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W C Mobley

Publications and source records attributed to W C Mobley.

At least 19 recordsLinked to original sources
PubMed2001

NGF signaling from clathrin-coated vesicles: evidence that signaling endosomes serve as a platform for the Ras-MAPK pathway.

The target-derived neurotrophic factor "nerve growth factor" (NGF) signals through TrkA to promote the survival, differentiation, and maintenance of neurons. How the NGF signal in axon terminals is conveyed to the cell body is unknown. The "signaling endosome hypothesis" envisions that NGF-TrkA complexes are internalized at the axon terminal and retrogradely transported to the cell body. Following NGF treatment, we found that clathrin-coated vesicles contained NGF bound to TrkA together with activated signaling proteins of the Ras-MAP kinase pathway. Evidence that these vesicles could signal was their ability in vitro to activate Elk, a downstream target of Erk1/2. Our results point to the existence of a population of signaling endosomes derived from clathrin-coated membranes in NGF-treated cells.

Animals↗
PubMed2001

Failed retrograde transport of NGF in a mouse model of Down's syndrome: reversal of cholinergic neurodegenerative phenotypes following NGF infusion.

Age-related degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive decline in Alzheimer's disease and Down's syndrome. With aging, the partial trisomy 16 (Ts65Dn) mouse model of Down's syndrome exhibited reductions in BFCN size and number and regressive changes in the hippocampal terminal fields of these neurons with respect to diploid controls. The changes were associated with significantly impaired retrograde transport of nerve growth factor (NGF) from the hippocampus to the basal forebrain. Intracerebroventricular NGF infusion reversed well established abnormalities in BFCN size and number and restored the deficit in cholinergic innervation. The findings are evidence that even BFCNs chronically deprived of endogenous NGF respond to an intervention that compensates for defective retrograde transport. We suggest that age-related cholinergic neurodegeneration may be a treatable disorder of failed retrograde NGF signaling.

Aging↗
PubMed2001

Nerve growth factor activates persistent Rap1 signaling in endosomes.

We investigated a role for endogenous Rap1, a small monomeric GTP-binding protein of the Ras family, in nerve growth factor (NGF) signaling in PC12 cells. Although both epidermal growth factor (EGF) and NGF caused transient activation of Ras, only NGF induced the activation of Rap1. Moreover, Rap1 activation was sustained for hours, an effect that matched the sustained activation of the mitogen-activated protein kinase (MAPK) pathway. To investigate the molecular basis for Rap1 activation, we examined complexes containing C3G, a guanine nucleotide exchange factor for Rap1, and CrkL, an adapter protein known to influence Rap1 signaling. NGF induced the formation of a long-lived complex containing C3G/CrkL/Shp2/Gab2/TrkA. Linking the complex to Rap1 activation, we coprecipitated activated TrkA and activated MAPK with activated Rap1 in NGF-treated cells. Confocal microscopy and subcellular fractionation showed that activated Rap1 and the other proteins of the signaling complex were present in endosomes. Pretreatment of PC12 cells with brefeldin A (BFA), which disrupts the Golgi and endosomal compartments, had little effect on Ras activation but strongly inhibited NGF-induced Rap1 activation and continuing MAPK activation. We propose that endosomes are a site from which NGF induces the prolonged activation of Rap1 and MAPK.

Adaptor Proteins, Signal Transducing↗
PubMed2001

The extracellular domain of p75NTR is necessary to inhibit neurotrophin-3 signaling through TrkA.

The TrkA receptor is activated primarily by nerve growth factor (NGF), but it can also be activated by high concentrations of neurotrophin 3 (NT-3). The pan-neurotrophin receptor p75(NTR) strongly inhibits activation of TrkA by NT-3 but not by NGF. To examine the role of p75(NTR) in regulating the specificity of TrkA signaling, we expressed both receptors in Xenopus oocytes. Application of NGF or NT-3 to oocytes expressing TrkA alone resulted in efflux of (45)Ca(2+) by a phospholipase C-gamma-dependent pathway. Coexpression of p75(NTR) with TrkA inhibited (45)Ca(2+) efflux in response to NT-3 but not NGF. The inhibitory effect on NT-3 activation of TrkA increased with increasing expression of p75(NTR). Coexpression of a truncated p75(NTR) receptor lacking all but the first 9 amino acids of the cytoplasmic domain inhibited NT-3 stimulation of (45)Ca(2+) efflux, whereas coexpression of an epidermal growth factor receptor/p75(NTR) chimera (extracellular domain of epidermal growth factor receptor with transmembrane and cytoplasmic domains of p75(NTR)) did not inhibit NT-3 signaling through TrkA. These studies demonstrated that the extracellular domain of p75(NTR) was necessary to inhibit NT-3 signaling through TrkA. Remarkably, p75(NTR) binding to NT-3 was not required to prevent signaling through TrkA, since occupying p75(NTR) with brain-derived neurotrophic factor or anti-p75 antibody (REX) did not rescue the ability of NT-3 to activate (45)Ca(2+) efflux. These data suggested a physical association between TrkA and p75(NTR). Documenting this physical interaction, we showed that p75(NTR) and TrkA could be coimmunoprecipitated from Xenopus oocytes. Our results suggest that the interaction of these two receptors on the cell surface mediated the inhibition of NT-3-activated signaling through TrkA.

Animals↗
PubMed2001

Nerve growth factor signaling, neuroprotection, and neural repair.

Nerve growth factor (NGF) was discovered 50 years ago as a molecule that promoted the survival and differentiation of sensory and sympathetic neurons. Its roles in neural development have been characterized extensively, but recent findings point to an unexpected diversity of NGF actions and indicate that developmental effects are only one aspect of the biology of NGF. This article considers expanded roles for NGF that are associated with the dynamically regulated production of NGF and its receptors that begins in development, extends throughout adult life and aging, and involves a surprising variety of neurons, glia, and nonneural cells. Particular attention is given to a growing body of evidence that suggests that among other roles, endogenous NGF signaling subserves neuroprotective and repair functions. The analysis points to many interesting unanswered questions and to the potential for continuing research on NGF to substantially enhance our understanding of the mechanisms and treatment of neurological disorders.

Animals↗
PubMed2000

NGF signals through TrkA to increase clathrin at the plasma membrane and enhance clathrin-mediated membrane trafficking.

Neurotrophin (NT) signals may be moved from axon terminals to neuron cell bodies via signaling endosomes-organelles in which NTs continue to be bound to their activated receptors. Suggesting that clathrin-coated membranes serve as one source of signaling endosomes, in earlier studies we showed that nerve growth factor (NGF) treatment increased clathrin at the plasma membrane and resulted in colocalization of clathrin with TrkA, the receptor tyrosine kinase for NGF. Strikingly, however, we also noted that most clathrin puncta at the surface of NGF-treated cells did not colocalize with TrkA, raising the possibility that NGF induces a general increase in clathrin-coated membrane formation. To explore this possibility further, we examined the distribution of clathrin in NGF- and BDNF-treated cells. NGF signaling in PC12 cells robustly redistributed the adaptor protein AP2 and the clathrin heavy chain (CHC) to surface membranes. Using confocal and epifluorescence microscopy, as well as biochemical assays, we showed the redistribution of clathrin to be attributable to the activation of TrkA. Significantly, NGF signaled through TrkA to induce an increase in clathrin-mediated membrane trafficking, as revealed in the increased endocytosis of transferrin. In that BDNF treatment increased AP2 and clathrin at the surface membranes of hippocampal neurons, these findings may represent a physiologically significant response to NTs. We conclude that NT signaling increases clathrin-coated membrane formation and clathrin-mediated membrane trafficking and speculate that this effect contributes to their trophic actions via the increased internalization of receptors and other proteins that are present in clathrin-coated membranes.

Adaptor Protein Complex 2↗
PubMed2000

Human nerve growth factor protects common marmosets against autoimmune encephalomyelitis by switching the balance of T helper cell type 1 and 2 cytokines within the central nervous system.

Multiple sclerosis is a demyelinating disorder of the central nervous system (CNS), in which an immune attack directed against myelin constituents causes myelin destruction and death of oligodendrocytes, the myelin-producing cells. Here, the efficacy of nerve growth factor (NGF), a growth factor for neurons and oligodendrocytes, in promoting myelin repair was evaluated using the demyelinating model of experimental allergic encephalomyelitis (EAE) in the common marmoset. Surprisingly, we found that NGF delayed the onset of clinical EAE and, pathologically, prevented the full development of EAE lesions. We demonstrate by immunocytochemistry that NGF exerts its antiinflammatory effect by downregulating the production of interferon gamma by T cells infiltrating the CNS, and upregulating the production of interleukin 10 by glial cells in both inflammatory lesions of EAE and normal-appearing CNS white matter. Thus, NGF, currently under investigation in human clinical trials as a neuronal trophic factor, may be an attractive candidate for therapy of autoimmune demyelinating disorders.

Animals↗
PubMed1999

Nerve growth factor signaling in caveolae-like domains at the plasma membrane.

Nerve growth factor (NGF) binding to its receptors TrkA and p75(NTR) enhances the survival, differentiation, and maintenance of neurons. Recent studies have suggested that NGF receptor activation may occur in caveolae or caveolae-like membranes (CLM). This is an intriguing possibility because caveolae have been shown to contain many of the signaling intermediates in the TrkA signaling cascade. To examine the membrane localization of TrkA and p75(NTR), we isolated caveolae from 3T3-TrkA-p75 cells and CLM from PC12 cells. Immunoblot analysis showed that TrkA and p75(NTR) were enriched about 13- and 25-fold, respectively, in caveolae and CLM. Binding and cross-linking studies demonstrated that the NGF binding to both TrkA and p75(NTR) was considerably enriched in CLM and that about 90% of high affinity binding to TrkA was present in CLM. When PC12 cells were treated with NGF, virtually all activated (i.e. tyrosine phosphorylated) TrkA was found in the CLM. Remarkably, in NGF-treated cells, it was only in CLM that activated TrkA was coimmunoprecipitated with phosphorylated Shc and PLCgamma. These results document a signaling role for TrkA in CLM and suggest that both TrkA and p75(NTR) signaling are initiated from these membranes.

Animals↗
PubMed1999

Apparent loss and hypertrophy of interneurons in a mouse model of neuronal ceroid lipofuscinosis: evidence for partial response to insulin-like growth factor-1 treatment.

The neuronal ceroid lipofuscinoses (NCL) are progressive neurodegenerative disorders with onset from infancy to adulthood that are manifested by blindness, seizures, and dementia. In NCL, lysosomes accumulate autofluorescent proteolipid in the brain and other tissues. The mnd/mnd mutant mouse was first characterized as exhibiting adult-onset upper and lower motor neuron degeneration, but closer examination revealed early, widespread pathology similar to that seen in NCL. We used the autofluorescent properties of accumulated storage material to map which CNS neuronal populations in the mnd/mnd mouse show NCL-like pathological changes. Pronounced, early accumulation of autofluorescent lipopigment was found in subpopulations of GABAergic neurons, including interneurons in the cortex and hippocampus. Staining for phenotypic markers normally present in these neurons revealed progressive loss of staining in the cortex and hippocampus of mnd/mnd mice, with pronounced hypertrophy of remaining detectable interneurons. In contrast, even in aged mutant mice, many hippocampal interneurons retained staining for glutamic acid decarboxylase. Treatment with insulin-like growth factor-1 partially restored interneuronal number and reduced hypertrophy in some subregions. These results provide the first evidence for the involvement of interneurons in a mouse model of NCL. Moreover, our findings suggest that at least some populations of these neurons persist in a growth factor-responsive state.

Aging↗
PubMed1999

EGF receptor signaling stimulates SRC kinase phosphorylation of clathrin, influencing clathrin redistribution and EGF uptake.

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor (EGFR) signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling.

Amino Acid Sequence↗
PubMed1999

Early BDNF, NT-3, and NT-4 signaling events.

Much more is known about nerve growth factor (NGF) signaling than that initiated by brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or NT-4. We sought to study early BDNF, NT-3, and NT-4 signaling events. Using TrkB-expressing cells, we found that BDNF and NT-4 individually induced tyrosine phosphorylation of TrkB in a dose-dependent fashion. At maximally effective concentrations, BDNF or NT-4 induced robust TrkB tyrosine phosphorylation at 5 min; this progressively declined at 15, 30, and 60 min. Using immunoprecipitation, PI3-kinase and tyrosine phosphorylated PLC-gamma1 and SHC were shown to be associated with tyrosine phosphorylated TrkB in response to both BDNF and NT-4. BDNF and NT-4 induced similar intensities of phosphorylation of TrkB and signaling intermediates at equivalent doses. NT-3 treatment of TrkC-expressing cells induced very similar patterns for induction of TrkC tyrosine phosphorylation and recruitment of signaling intermediates. BDNF, NT-3, and NT-4 caused rapid tyrosine phosphorylation of ERK and SNT. These data suggest that the earliest signaling events for BDNF, NT-3, and NT-4 are very similar to those for NGF.

3T3 Cells↗
PubMed1999

Targeted disruption of the Cln3 gene provides a mouse model for Batten disease. The Batten Mouse Model Consortium [corrected].

Batten disease, a degenerative neurological disorder with juvenile onset, is the most common form of the neuronal ceroid lipofuscinoses. Mutations in the CLN3 gene cause Batten disease. To facilitate studies of Batten disease pathogenesis and treatment, a murine model was created by targeted disruption of the Cln3 gene. Mice homozygous for the disrupted Cln3 allele had a neuronal storage disorder resembling that seen in Batten disease patients: there was widespread and progressive intracellular accumulation of autofluorescent material that by EM displayed a multilamellar rectilinear/fingerprint appearance. Inclusions contained subunit c of mitochondrial ATP synthase. Mutant animals also showed neuropathological abnormalities with loss of certain cortical interneurons and hypertrophy of many interneuron populations in the hippocampus. Finally, as is true in Batten disease patients, there was increased activity in the brain of the lysosomal protease Cln2/TPP-1. Our findings are evidence that the Cln3-deficient mouse provides a valuable model for studying Batten disease.

Animals↗
PubMed1998

Crystal structure of neurotrophin-3 homodimer shows distinct regions are used to bind its receptors.

Neurotrophin-3 (NT-3) is a cystine knot growth factor that promotes the survival, proliferation, and differentiation of developing neurons and is a potential therapeutic for neurodegenerative diseases. To clarify the structural basis of receptor specificity and the role of neurotrophin dimerization in receptor activation, the structure of the NT-3 homodimer was determined using X-ray crystallography. The orthorhombic crystals diffract to 2.4 A, with dimer symmetry occurring about a crystallographic 2-fold axis. The overall structure of NT-3 resembles that of the other neurotrophins, NGF and BDNF; each protomer forms a twisted four-stranded beta sheet, with three intertwined disulfide bonds. There are notable differences, however, between NT-3 and NGF in the surface loops and in three functionally important regions, shown in previous mutagenesis studies to be critical for binding. One such difference implies that NT-3's binding affinity and specificity depend on a novel hydrogen bond between Gln 83, a residue important for binding specificity with TrkC, and Arg 103, a residue crucial for binding affinity with TrkC. NT-3's extensive dimer interface buries much of the otherwise solvent-accessible hydrophobic surface area and suggests that the dimeric state is stabilized through the formation of this hydrophobic core. A comparison of the dimer interface between the NT-3 homodimer and the BDNF/NT-3 heterodimer reveals similar patterns of hydrogen bonds and nonpolar contacts, which reinforces the notion that the evolutionarily conserved neurotrophin interface resulted from the need for receptor dimerization in signal initiation.

Amino Acid Sequence↗
PubMed1998

Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities.

A mouse model for Down syndrome, Ts1Cje, has been developed. This model has made possible a step in the genetic dissection of the learning, behavioral, and neurological abnormalities associated with segmental trisomy for the region of mouse chromosome 16 homologous with the so-called "Down syndrome region" of human chromosome segment 21q22. Tests of learning in the Morris water maze and assessment of spontaneous locomotor activity reveal distinct learning and behavioral abnormalities, some of which are indicative of hippocampal dysfunction. The triplicated region in Ts1Cje, from Sod1 to Mx1, is smaller than that in Ts65Dn, another segmental trisomy 16 mouse, and the learning deficits in Ts1Cje are less severe than those in Ts65Dn. In addition, degeneration of basal forebrain cholinergic neurons, which was observed in Ts65Dn, was absent in Ts1Cje.

Animals↗
PubMed1998

Evidence for normal aging of the septo-hippocampal cholinergic system in apoE (-/-) mice but impaired clearance of axonal degeneration products following injury.

The association of the epsilon4 allele of apoE with increased risk for Alzheimer's disease (AD) and with poor clinical outcome after certain acute brain injuries has sparked interest in the neurobiology of apoE. ApoE (-/-) mice provide a tool to investigate the role of apoE in the nervous system in vivo. Since integrity of the basal forebrain cholinergic system is severely compromised in AD, with severity of dysfunction correlating with apoE4 gene dosage, the present study tested the hypothesis that apoE is required to maintain the normal integrity of basal forebrain cholinergic neurons (BFCNs). Histological and biochemical analyses of the septo-hippocampal cholinergic system were performed in apoE (-/-) mice during aging and following injury. Using unbiased quantitative methods, there was little or no evidence for defects in the septo-hippocampal cholinergic system, as assessed by p75(NTR)-immunoreactive neuron number and size in the medial septum, cholinergic fiber density in the hippocampus, and choline acetyltransferase activity in the hippocampus, cortex, and striatum in aged apoE (-/-) mice (up to 24 months of age) as compared to age-matched wild-type mice of the same strain. In addition, cholinergic neuronal survival and size following fimbria-fornix transection in apoE (-/-) mice did not differ from controls. However, following entorhinal cortex lesion, there was persistence of degeneration products in the deafferented hippocampus in apoE (-/-) mice. These data suggest that although apoE is not required for the maintenance of BFCNs in vivo, it may play a role in the clearance of cholesterol-laden neurodegeneration products following brain injury.

Aging↗