is butyrolactone I, which did not prevent retinal ganglion cell death. The specific p38MAPK inhibitor SB-203580 did not prevent apoptosis in retinal ganglion cells. Death of these cells in the absence of neurotrophic factors was accompanied by morphological changes indicative of apoptosis, including nuclear condensation and fragmentation. Treatment with olomoucine or roscovitine not only prevented these apoptotic changes in retinal ganglion cells but also blocked neurite outgrowth. The survival-promoting activity of olomoucine correlated with its in vitro IC50 for c-Jun N-terminal kinase-1 and its potency to repress c-jun induction in live PC12 cells. Roscovitine was more potent in rescuing neurons than in inhibiting Jun kinase. Thus, the antiapoptotic action of roscovitine might be due to inhibition of additional kinases.
View details for Web of Science ID 000072564100008
View details for PubMedID 9523556
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.
View details for Web of Science ID 000072219400006
View details for PubMedID 9514200
In order to obtain the basic knowledge necessary to develop therapeutical intervention for blindness due to the damaged retina and optic nerve, the mechanism of retinal degeneration and regeneration in an amphibian model, Cynops pyrrhogaster, was studied. In the retinal degenerative process following enucleation and reimplantation of the eye ball, evidence was found for active cell death of neural retinal cells. As the degeneration proceeded, Musashi, an ribonucleic acid (RNA)-binding protein, started its expression in the daughter cells of proliferating retinal pigment epithelium (RPE) cells, messenger RNA (mRNA) expression of proneural genes with basic helix-loop-helix motif was then detected in the newly developing retina. These results suggest that transdifferentiation of RPE cells to neural retina involves at least partial cascade, if not entirely, of neural induction from uncommitted ectodermal tissue. Search for genes that are required for transdifferentiation of RPE cells to neural retinal cells, in addition to those mentioned above, will provide the basic knowledge for successful retinal transplantation and retinal regeneration in higher vertebrates.
View details for PubMedID 9436360
The role of the nerve growth factor (NGF) carboxyl terminus in the function of NGF is not well understood. Previous work showed that deletion of residues 112-120 abolished NGF bioactivity. Several mutagenesis studies, however, have localized the binding sites of the two NGF receptors, p75 and TrkA, to other regions of the NGF molecule. To investigate the role of the NGF COOH terminus, we performed a detailed structure-function analysis of this region by deleting stepwise each of the nine COOH-terminal residues as well as constructing six point mutants. We found that point mutations within the 111-115 region, but not deletion of residues 116-120, significantly decreased NGF bioactivity, as determined by TrkA tyrosine phosphorylation and neurite outgrowth from PC12 cells. Mutation of the absolutely conserved Leu112 led to severely disrupted p75 binding on A875 cells but had only a modest effect on TrkA binding to MG87-TrkA fibroblasts. This suggests that the p75 binding surface is more extended than previously believed and includes not only charged residues within loops 1 and 5 but also spatially discontinuous, uncharged residues in a region where the NH2 and COOH termini are in close proximity. Unexpectedly, deletion of COOH-terminal residues beyond Ala116 led to significantly decreased stability. These results demonstrate that residues 111-115, but not residues 116-120, are important for both the structural stability and biological activity of NGF.
View details for Web of Science ID A1997YF68400060
View details for PubMedID 9361001
The neurotrophin receptor TrkB plays a key role in promoting cell survival and differentiation in the nervous system. Two adhesive motifs in the extracellular domain of TrkB have been proposed based on its predicted secondary structure. To investigate the potential adhesive function of trkB, a full length trkB cDNA was stably transfected into NIH 3T3 cells and TrkB-expressing clones isolated. Transfectant clones producing different levels of TrkB protein were subjected to a homotypic aggregation assay. Results showed that parental cells were non-adhesive during the assay while TrkB-expressing cells displayed varying degrees of aggregation depending on the amount of TrkB protein expressed. The observed adhesion was Ca(2+)-, Mg(2+)-, and temperature-dependent, characteristics shared by the cadherin family of adhesion molecules. The transfected cell lines also expressed cadherin in proportion to TrkB expression and both molecules were required for cell adhesion. Double immunofluorescence staining studies showed that TrkB was colocalized with cadherin and catenin at cell-cell contact sites. Whether TrkB and cadherin mediate adhesion separately or synergistically remains to be determined.
View details for Web of Science ID A1997XN88700003
View details for PubMedID 9260739
A nonconservative leucine to proline mutation in peripheral myelin protein 22 (PMP22) causes the Trembler-J (TrJ) neuropathy in mice and humans. The expression levels and localization of the PMP22 protein in the TrJ mouse have not been previously determined. The aim of our studies was to reevaluate the extent of myelin deficit in genotyped heterozygous and homozygous animals and to examine how the TrJ mutation alters the normal in vivo post-translational processing of PMP22. Morphological studies show evidence for primary dysmyelination and myelin instability in affected animals. As expected, Western blot analysis indicates that in adult heterozygous TrJ animals, the level of PMP22 is markedly decreased, similar to myelin basic protein and protein zero, whereas myelin-associated glycoprotein is largely unaffected. The decrease in myelin protein expression is associated with an increase in lysosomal biogenesis, suggestive of augmented endocytosis or autophagy. Double-immunolabeling experiments show the accumulation of PMP22 in endosomal/lysosomal structures of TrJ Schwann cells, and chloroquine treatment of nerve segments indicates that the degradation of protein zero, PMP22, and myelin basic protein is augmented in TrJ nerves. These studies suggest that the TrJ mutation alters myelin stability and that the mutant protein is likely degraded via the lysosomal pathway.
View details for Web of Science ID A1997XA05600023
View details for PubMedID 9151736
The varied roles that neurotrophins play in the development and activity-dependent plasticity of the nervous system presumably require that the sites and quantity of neurotrophin release be precisely regulated. As a step toward understanding how different neurotrophins are sorted and secreted by neurons, we expressed nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin-3 in cell lines used as models for neuronal protein sorting. All three neurotrophins were secreted by a regulated pathway in transfected AtT-20 and PC12 neuroendocrine cells, with a 3-6-fold increase in neurotrophin release in response to 8-bromo-cAMP or depolarization, respectively. To determine if the propeptide directs the intracellular sorting of mature NGF, we examined mutants in which regions spanning the propeptide were deleted. These mutants underwent regulated release in every case in which expression could be detected. Similarly, NGF sorting was not significantly altered by mutations which specifically abolished N-glycosylation or proteolytic processing sites within the NGF precursor. Finally, we found that all three neurotrophins were secreted 65-75% basolaterally by polarized Madin-Darby canine kidney epithelial cells. These findings suggest that the determinants of regulated neurotrophin secretion lie within the mature neurotrophin moiety and that NGF, brain-derived neurotrophic factor, and neurotrophin-3 are likely to be sorted similarly and released in a regulated manner by neurons.
View details for Web of Science ID A1996VL69300056
View details for PubMedID 8810312
Peripheral Myelin Protein-22 (PMP22) is a membrane glycoprotein which represents up to 5% of total protein in myelin of peripheral nerves. Mutations affecting the PMP22 gene have been linked to the inherited peripheral neuropathies Charcot-Marie-Tooth disease type 1A (CMT1A; duplications and point mutations), Dejerine-Sottas syndrome (DSS; point mutations), and hereditary neuropathy with liability to pressure palsies (HNPP; deletions). In this study, we determined the ultrastructural distribution of PMP22 and other myelin proteins in normal human peripheral nervous system (PNS) nerves and in CMT1 patients with or without the CMT1A duplication on chromosome 17. Our results demonstrate that PMP22, P0 protein, and myelin basic protein are present in compact myelin of all patients examined. PMP22 was also present in the plasma membrane of Schwann cells of unmyelinated fibers and onion bulbs. Although the precise biological role of PMP22 remains to be discovered, our results support the hypothesis that this protein serves multiple functions in Schwann cells.
View details for Web of Science ID A1996TZ88900004
View details for PubMedID 8786387
Prompted by the recent discovery that neurotrophins, which are known to be biologically active as noncovalently linked homodimers, can also be induced to form biologically active heterodimers in vitro, we have investigated the biosynthesis of neurotrophin heterodimers by transfected mammalian cells. When COS cells were cotransfected with expression plasmids for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3), the appropriate heterodimers were detected in the conditioned medium by immunoprecipitation and, in the case of NGF.NT-3, using a two-site enzyme-linked immunosorbent assay. Heterodimer formation occurred predominantly intracellularly and did not require precursor cleavage, because heterodimers containing pro-NGF and pro-BDNF were detected in the conditioned medium. When rat C6 glioma cells or mouse AtT-20 neuroendocrine cells were cotransfected with expression plasmids for NGF and NT-3, NGF.NT-3 heterodimer was detected at levels comparable with those of homodimeric NGF and NT-3, indicating that heterodimer formation can occur at significant levels in a variety of cell types. These data provide evidence that NGF, BDNF, and NT-3 are capable of forming heterodimers when coexpressed in mammalian cells and suggest that such heterodimers are likely to be formed in vivo when a single cell expresses multiple neurotrophins.
View details for Web of Science ID A1995QY73600087
View details for PubMedID 7744882
RNase protection analysis using p75, trk A, and trk B RNA probes was used to examine mRNA expression in rat tissues, with particular emphasis on the immune system. Every tissue examined, with the exception of postnatal day 0 spleen, expressed p75 mRNA. Trk A mRNA was observed in tissues previously reported to be negative for the trk A receptor, such as kidney, thymus, lymph node, muscle, and lung. Neuronal tissues expressed only the long form of trk A, whereas nonneuronal tissues expressed both trk A forms. Trk B mRNA was expressed by the same tissues as trk A, plus heart and spleen. Neuronal tissues expressed full-length and truncated trk B, whereas nonneuronal tissues only expressed truncated trk B. During development of the thymus p75 mRNA levels increased and trk A mRNA levels decreased. Similarly, for the spleen, p75 mRNA levels increased and those of trk B decreased during development. The expression of p75, trk A and trk B was localized primarily to the stroma of the thymus and spleen, but there was some expression by the splenocytes and thymocytes. The widespread expression of neurotrophin receptors in areas not known to be targets for neurotrophins suggests broader functions for neurotrophins outside of the nervous system.
View details for Web of Science ID A1995QN15000043
View details for PubMedID 7891106
The binding of NGF (nerve growth factor) to the rat low affinity nerve growth factor receptor (p75NGFR) has been studied by site-directed mutagenesis of the receptor. Introduction of non-native N-glycosylation sites within the binding domain indicates that the second of the characteristic cysteine-rich repeats may be particularly important to NGF binding. Two mutants of the second repeat, S42N and S66N, are glycosylated and bind NGF at a drastically reduced level, while still maintaining a conformation recognized by the monoclonal antibody against p75, MC192. Alanine substitution at these sites does not affect NGF binding. Two other mutations that result in local structural changes in the second repeat also greatly decrease binding. One of these altered residues, Ser50, appears to play an essential structural role, since it cannot be replaced by Asn, Ala, or Thr without loss of both NGF binding and MC192 recognition on a Western. Glycosylation of selected sites in the other repeats has little effect on NGF binding or antibody recognition. The introduction of non-native N-glycosylation sites may provide a generally useful scanning technique for the study of protein-protein interactions.
View details for Web of Science ID A1995QK08400063
View details for PubMedID 7876230
When PC12 cells are primed with nerve growth factor (NGF) for periods of > or = 1 week, they acquire the ability to regenerate neurites rapidly in response to NGF. It is not known how NGF promotes this regeneration, but it does not require ongoing RNA synthesis. Previous studies have suggested that NGF directs the accumulation of precursor molecules that are rapidly assembled to form the regenerated neurites. To address the nature of these precursor molecules, we have treated PC12 cells with macromolecular synthesis inhibitors during the priming and regeneration phases of neurite growth. Here we show that NGF promotes neurite regeneration by inducing the synthesis of new proteins. These proteins are encoded by short-lived mRNAs that are generated during the NGF priming period. The isolation and identification of these mRNAs will allow a further understanding of how NGF promotes neurite regeneration.
View details for Web of Science ID A1995QD02700010
View details for PubMedID 7830047
Interleukin-6 (IL-6) was produced by the spontaneously immortal Schwann cell clone, iSC, when cocultured with PC12 cells. The iSC cell-derived IL-6 in coculture conditioned media caused the neuronal differentiation of naive PC12 cells and this bioactivity was neutralized by preincubation of conditioned media with antisera to IL-6. Cocultured iSC transcribe IL-6 message as confirmed by northern analysis. Stimuli that induce IL-6 production in the hematopoietic lineage induced transcription and production of IL-6 by iSC cells. Lipopolysaccharide, tumor necrosis factor-alpha, IL-1 alpha, IL-6, and serum withdrawal induced iSC cell IL-6 mRNA. The kinetics of IL-6 production was confirmed in the mouse IL-6-dependent B9 bioassay and that activity could be neutralized with antisera to IL-6. Expression of both the IL-6 receptor and the gp130 signal transduction component by iSC as determined by northern analysis suggests an autocrine regulatory mechanism. The observed iSC production of IL-6 in vitro led to an investigation of the sciatic nerve crush model of Schwann cell activation in vivo. In the initial 12 h after crush injury, IL-6 message is induced. IL-6 mRNA expression was highest distal to the crush injury. Our in vitro data demonstrate that iSC cells produce IL-6 in response to PC12 cell coculture and to stimuli that induce IL-6 production in the hematopoietic lineage. The induction of IL-6 message distal to a crush injury suggests another mechanism by which Schwann cells facilitate peripheral nerve regeneration.
View details for Web of Science ID A1995QD02700046
View details for PubMedID 7830079
The low affinity neurotrophin receptor, termed p75LNTR, plays a role in increasing the amount of nerve growth factor that becomes bound to the tyrosine kinase receptor, trkA (Barker, P. A., and Shooter, E. M. (1994) Neuron 13: 203-215), possibly by increasing the nerve growth factor concentration in the microenvironment surrounding the trkA receptor. Because protein acylation may be a means by which cell surface receptor distribution may be regulated, we have determined the acylation status of p75LNTR. We find that p75LNTR expressed in PC12, PCNA, or transfected COS cells is metabolically labeled with [3H]palmitic acid. This modification occurs post-translationally, and the incorporated fatty acid is removed by hydroxylamine treatment at pH 7 or 11 and by sulfhydryl reducing agents, suggesting a thioester linkage to palmitic acid. Consistent with this, p75LNTR in which the juxtamembrane cysteine present at position 279 is substituted with alanine is expressed but cannot be metabolically labeled with [3H]palmitic acid. Substitution of other cysteine residues present in the transmembrane or intracellular domain of the receptor has no effect on protein acylation, suggesting that only Cys279 is esterified to palmitate.
View details for Web of Science ID A1994PU52500092
View details for PubMedID 7982984
The role of the low affinity neurotrophin receptor, p75LNTR, in NGF-mediated signal transduction has been examined. Our results show that treatment of PC12 cells with MC192, a monoclonal antibody directed against p75LNTR, results in reduced NGF binding to TrkA and attenuated TrkA activation. Use of mutant NGF that binds TrkA but not p75LNTR shows that the MC192 effect requires that NGF bind the p75LNTR receptor. To explore the possibility that MC192 disrupts some normal functional role of p75LNTR, BDNF was used to block binding of NGF to p75LNTR on PC12 cells. By preventing NGF binding to p75LNTR, NGF binding to TrkA and NGF-mediated signal transduction were reduced. We propose that p75LNTR normally acts to increase binding of NGF to TrkA, possibly by increasing the local NGF concentration in the microenvironment surrounding the cell surface TrkA receptor.
View details for Web of Science ID A1994NZ24700016
View details for PubMedID 7519025
The binding domain of the low affinity nerve growth factor receptor (p75NGFR) is built from four "cysteine repeats," with almost identical patterns of half-cystine residues. To study the pattern of disulfide bridging within each cysteine repeat, we have mutated pairs of cysteine residues, primarily in the fourth repeat, and have tested the relative ability of each mutant to be cross-linked to the nerve growth factor after transient expression in COS 7 cells. The results give an indication of the relative importance of different disulfide bonds for the stability of the active conformation. Immunofluorescent staining shows that all of these proteins, even those that may be partially misfolded, are expressed to varying degrees on the surface of COS cells. They can also be detected by the monoclonal antibody MC192 on a Western blot following electrophoresis on a nondenaturing gel. Some cannot, however, be detected by MC192 on a Western blot after heat denaturation in SDS. The ability to refold under these conditions correlates in general with the ability of the mutant protein to bind nerve growth factor on the surface of COS cells.
View details for Web of Science ID A1994NF96600071
View details for PubMedID 8157676
Schwann cells support and facilitate axonal growth during development and successful regeneration in the peripheral nerve. In the regenerating rat sciatic nerve, Schwann cells provide a trophic milieu for primary sensory, sympathetic, and motoneurons. We have characterized a neurotrophic activity produced by adult rat sciatic nerve Schwann cells and a spontaneously immortal Schwann cell clone (iSC). This activity elicits neurite outgrowth from chick embryo explants of both CNS and PNS. The iSC activity has been concentrated by cation-exchange chromatography and compared to known neurotrophins in bioassay. Pooled bound fractions elicit neurite outgrowth from sympathetic, ciliary and motoneurons. In collagen matrix cocultures of iSC and E4 ventral horn (before motor axon extension to muscle targets), the iSC activity can direct the initial axonal extension from motoneurons. The data presented suggest that Schwann cell-produced activity may mediate motoneuron axonal extension before contact with their peripheral source of neurotrophin.
View details for Web of Science ID A1994MR52400003
View details for PubMedID 8145301
p21ras is believed to be involved in the neuronal differentiation of cells responsive to nerve growth factor (NGF). We show that NGF stimulates the activation of p21ras in embryonic sensory neurons and in PC12 cells. In the initial 5 min of exposure to NGF, the activation is concentration-dependent. In the sensory neurons and PC12 cells, the apparent maximal activation was reached at 50 and 10 ng/ml, respectively, with half-maximal activation at approximately 5 and 2-3 ng/ml, respectively. Kinetic analysis at low concentrations of NGF showed that p21ras activation slowly increases with time in both types of cells, while high concentrations result in rapid activation within 5 min. These results indicate that NGF regulates the activation state of p21ras in these cells and provides evidence suggesting that activation of p21ras is involved in NGF signal transduction. Treatment of PC12 cells with brain-derived neurotrophic factor or neurotrophin-3 (NT-3) failed to activate p21ras, suggesting that binding alone to p75LNGFR is insufficient for ras activation. Treatment with the kinase inhibitor, K252a, which inhibits the NGF tyrosine kinase receptor p140trk, abolished ras activation, suggesting that p140trk is the major mediator of p21ras activation by NGF.
View details for Web of Science ID A1993MK10000011
View details for PubMedID 8244964
A series of mouse nerve growth factor (NGF) deletion mutants have been constructed using in vitro mutagenesis to define domains of the protein essential for its activity. Deletions of the amino or carboxyl termini of mature NGF or of an internal domain, which normally produces a surface-exposed reverse turn, have been analyzed. Mutants with deletions in the amino terminus or in the reverse turn retain significant biological activity, whereas, in contrast, a mutant NGF lacking the seven most carboxyl-terminal amino acids is appropriately synthesized but shows no measurable biological activity. These results suggests that the flexible carboxyl tail of NGF, and perhaps other neurotrophins, plays a crucial role in mediating receptor recognition and/or ligand binding.
View details for Web of Science ID A1993MF51500041
View details for PubMedID 8226840
Molecular genetic studies have established that mutations in the gene encoding the 22-kDa peripheral myelin protein (PMP-22) are responsible for hereditary peripheral neuropathies in the trembler mouse and in a subset of humans with Charcot-Marie-Tooth disease, type 1a. The function of the PMP-22 protein remains unknown. Several studies on myelin proteins in the PNS have indicated that the L2/HNK-1 epitope, which is believed to be both a ligand for cellular adhesion and a target for autoimmune monoclonal IgM neuritis, may be found on heretofore unidentified proteins with a molecular mass of 19-28 kDa. In this report, we provide immunological evidence that at least one of these proteins is PMP-22.
View details for Web of Science ID A1993MD35000049
View details for PubMedID 7693873
The adult rat dorsal root ganglion (DRG) produces mRNA for the neurotrophic factors nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and contains large populations of neurons responsive to these factors. We report that following a focal crush injury of the sciatic nerve, NGF mRNA expression increases threefold and BDNF mRNA two-fold, in the ipsilateral L4 and L5 DRGs. The mRNAs encoding the cognate neurotrophin receptors, p75NGFR, trkA, and trkB were expressed in the DRG throughout the post-injury time course, suggesting that DRG neurons remain responsive to both NGF and BDNF. p75NGFR mRNA levels became transiently depressed in the DRG during the first several days after the lesion but returned to normal within 1 week. trkB mRNA was expressed in the normal sciatic nerve and levels were not altered by nerve crush. RNase protection assays detected both full-length and truncated trkB transcripts in the DRG, but only truncated trkB mRNA, lacking the tyrosine kinase domain, was detected in the sciatic nerve. Likewise, trkA transcripts were not detected by RNase protection in normal sciatic nerve or in a segment of nerve distal to the crush site. These results are consistent with a model in which regenerating sensory neurons are supported by neurotrophic factors synthesized within the DRG.
View details for Web of Science ID A1993ME27200001
View details for PubMedID 8271314
Successful peripheral nerve regeneration and functional recovery require the reestablishment of the neuron-Schwann cell relationship in the regenerating rat sciatic nerve, neurons differentially regulate Schwann cell genes. The message for the low-affinity NGF receptor, p75NGFR, is induced in Schwann cells distal to the injury and is repressed as regenerating axons make contact with these cells. The inverse is true for mRNA of the myelin gene P0; expression decreases distal to injury and increases as new axons contact Schwann cells and a program of myelination is initiated. Using an in vitro co-culture paradigm in which primary neurons and adult Schwann cells are separated by a microporous membrane, we show that axon contact is not an absolute requirement for neuronal regulation of Schwann cell genes. In this system neurons but not other cell types, repress the expression of Schwann cell p75NGFR while inducing the expression of the POU domain transcription factor, suppressed cAMP inducible POU, and myelin P0. These results demonstrate that regenerating axons can direct the Schwann cell genetic program from a distance through diffusible molecules.
View details for Web of Science ID A1993LZ63100022
View details for PubMedID 8408202
Myelin formation and maintenance requires complex interactions between neurons and glia, and between the integral protein and lipid components of the myelin sheath. Many of the underlying mechanisms may be examined by studying the perturbations caused by spontaneous and targeted mutations in myelin protein genes. This review summarizes the progress in our understanding of these mutations with an emphasis on integrating the recent advances in the genetics of myelin into a more generalized view of myelin organization and function.
View details for PubMedID 7505139
To elucidate the function of the two nerve growth factor (NGF) receptors, p75NGFR and p140trk, chimeric molecules were constructed of tumor necrosis factor (TNF) and NGF receptors. Rat PC12 pheochromocytoma cells transiently transfected with TNF-p140trk chimeras, which contain the extracellular domain of TNF receptor and the transmembrane and cytoplasmic domains of p140trk, showed TNF-dependent neuronal differentiation and cell survival. The activity of TNF-p140trk chimeras was completely blocked by the tyrosine kinase inhibitor K252a, and TNF was unable to induce neurite elongation in PC12 cells transfected with a tyrosine kinase-defective chimeric receptor. The TNF-p75NGFR chimeras, which contain the cytoplasmic domain of p75NGFR, were nonfunctional. Our results suggest that p140trk may function as ligand-activated homodimers and that ligand-mediated activation of the cytoplasmic domain of p140trk alone is sufficient for inducing a neuronal phenotype.
View details for Web of Science ID A1993LX75000087
View details for PubMedID 7690970
The trkA receptor functions as a signal transducing receptor for nerve growth factor. In this report, we show that alternative splicing results in the production of two distinct trkA isoforms in both rats and humans. These isoforms differ by virtue of a 6-amino acid insertion in their extracellular domain, the placement of which corresponds exactly with the breakpoint found in several human trkA oncogenes. When tested in fibroblasts, the presence (trkAII) or absence (trkAI) of the 6-amino acid insert does not affect the receptor's ligand binding specificity or its ability to transduce functional signals in response to nerve growth factor. In rats and humans, trkAII is the only isoform expressed within neuronal tissues at appreciable levels whereas trkAI, the form of trkA originally cloned, appears to be expressed mainly in non-neuronal tissues.
View details for Web of Science ID A1993LL75900088
View details for PubMedID 8325889
Successful mammalian peripheral nerve regeneration is dependent on activated Schwann cells. Schwann cells facilitate neuronal regrowth through the production of tropic cell membrane molecules, neurotrophins, and extracellular matrix components. To better understand Schwann cell function in the regenerating nerve, we have designed a method of isolating proliferating adult Schwann cells from the injured rat sciatic nerve. Relying on the mitotic signal that is present after a crush injury, we can obtain sufficient numbers of dividing Schwann cells within one week of initial culture. A spontaneously immortal Schwann cell clone (iSC) was observed in and isolated from one of these primary cultures. These cells were transformed at a time of maximal Schwann cell activation in response to injury. Both the primary Schwann cells and the iSC have been characterized as Schwann cells by morphology, immunohistochemistry and gene expression.
View details for Web of Science ID A1992JU51800005
View details for PubMedID 1280693
Human neuroblastoma SH-SY5Y cells differentiate terminally in culture upon exposure to nerve growth factor (NGF) for 4-5 weeks. The neuronal phenotypic properties acquired in response to prolonged NGF treatment include morphological differentiation, cessation of mitotic activity, neuronal marker expression, increased membrane electrical potentials, and a survival dependence upon NGF for trophic support (Jensen, L.M. (1987) Dev. Biol. 120, 56-64). Thus, differentiated cultures survive indefinitely in the continued presence of NGF, however, withdrawal of NGF from differentiated cultures effects the loss of cellular viability within 3-6 days. Here, we show that death of differentiated SH-SY5Y cells caused by NGF deprivation is characteristic of apoptosis. To compare the differentiation promoting and the neurotrophic properties of NGF, whole SH-SY5Y cell extracts were analyzed by two-dimensional polyacrylamide gel electrophoresis using isoelectric focusing and nonequilibrium pH gradient electrophoresis gels in the first dimension. Steady-state levels of polypeptides extracted from whole-cell lysates of naive (untreated) cells, terminally differentiated cells, and NGF-deprived differentiated cells were compared. Over 1,000 spots from each were analyzed using computer-aided spot matching and densitometry. We noted 25 polypeptides that decreased during differentiation, including 15 that decreased by a factor of 10 or more. The levels of five polypeptides were induced from very low or undetectable levels in naive cells. Withdrawal of NGF from terminally differentiated cells produced alterations in steady-state protein patterns substantially distinct from those occurring during differentiation. While levels of most proteins do not appear affected early after NGF withdrawal, others rapidly return to levels comparable with those of the naive state and some changes occurring with differentiation are enhanced further upon NGF withdrawal. Three polypeptides were regulated uniquely by NGF withdrawal, including two that were induced, on average, 20- and 28-fold and another that was depressed more than 7-fold after NGF deprivation, before cell death. These data indicate that NGF elicits both constitutive and nonconstitutive changes in gene expression and suggest that the differentiation promoting and the neurotrophic properties of NGF correlate with the regulation of different gene products.
View details for Web of Science ID A1992JP59300048
View details for PubMedID 1527053
The neurotrophins, of which nerve growth factor (NGF) is the best known example, support the survival and differentiation of chick embryo sensory neurons at extremely low concentrations, 10(-12) M or less. These same neurons display two different classes of neurotrophin receptors with dissociation constants of 10(-11) M and 10(-9) M, respectively, implying that only low occupancy of the higher affinity receptor is required to mediate the biological actions of the neurotrophins. Two structurally unrelated receptors have now been characterized for NGF, and one of them, p75NGFR, serves as a receptor for all the known neurotrophins. This is the receptor with a dissociation constant of 10(-9) M. The second NGF receptor is a member of the trk family of tyrosine kinase receptors, p140trkA. Other members, p145trkB and p145trkC, are receptors for brain-derived neurtrophic factor (BDNF) and neurotrophin-4 (NT-4) and neurotrophin-3 (NT-3), respectively, when assayed in fibroblasts. The specificity of neurotrophin binding to these receptors appears to be much higher in neurons than in the non-neuronal cells. The receptor p140trkA has many of the properties of the higher affinity class of NGF receptors, and is able to mediate survival and differentiation of the PC12 cell line, and cell growth and transformation in fibroblast cells. On the other hand, expression of p75NGFR in several types of cells displaying p140trkA induces a component of higher affinity NGF binding not seen in its absence. Since it is unlikely that p75NGFR and p140trkA interact at the level of the receptors, the crosstalk between receptors probably occurs through their signal transduction mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1992JJ75800004
View details for PubMedID 1382329
Peripheral myelin protein PMP-22 is a potential growth-regulating myelin protein that is expressed by Schwann cells and predominantly localized in compact peripheral myelin. A point mutation in the Pmp-22 gene of inbred trembler (Tr) mice was identified and proposed to be responsible for the Tr phenotype, which is characterized by paralysis of the limbs as well as tremors and transient seizures. In support of this hypothesis, we now report the fine mapping of the Pmp-22 gene to the immediate vicinity of the Tr locus on mouse chromosome 11. Furthermore, we have found a second point mutation in the Pmp-22 gene of trembler-J (TrJ) mice, which results in the substitution of a leucine residue by a proline residue in the putative first transmembrane region of the PMP-22 polypeptide. Tr and TrJ were previously mapped genetically as possible allelic mutations giving rise to similar, but not identical, phenotypes. This finding is consistent with the discovery of two different mutations in physicochemically similar domains of the PMP-22 protein. Our results strengthen the hypothesis that mutations in the Pmp-22 gene can lead to heterogeneous forms of peripheral neuropathies and offer clues toward possible explanations for the dominant inheritance of these disorders.
View details for Web of Science ID A1992HU97700034
View details for PubMedID 1374899
The binding domain of p75NGFR contains four "repeats" of a 6-cysteine pattern. To test whether these repeats have any structural or functional independence, each repeat has been separately deleted. In each case, deletion led to the loss of most nerve growth factor (NGF) binding activity. The epitopes of two monoclonal antibodies, MC192 and 271c, could be distinguished, however. Repeat IV was found to be unnecessary for binding MC192, whereas Repeat I was not required for binding 217c. This suggests that either terminal repeat can be removed without loss of native-like structure in the remaining repeats. Trp155 in the fourth repeat forms an essential part of the 217c epitope but is not required for either MC192 or NGF binding. Deletion of the linker region between the membrane-spanning domain and the cysteine-rich domain does not affect the binding of NGF or MC192, and has only a slight, if any, effect on 217c binding. Cyclic permutation of the four repeats failed to yield protein capable of binding NGF or MC192.
View details for Web of Science ID A1992HQ18500062
View details for PubMedID 1373724
We have recently described a novel cDNA, SR13 (Welcher, A. A., U. Suter, M. De Leon, G. J. Snipes, and E. M. Shooter. 1991. Proc. Natl. Acad. Sci. USA. 88:7195-7199), that is repressed after sciatic nerve crush injury and shows homology to both the growth arrest-specific mRNA, gas3 (Manfioletti, G., M. E. Ruaro, G. Del Sal, L. Philipson, and C. Schneider, 1990. Mol. Cell Biol. 10:2924-2930), and to the myelin protein, PASII (Kitamura, K., M. Suzuki, and K. Uyemura. 1976. Biochim. Biophys. Acta. 455:806-816). In this report, we show that the 22-kD SR13 protein is expressed in the compact portion of essentially all myelinated fibers in the peripheral nervous system. Although SR13 mRNA was found in the central nervous system, no corresponding SR13 protein could be detected by either immunoblot analysis or by immunohistochemistry. Northern and immunoblot analysis of SR13 mRNA and protein expression during development of the peripheral nervous system reveal a pattern similar to other myelin proteins. Furthermore, we demonstrate by in situ mRNA hybridization on tissue sections and on individual nerve fibers that SR13 mRNA is produced predominantly by Schwann cells. We conclude that the SR13 protein is apparently exclusively expressed in the peripheral nervous system where it is a major component of myelin. Thus, we propose the name Peripheral Myelin Protein-22 (PMP-22) for the proteins and cDNA previously designated PASII, SR13, and gas3.
View details for Web of Science ID A1992HL81800020
View details for PubMedID 1556154
The autosomal dominant trembler mutation (Tr), maps to mouse chromosome 11 (ref. 2) and manifests as a Schwann-cell defect characterized by severe hypomyelination and continuing Schwann-cell proliferation throughout life. Affected animals move clumsily and develop tremor and transient seizures at a young age. We have recently described a potentially growth-regulating myelin protein, peripheral myelin protein-22 (PMP-22; refs 7, 8), which is expressed by Schwann cells and found in peripheral myelin. We now report the assignment of the gene for PMP-22 to mouse chromosome 11. Cloning and sequencing of PMP-22 complementary DNAs from inbred Tr mice reveals a point mutation that substitutes an aspartic acid residue for a glycine in a putative membrane-associated domain of the PMP-22 protein. Our results identify the PMP-22 gene as a likely candidate for the mouse trembler locus and will encourage the search for mutations in the corresponding human gene in pedigrees with hypertrophic neuropathies such as Charcot-Marie-Tooth and Dejerine-Sottas diseases (hereditary motor and sensory neuropathies I and III).
View details for Web of Science ID A1992HJ94400058
View details for PubMedID 1552943
Two distinct nerve growth factor receptor (NGFR) complexes are present on NGF-responsive cell types; these correspond to 100 kDa and 158 kDa for the fast (fNGFR) and the slow (sNGFR) NGFRs, respectively. Previous studies indicate that each complex is derived from a separate gene product and that the sNGFR contains tyrosine kinase activity. The cDNA encoding the fNGFR has previously been cloned. In this report, a rat trk protooncogene cDNA has been isolated from PC12 cells and Trk has been shown to bind NGF, generating a complex of 158 kDa. Characterization of NGF-Trk interactions indicates that Trk and NGF dissociate more slowly than do NGF and the fNGFR. Moreover, NGF-bound Trk is not destroyed by trypsin digestion whereas the NGF-fNGFR complex is sensitive to trypsin digestion. These observations suggest that the trk protooncogene product, expressed in the absence of the fNGFR, binds NGF with properties characteristic of the sNGFR, which was identified as the high-affinity NGFR on primary neurons and PC12 cells.
View details for Web of Science ID A1992HJ05300078
View details for PubMedID 1312719
Despite their extensive sequence identities at the amino acid level (approximately 55%), NGF and brain-derived neurotrophic factor (BDNF) display distinct neuronal specificity toward neurons of both the PNS and CNS. To explore which region(s) within these neurotrophic factors might determine their differential actions on various subpopulations of peripheral neurons, a systematic series (homolog-scanning mutagenesis) of chimeric NGF/BDNF molecules was prepared using PCR overlap-extension techniques. After expression in COS-7 cells, the chimeric proteins were tested for their biological activities in neurite outgrowth and neuronal survival assays. This approach led to the functional expression of 12 NGF/BDNF chimeras. Surprisingly, despite replacing successive amino acid segments throughout the entire length of NGF with the corresponding parts of BDNF, all chimeras displayed full NGF-like activity in bioassays carried out with PC12 cells, embryonic chick dorsal root ganglion explants, sympathetic ganglion explants, and dissociated cultures of dorsal root ganglion neurons. Most of the chimeras additionally showed BDNF-like activity as defined by neurite outgrowth on chick nodose ganglion explants. However, none of the chimeras supported the survival of dissociated nodose ganglion neurons. Our results suggest that NGF and BDNF must share very similar higher-order protein structures, and we propose that the overall structure or conformation of NGF, in contrast to short amino acid "active-site" segments, may determine its exact neuronal specificity.
View details for Web of Science ID A1992HA64400027
View details for PubMedID 1729439
By using in vitro DNA mutagenesis, we replaced the tryptophan residue at position 21 in mouse nerve growth factor (NGF) with either phenylalanine, leucine or serine. Yield, biological activity, immunological reactivity and receptor binding of the recombinant proteins were determined. All three mutants were produced at considerably lower yields than wild-type NGF, with the serine mutant being undetectable. The results of competitive binding assays show that tryptophan-21 is involved in recognition of the fast NGF receptor of PC12 cells. However, specific biological activity of NGF is not altered by the replacement of tryptophan-21. Our results therefore suggest that biological activity of NGF is not directly coupled to binding to the fast NGF receptor.
View details for Web of Science ID A1991GX98500016
View details for PubMedID 1686097
The three members of the neurotrophin family (NGF, BDNF and NT-3) are synthesized as large precursor proteins which undergo proteolytic processing to yield biologically active, mature neurotrophic factors. We have used in vitro mutagenesis to examine the pro-region in the NGF precursor protein as a first step towards a general understanding of the role of propeptides in the biosynthesis of neurotrophins. Our results demonstrate that only two small domains within the NGF propeptide are required for the expression and secretion of properly processed and biologically active, recombinant mouse NGF in COS-7 cells. Domain I plays an important role in the expression of active NGF while domain II is involved in proteolytic processing. Both domains are partially conserved between the propeptides of NGF proteins isolated from different species as well as BDNF and NT-3.
View details for Web of Science ID A1991GB44800008
View details for PubMedID 1868828
Striking features of the cellular response to sciatic nerve injury are the proliferation of Schwann cells in the distal nerve stump and the downregulation of myelin-specific gene expression. Once the axons regrow, the Schwann cells differentiate again to reform the myelin sheaths. We have isolated a rat cDNA, SR13, which is strongly downregulated in the initial phase after sciatic nerve injury. This cDNA encodes a glycoprotein that shares striking amino acid similarity with a purified myelin protein and is specifically precipitated by a myelin-specific antiserum. Immunohistochemistry experiments using peptide-specific polyclonal antibodies localize the SR13 protein to the myelin sheath of the sciatic nerve. Computer-aided sequence analysis identified a pronounced homology of SR13 to a growth arrest-specific mRNA (Gas-3) that is expressed in resting but not in proliferating 3T3 mouse fibroblasts. SR13 is similarly downregulated during Schwann cell proliferation in the rat sciatic nerve. The association of the SR13 as well as the Gas-3 mRNA with nonproliferating cells in two different experimental systems suggests a common role for these molecules in maintaining the quiescent cell state.
View details for Web of Science ID A1991GB70100060
View details for PubMedID 1714591
Mammalian peripheral nerve fibres can regenerate after injury. In an attempt toward a better understanding of the underlying molecular events, we have isolated novel and known rat cDNA sequences, the expression of which are regulated during sciatic nerve regeneration. For this purpose, cDNA libraries were constructed from either the nerve segment distal to the crush site or the corresponding contralateral uninjured nerve of the same animals. These libraries were screened by differential hybridization and several transcriptionally repressed and induced sequences were isolated. Out of 2,000 cDNA clones screened from the distal library, 11 sequences were found to be induced in the distal nerve segment. This set of induced cDNAs included the rat homolog of vimentin, 28 S and 18 S ribosomal RNA species, and two novel sequences. Of 5,000 screened colonies of the contralateral library, 30 colonies contained sequences that were repressed in the distal segment after nerve crush. They were identified as myelin basic protein, myelin P0, alpha-globin, cytochrome oxidase subunit 1, creatine kinase (muscle type, M) and collagen type I. In addition, five novel sequences were found that were dramatically repressed after sciatic nerve crush. Representative clones were tested by northern blot analysis to study their time course of transcriptional regulation during nerve regeneration. The observed patterns suggest that the regeneration phenomenon shows complex gene regulation in which the nonneuronal cells of the distal segment play an important role. Further characterization of the isolated regulated known and unknown sequences will increase our understanding of the molecular events associated with neuronal regeneration.
View details for Web of Science ID A1991GE46300003
View details for PubMedID 1724268
Antibodies directed against nerve growth factor (NGF) immunoprecipitate a tyrosine kinase activity from NGF-treated PC12 rat pheochromocytoma cells. Based on several criteria, this activity has been correlated with the high-affinity and not the low-affinity NGF-receptor complex. The in vitro kinase activity and the tyrosine phosphorylation of the high-affinity complex can be blocked by an agent that inhibits NGF (and not epidermal growth factor)-induced tyrosine phosphorylation in PC12 cells, as well as NGF-induced neuronal differentiation of PC12 cells. These observations suggest that the high-affinity NGF-receptor complex is a substrate of tyrosine kinase activity. Phosphorylation reactions by the complex, performed in the absence of added substrate, label a single phosphopeptide of 130-135 kDa. This observation suggests that this phosphopeptide may represent the phosphorylation of the receptor kinase or the phosphorylation of a coimmunoprecipitating substrate, and possible signal-transducing molecule, of the high-affinity NGF-receptor complex.
View details for Web of Science ID A1991FU90100079
View details for PubMedID 1712109
The changes in the levels of S100 beta (a protein that stimulates neurite extension and neuronal survival) and 42A and 42C (S100-like proteins whose mRNAs are induced in PC12 cells by nerve growth factor) during development and after rat sciatic nerve lesions were analyzed. S100 beta, 42A, and 42C mRNAs showed differential regulation during development. S100 beta mRNA was present both in sciatic nerve and brain, and increased more than 11-fold during the first 3 wk of nerve postnatal development. 42A and 42C mRNAs were essentially restricted to sciatic nerve, with little found in either embryonic or adult brain. The levels of 42C and 42A mRNAs in sciatic nerve increased 4- and 14-fold, respectively, by postnatal day 23 compared to postnatal day 2. 42A, 42C, and S100 beta mRNAs also showed a differential regulation during sciatic nerve degeneration and regeneration. Axotomized and control sciatic nerves were examined by Northern blots at various times after a crush or cut injury. 42A and 42C mRNA levels increased rapidly in the distal segment of axotomized nerve, remained two- to five-fold higher than controls at day 14 after injury but returned to control levels by 40 days. In contrast, S100 beta mRNA showed a three-fold decrease in the axotomized nerve between days 1 and 3 after injury, and slowly returned towards control levels over the next few weeks. The decrease in S100 beta mRNA was reflected by a corresponding decrease in S100 beta protein levels. The induction of 42A and 42C mRNAs and repression of S100 beta mRNA remained if nerve regeneration was prevented.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1991FV00900003
View details for PubMedID 1890696
The 7S NGF complex from the male mouse submaxillary gland consists of the alpha, gamma, and beta subunits in the ratio alpha 2 gamma 2 beta. The beta (NGF) subunit contains all the known biolocial activity of 7S NGF. The alpha and gamma subunits are both members of glandular kallikrein gene family, yet only gamma subunit has protease activity. The gamma subunit plays a role in the processing of preproNGF to its mature form, while the role of the alpha subunit is not yet understood. Despite the fact that 7S NGF has been extensively characterized, no other NGF complex has been characterized, nor have the alpha or gamma subunits been observed in tissues which express NGF. We have therefore purified and characterized the NGF complex from the submaxillary glands of the rat Mastomys natalensis in order to more fully understand the roles of the alpha and gamma subunits. The NGF complex from M. natalensis contains subunits similar to those found in mouse 7S NGF. Although similar, there are significant differences between mouse and M. natalensis NGF complexes, especially in the degree of post-translational modification of the gamma and NGF subunits, the expression of esterase activity and the ease with which the complexes dissociate. Evidence is presented that suggests that the NGF complex from M. natalensis may consist of subunits in the ratio alpha 2 gamma beta. The amino acid sequence of the M. natalensis NGF suggests some, but not all, ways in which these differences arise.
View details for Web of Science ID A1991GE21100003
View details for PubMedID 1791910
Current research into regeneration of the nervous system has focused on defining the molecular events that occur during regeneration. One well-characterized system for studying nerve regeneration is the sciatic nerve of rat. Numerous studies have characterized the sequence of events that occur after a crush injury to the sciatic nerve (Cajal 1928; Hall 1989). These events include axon and myelin breakdown, changes in the permeability of the blood vessels, proliferation of Schwann cells, invasion of macrophages, and the phagocytosis of myelin fragments by Schwann cells and macrophages. The distal segment of the injured sciatic nerve provides a supportive environment for the regeneration of the nerve fibres (Cajal 1928; David & Aguayo 1981). Within a period of weeks, the injured sciatic nerve is able to regrow and successfully reinnervate the appropriate targets. Some of the molecules that provide trophic support for the regrowing nerve fibres have been identified, including nerve growth factor (NGF) (Heumann et al. 1987) and glial maturation factor beta (Bosch et al. 1989). Another class of molecules show changes in their rates of synthesis during regeneration, including both proteins (Skene & Shooter 1983; Muller et al. 1986) and mRNA species (Trapp et al. 1988; Meier et al. 1989). To better understand nerve regeneration, we have taken two, parallel molecular approaches to study the events associated with regeneration. The first of these is to study in detail the mechanism of action of a molecule that has been implicated in the regeneration process, nerve growth factor. The second approach is to identify novel gene sequences which are regulated during regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1991FH55100008
View details for PubMedID 1677474
A structural analysis of the rat low-affinity nerve growth factor (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse fibroblasts or COS cells, and the ability of the mutant receptor to bind NGF was assayed. In the first mutant, all but 16 amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a Kd comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extracellular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terminal deletions, in which either the first cysteine-rich sequence of the first and part of the second cysteine-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteine-rich sequences of the NGF receptor contain the NGF binding domain.
View details for Web of Science ID A1991EQ54400035
View details for PubMedID 1846035
Nerve growth factor (NGF) receptors have been investigated by means of affinity labeling with 125I-NGF and chemical cross-linking. Two distinct NGF-receptor complexes are detected on PC12 cells; these correspond to 100 kd and 158 kd for the low-affinity (LNGFR) and the high-affinity (HNGFR) receptors, respectively. Interestingly, three different antibodies directed against distinct epitopes on the LNGFR immunoprecipitate the low-but not the high-affinity NGF-receptor complex. Although the identities of the signaling molecules in the HNGFR are unknown, antibodies to the src, ras, raf-1, and yes products fail to immunoprecipitate either receptor complex, suggesting that these molecules are not a part of, or tightly coupled to, either receptor type. Phosphotyrosine residues are found exclusively on the HNGFR complex, suggesting that tyrosine phosphorylation may be one of the initiating events in the NGF-induced signal transduction cascade.
View details for Web of Science ID A1991EU27800015
View details for PubMedID 1702649
The cloned DNA coding for 42C protein (light chain of calpactin I), whose mRNA is induced in PC12 cells by treatment with nerve growth factor (NGF), was reintroduced into these cells. A cell line was obtained in which the outgrowth of processes in the absence of added NGF, similar to that induced in the parental PC12 cells by the factor, was accompanied by high levels of 42C RNA. The apparent reason for this constitutive overexpression of 42C is the stable integration of multiple copies of the 42C DNA into the cell genome. These results further support the notion that S-100 protein family, of which 42C is a member, may play an important role in development.
View details for Web of Science ID A1990EH87900003
View details for PubMedID 2151454
Nerve growth factor and its receptor (NGFR) are known to be present in diverse embryonic and neonatal central nervous system tissues, including the cerebral cortex. However, the identity of the cortical cells expressing NGFR immunoreactivity has not been established. We have used immunolabeling coupled with [3H]thymidine autoradiography to identify such cells in ferret and cat brain. Polyclonal antibodies raised against a synthetic peptide corresponding to a conserved amino acid sequence of the NGFR were used for this purpose. Western (immunologic) blot analyses show that these antibodies specifically recognize NGFR and precursor proteins. In both species, NGFR immunoreactivity is primarily associated with the early generated and transient subplate neuron population of the developing neocortex, as indicated by the following evidence: the immunoreactive cells (i) are located directly beneath the developing cortical plate, (ii) frequently have the inverted pyramid shape characteristic of subplate neurons, and (iii) can be labeled by an injection of [3H]thymidine on embryonic day (E) 28, a time when only subplate neurons are being generated. Intense NGFR immunostaining is seen on the cell bodies of these neurons as early as E30, several days after their last round of cell division, and this immunostaining remains strong for approximately 3 weeks. The NGFR immunoreactivity begins to decline around E52 and has disappeared from the region altogether by E60, at which time subplate neurons begin to die. The cellular localization and timing of expression suggest that the NGFR may play a role in the maintenance of subplate neurons and in the maturation of the cerebral cortex.
View details for Web of Science ID A1990CH19000040
View details for PubMedID 2153287
The effect of removal of PC12 cell nuclei on neurite outgrowth was studied. Enucleation (80-90%) was accomplished in the presence of cytochalasin B using a centrifugation technique that exploited the very tight adhesivity of PC12 cells for a substratum composed of an extracellular matrix secreted by bovine corneal endothelial cells in response to epidermal growth factor treatment. Neither nucleated nor enucleated PC12 cells showed significant neurite outgrowth on this particular matrix in the absence of nerve growth factor. In the presence of nerve growth factor both PC12 cell types initiated neurite outgrowth, but whereas neurites from nucleated cells grew continuously for two days, those from enucleated cells reached a maximum length after one day. The results suggest that neurite initiation but not continued neurite growth or stabilization can occur in the absence of transcription.
View details for Web of Science ID A1989AX81700010
View details for PubMedID 2808539
The synthesis of a 37-kilodalton (kDa) protein which has been shown recently to be identical with apolipoprotein E (apo-E) was increased after sciatic nerve injury of the rat. When regeneration of the nerve was allowed, its synthesis returned to control levels at about 8 weeks post injury. In this report it is shown that similar time-course studies of the protein in the rat optic nerve revealed a delayed increase of the protein but a comparably high level of synthesis at 3 weeks post injury. This level was maintained up to at least 18 weeks after crush. Furthermore, two-dimensional electrophoresis revealed that the characteristic "trailing" of the protein is due to its sialylation, because it was reduced after neuraminidase treatment. This treatment, however, detected a neuraminidase-resistant heterogeneous form in CNS tissue and a homogeneous form in peripheral nervous tissue. The trailing persisted up to 18 days of culture of optic nerve explants, of CNS glial cells, and of peritoneal macrophages, but disappeared during the first culture days of sciatic nerve explants and was not observed in Schwann cell culture media. Incorporation studies with 35SO4 revealed that apo-E was the major sulfated protein in culture media conditioned by CNS glial cells, whereas sulfation of the protein was undetectable in Schwann cell cultures. Because macrophages are likely to be the major source of apo-E in both peripheral and central glial cell cultures as well as in injured optic and sciatic nerves, it is hypothesized that resident cells of sciatic nerves secrete potent sulfatases. As a result, sialic acid residues may be more susceptible to degradation.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1989AL01000037
View details for PubMedID 2668445
Nerve growth factor (NGF) induces the chromaffin cell line PC12 to differentiate into cells with many of the properties of sympathetic neurons. We investigated the early differentiative phase and identified a gene, PC4, rapidly and transiently induced by NGF in PC12 cells. PC4 cDNA is homologous to the partial sequence of a putative mouse beta-interferon and encodes a protein related to a lymphokine, the rat gamma-interferon protein. Nonetheless, PC4 appears devoid of antiviral activity. PC4 is expressed in proliferating and differentiating tissues, such as amnion, placenta, and brain at embryonic day 13.5. The relationship of PC4 to interferons and lymphokines suggests that it could play a role in regulating gene activity in the pathways induced by NGF.
View details for Web of Science ID A1989T788900074
View details for PubMedID 2467301
The in vitro translation of the capped nerve growth factor (NGF) mRNA in wheat germ extracts produced a major 34 kDa NGF precursor with translation starting at the first methionine residue and a minor 29 kDa precursor with translation starting probably at the second methionine. Digestion with the gamma-subunit generated the 22 and 18 kDa intermediates as well as a very small amount of NGF indicating that the gamma-subunit is able to carry out all the processing steps in NGF biosynthesis. The digestion also produced a large amount of a peptide from the N-terminus of the NGF precursors. The discrepancy between the yield of this peptide and of mature NGF emphasizes that inappropriate formation or the lack of formation of disulfide bridges in the NGF precursors, resulting in rapid proteolysis, probably accounts for the low yield of the NGF peptide chains.
View details for Web of Science ID A1989T356900007
View details for PubMedID 2716498
The kallikrein genes and their expression in the salivary glands of mouse, the African rat Mastomys natalensis and human were compared. The Mastomys kallikrein genes comprise a family of genes similar to those of mouse. Androgen markedly enhances transcription of glandular nerve growth factor (NGF) and kallikrein in both male and female Mastomys suggesting the presence of testosterone regulated kallikrein genes for growth factor precursor-processing in both sexes. In contrast, although a kallikrein transcript was detected in human salivary glands of the same size as the mouse or Mastomys transcript no difference in the amount of transcript was seen in adult male or female. The absence of kallikrein genes regulated by testosterone and of NGF transcripts in the human implies that there is no human equivalent of the mouse salivary 7S NGF complex.
View details for PubMedID 3133079
Differential hybridization of a cDNA library from rat pheochromocytoma PC12 cells with cDNA probes from naive PC12 cells and from PC12 cells exposed to nerve growth factor for 7 days identified cDNA sequences of two genes induced by NGF. The mRNA species detected by these cDNA sequences, designated 42A and 42C, reached maximal levels after 24 hr of treatment with NGF and were still significantly higher than control levels after 7 days. Epidermal growth factor transiently induced both mRNAs but at much lower levels. The mRNAs code for 95- (42C) and 101- (42A) amino acid residue peptides whose sequences are homologous to those of a family of calcium-binding proteins including the S-100 protein. The conservation of primary and secondary structure between 42A, 42C, and the other proteins suggests a possible role for them in the regulation of cell growth and differentiation.
View details for Web of Science ID A1988M196200069
View details for PubMedID 3422491
One of the major roles of nerve growth factor (NGF) is to mediate the selective survival of a proportion of the developing sympathetic and sensory neurones as they innervate their particular target tissues. The underlying basis of this phenomenon is the synthesis of limited amounts of NGF in the target, its secretion around, and uptake by, the nerve terminal and its retrograde transport along axons to the neuronal cell bodies. The cascades of reactions which lead to neuronal survival and maintenance are initiated by signal transduction somewhere in this pathway. Retrograde transport and the initial signal transduction step begin when NGF binds to NGF receptors on the nerve terminal. Receptor-mediated internalization and the survival and maintenance function of NGF are mediated by the higher affinity receptors. These receptors have relative molecular masses of approx. 145,000 and are trypsin-resistant when occupied. In contrast, the larger population of lower affinity receptors have relative molecular masses of 85,000 and are rapidly degraded by trypsin. Clustering of the lower affinity receptors by a variety of agents gives them many of the characteristics of the higher affinity receptors, suggesting receptor interconversion may play a role in NGF actions. The structure of the lower affinity NGF receptor, determined by gene transfer and cloning, shows it to be a unique, heavily glycosylated protein. The extracellular domain is rich in cysteine-containing repeat units while the intracellular domain lacks the consensus sequence for an endogenous kinase activity. It is likely that the higher affinity receptor contains this protein as the NGF binding subunit together with a second protein which determines both the nature of the signal transduction mechanism and the process of internalization.
View details for Web of Science ID A1987K564500013
View details for PubMedID 2828509