Comparison of nerve growth factor receptor binding models using heterodimeric muteins.

Nerve growth factor (NGF) is a homodimer that binds to two distinct receptor types, TrkA and p75, to support survival and differentiation of neurons. The high-affinity binding on the cell surface is believed to involve a heteroreceptor complex, but its exact nature is unclear. We developed a heterodimer (heteromutein) of two NGF muteins that can bind p75 and TrkA on opposite sides of the heterodimer, but not two TrkA receptors. Previously described muteins are Δ9/13 that is TrkA negative and 7-84-103 that is signal selective through TrkA. The heteromutein (Htm1) was used to study the heteroreceptor complex formation and function, in the putative absence of NGF-induced TrkA dimerization. Cellular binding assays indicated that Htm1 does not bind TrkA as efficiently as wild-type (wt) NGF but has better affinity than either homodimeric mutein. Htm1, 7-84-103, and Δ9/13 were each able to compete for cold-temperature, cold-chase stable binding on PC12 cells, indicating that binding to p75 was required for a portion of this high-affinity binding. Survival, neurite outgrowth, and MAPK signaling in PC12 cells also showed a reduced response for Htm1, compared with wtNGF, but was better than the parent muteins in the order wtNGF > Htm1 > 7-84-103 >> Δ9/13. Htm1 and 7-84-103 demonstrated similar levels of survival on cells expressing only TrkA. In the longstanding debate on the NGF receptor binding mechanism, our data support the ligand passing of NGF from p75 to TrkA involving a transient heteroreceptor complex of p75-NGF-TrkA.

Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration.

PURPOSE: Neurotrophins, including nerve growth factor (NGF), are secreted by glia as a pro-form (proNGF) that is normally cleaved into the mature ligand. Increases of proNGF has been well documented in retinal neurodegenerative diseases. Since systemic overexpression of proNGF exhibits embryonic lethality, we aimed to establish a model that specifically and stably overexpresses a cleavage-resistant mutant of proNGF (proNGF123) plasmid in the retina using electroporation. METHODS: Male Sprague-Dawley rats were injected intravitreally with pGFP or pGFP-proNGF123 plasmids, then electroporated with various settings for optimization. Retinal cell death and ganglion cell count were assessed by TUNEL and immunostaining with anti-Brn3. Expression of proNGF, NGF, and their receptors was examined by western blot. Retinal vascular permeability was assessed by extravasation of bovine serum albumin-fluorescein. Development of acellular capillaries was assessed by periodic acid-Schiff and hematoxylin staining. RESULTS: Successful pGFP-proNGF123 gene delivery and expression of proNGF was demonstrated by western blot and extensive proNGF immunostaining in retina sections. Overexpression of proNGF reduced NGF expression while inducing the expression of neurotrophin receptors, including p75(NTR) and tyrosine receptor kinase A, but not sortilin. Overexpression of proNGF resulted in ~50% reduction in ganglion cell count and fivefold increase in TUNEL-positive cells in rat retina. In addition, overexpression of proNGF induced breakdown of the blood-retina barrier evident by twofold increase in extravasation of bovine serum albumin-fluorescein after 1 week and induced the development of acellular capillaries after 4 weeks. CONCLUSIONS: Electroporation can successfully incorporate and express biologically active cleavage-resistant proNGF locally in rat retinas. Overexpression of cleavage-resistant proNGF can be a useful tool to investigate specific molecular mechanisms by which proNGF causes neurodegeneration and vascular injury in the retina.

Ligand-dependent TrkA activity in brain differentially affects spatial learning and long-term memory.

In the central nervous system, the nerve growth factor (NGF) receptor TrkA is expressed primarily in cholinergic neurons that are implicated in spatial learning and memory, whereas the NGF receptor p75(NTR) is expressed in many neuronal populations and glia. We asked whether selective TrkA activation may have a different impact on learning, short-term memory, and long-term memory. We also asked whether TrkA activation might affect cognition differently in wild-type mice versus mice with cognitive deficits due to transgenic overexpression of mutant amyloid-precursor protein (APP mice). Mice were treated with wild-type NGF (a ligand of TrkA and p75(NTR)) or with selective pharmacological agonists of TrkA that do not bind to p75(NTR). In APP mice, the selective TrkA agonists significantly improved learning and short-term memory. These improvements are associated with a reduction of soluble Aß levels in the cortex and AKT activation in the cortex and hippocampus. However, this improved phenotype did not translate into improved long-term memory. In normal wild-type mice, none of the treatments affected learning or short-term memory, but a TrkA-selective agonist caused persistent deficits in long-term memory. The deficit in wild-type mice was associated temporally, in the hippocampus, with increased AKT activity, increased brain-derived neurotrophic factor precursor, increased neurotrophin receptor homolog-2 (p75-related protein), and long-term depression. Together, these data indicate that selective TrkA activation affects cognition but does so differently in impaired APP mice versus normal wild-type mice. Understanding mechanisms that govern learning and memory is important for better treatment of cognitive disorders.

Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism.

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75(NTR). During retinal injury, endogenous NGF, TrkA, and p75(NTR) are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75(NTR) affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75(NTR) agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75(NTR) action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α(2)-macroglobulin. Antagonists of p75(NTR) inhibit TNF-α and α(2)-macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.

Identification of critical residues within the conserved and specificity patches of nerve growth factor leading to survival or differentiation.

Afflicted neurons in Alzheimer disease have been shown to display an imbalance in the expression of TrkA and p75(NTR) at the cell surface, and administration of nerve growth factor (NGF) has been considered and attempted for treatment. However, wild-type NGF causes extensive elaboration of neurites while providing survival support. This study was aimed at developing recombinant NGF muteins that did not support neuritogenesis while maintaining the survival response. Critical residues were identified at the ligand-receptor interface by point mutagenesis that played a greater importance in neuritogenesis versus survival. By combining point mutations, two survival-selective recombinant NGF muteins, i.e./7-84-103 and KKE/7-84-103, were generated. Both muteins reduced neuritogenesis in PC12 (TrkA(+)/p75(NTR+)) cells by >90%, while concurrently retaining near wild-type survival activity in MG139 (TrkA(+) only) and PCNA fibroblast (p75(NTR+)-only) cells. Additionally, survival in both naive and terminally differentiated PC12 cells was shown to be intermediate between NGF and negative controls. Dose-response curves with 7-84-103 showed that the differentiation curve was shifted by about 100-fold, whereas the EC(50) for survival was only increased by 3.3-fold. Surface plasmon resonance analysis revealed a 200-fold decrease in binding of 7-84-103 to TrkA. The retention of cell survival was attributed to maintenance of signaling through the Akt survival pathway with reduced MAPK signaling for differentiation. The effect of key mutations along the NGF receptor interface are transmitted inside the cell to enable the generation of survival-selective recombinant NGF muteins that may represent novel pharmacologic lead agents for the amelioration of Alzheimer disease.

Biological activity of nerve growth factor precursor is dependent upon relative levels of its receptors.

Nerve growth factor (NGF) is produced as a precursor called pro-nerve growth factor (proNGF), which is secreted by many tissues and is the predominant form of NGF in the central nervous system. In Alzheimer disease brain, cholinergic neurons degenerate and can no longer transport NGF as efficiently, leading to an increase in untransported NGF in the target tissue. The protein that accumulates in the target tissue is proNGF, not the mature form. The role of this precursor is controversial, and both neurotrophic and apoptotic activities have been reported for recombinant proNGFs. Differences in the protein structures, protein expression systems, methods used for protein purification, and methods used for bioassay may affect the activity of these proteins. Here, we show that proNGF is neurotrophic regardless of mutations or tags, and no matter how it is purified or in which system it is expressed. However, although proNGF is neurotrophic under our assay conditions for primary sympathetic neurons and for pheochromocytoma (PC12) cells, it is apoptotic for unprimed PC12 cells when they are deprived of serum. The ratio of tropomyosin-related kinase A to p75 neurotrophin receptor is low in unprimed PC12 cells compared with primed PC12 cells and sympathetic neurons, altering the balance of proNGF-induced signaling to favor apoptosis. We conclude that the relative level of proNGF receptors determines whether this precursor exhibits neurotrophic or apoptotic activity.

Differences in salicylic acid glucose conjugations by UGT74F1 and UGT74F2 from Arabidopsis thaliana.

Salicylic acid (SA) is a signaling molecule utilized by plants in response to various stresses. Through conjugation with small organic molecules such as glucose, an inactive form of SA is generated which can be transported into and stored in plant vacuoles. In the model organism Arabidopsis thaliana, SA glucose conjugates are formed by two homologous enzymes (UGT74F1 and UGT74F2) that transfer glucose from UDP-glucose to SA. Despite being 77% identical and with conserved active site residues, these enzymes catalyze the formation of different products: UGT74F1 forms salicylic acid glucoside (SAG), while UGT74F2 forms primarily salicylic acid glucose ester (SGE). The position of the glucose on the aglycone determines how SA is stored, further metabolized, and contributes to a defense response. We determined the crystal structures of the UGT74F2 wild-type and T15S mutant enzymes, in different substrate/product complexes. On the basis of the crystal structures and the effect on enzyme activity of mutations in the SA binding site, we propose the catalytic mechanism of SGE and SAG formation and that SA binds to the active site in two conformations, with each enzyme selecting a certain binding mode of SA. Additionally, we show that two threonines are key determinants of product specificity.

Deacetylation of p53 after nerve growth factor treatment in PC12 cells as a post-translational modification mechanism of neurotrophin-induced tumor suppressor activation.

The tumor suppressor protein p53 is a transcription factor that regulates the response to cellular insults such as DNA damage and growth factor withdrawal. Transcriptional activity of p53 requires post-translational modification by phosphorylation and acetylation. This study used site-specific antibodies to demonstrate that nerve growth factor (NGF) treatment of PC12 cells results in p53 deacetylation at lysine (Lys) 382. Histone deacetylase (HDAC) activity, measured by a direct fluorescent assay, was increased after NGF treatment and peaked before p53 deacetylation. Inhibition of HDAC by trichostatin blocked the deacetylation of p53 and its transcriptional activity toward a reporter gene construct. Comparison of PC12 with PC12 cells containing a temperature-sensitive, dominant-negative construct showed that p53 deacetylation required functional p53. Inhibitors of MAP kinase that block p53 transactivation and inhibitors of TrkA receptor also abolished HDAC activation, indicating that deacetylation of p53 is an NGF-dependent post-translational mechanism of p53 activation. Finally, NGF or serum withdrawal did not lead to p53 deacetylation. A model is proposed in which the acetylation status of Lys 382 of p53 discriminates between cell cycle arrest and apoptosis.

Tumour necrosis factor-alpha- vs. growth factor deprivation-promoted cell death: different receptor requirements for mediating nerve growth factor-promoted rescue.

Physiological and pathological aging of the central nervous system (CNS) is characterized by functional neuronal impairments which may lead to perturbed cell homeostasis and eventually to neuronal death. Many toxic events may underlie age-related neurodegeneration. These include the effects of beta amyloid, Tau and mutated presenilin proteins, free radicals and oxidative stress, pro-inflammatory cytokines and lack of growth factor support, which can be individually or collectively involved. Taken individually, these toxicants can induce very diverse cell responses, thus requiring individually targeted corrective interventions upstream of common cell death (apoptotic) pathways. Recent preliminary evidence suggests that the pro-inflammatory cytokine tumour necrosis factor alpha (TNFalpha) and growth factor withdrawal can both activate a common apoptotic pathway in nerve growth factor (NGF)-responsive PC12 cells involving caspase 3, albeit through very distinct upstream pathways: the former through active signalling and the latter through passive or lack of survival signalling. Here, we show that NGF can rescue PC12 cells from both growth factor withdrawal- and TNFalpha-promoted cell death. However, NGF rescue from growth factor withdrawal requires NGF signalling through the high-affinity tyrosine kinase receptor (TrkA), while NGF rescue from TNFalpha-promoted cell death requires NGF signalling through the low-affinity p75NTR receptor. These results strengthen the idea that prevention of age- or pathology-associated neurodegeneration may require varied molecular approaches reflecting the diversity of the toxicants involved, possibly acting simultaneously.

Nerve growth factor: structure, function and therapeutic implications for Alzheimer's disease.

Over the past decade, neurotrophic factors have generated much excitement for their potential as therapy for neurological disorders. In this regard, nerve growth factor (NGF), the founding member of the neurotrophin family, has generated great interest as a potential target for the treatment of Alzheimer's disease (AD). This interest is based on the observation that cholinergic basal forebrain (CBF) neurons which provide the major source of cholinergic innervation to the cerebral cortex and hippocampus undergo selective and severe degeneration in advanced AD and that these neurons are dependent upon NGF and its receptors for their survival. In fact, NGF transduces its effects by binding two classes of cell surface receptors, TrkA and p75(NTR), both of which are produced by CBF neurons. This review focuses on NGF/receptor binding, signal transduction, regulation of specific cellular endpoints, and the potential use of NGF in AD. Alterations in NGF ligand and receptor expression at different stages of AD are summarized. Recent results suggest that cognitive deficits in early AD and mild cognitive impairment (MCI) are not associated with a cholinergic deficit. Thus, the earliest cognitive deficits in AD may involve brain changes other than simply cholinergic system dysfunction. Recent findings indicate an early defect in NGF receptor expression in CBF neurons; therefore treatments aimed at facilitating NGF actions may prove highly beneficial in counteracting the cholinergic dysfunction found in end-stage AD and attenuating the rate of degeneration of these cholinergic neurons.

During glaucoma, alpha2-macroglobulin accumulates in aqueous humor and binds to nerve growth factor, neutralizing neuroprotection.

PURPOSE: Glaucoma is an optic neuropathy caused by the chronic and progressive death of retinal ganglion cells (RGCs), resulting in irreversible blindness. Ocular hypertension is a major risk factor, but RGC death often continues after ocular hypertension is normalized, and can take place with normal tension. Continuous RGC death was related in rodents and humans to the local upregulation of neurotoxic proteins, such as TNF-α. In rat models of glaucoma, ocular hypertension also upregulates the expression of α2-macroglobulin, which is neurotoxic. α2-macroglobulin upregulation in the retina is long-lived, even after high IOP is reduced with medication. α2-macroglobulin is examined as a possible biomarker in human glaucoma, and a possible neurotoxic mechanism of action is sought. METHODS: Quantitative Western blotting of α2-macroglobulin in samples obtained from aqueous humor (human and rat) and retina (rat) was conducted. Ex vivo neuronal survival assays and nerve growth factor-α2-macroglobulin binding studies using surface plasmon resonance were used. RESULTS: Increased soluble α2-macroglobulin protein is also present in the aqueous humor in a rat glaucoma model, as well as in the aqueous humor of human glaucoma patients but not in cataract patients. One mechanism by which α2-macroglobulin is neurotoxic is by inhibiting the neuroprotective activity of nerve growth factor via TrkA receptors. CONCLUSIONS: This work further documents a potential novel mechanism of RGC death and a potential biomarker or therapeutic target for glaucoma.

ProNGF mediates death of Natural Killer cells through activation of the p75NTR-sortilin complex.

The common neurotrophin receptor P75NTR, its co-receptor sortilin and ligand proNGF, have not previously been investigated in Natural Killer (NK) cell function. We found freshly isolated NK cells express sortilin but not significant amounts of P75NTR unless exposed to interleukin-12 (IL-12), or cultured in serum free conditions, suggesting this receptor is sequestered. A second messenger associated with p75NTR, neurotrophin-receptor-interacting-MAGE-homologue (NRAGE) was identified in NK cells. Cleavage resistant proNGF123 killed NK cells in the presence of IL-12 after 20h and without IL-12 in serum free conditions at 48h. This was reduced by blocking sortilin with neurotensin. We conclude that proNGF induced apoptosis of NK cells may have important implications for limiting the innate immune response.

Proneurotrophins require endocytosis and intracellular proteolysis to induce TrkA activation.

The uncleaved, pro-form of nerve growth factor (proNGF) functions as a pro-apoptotic ligand for the p75 neurotrophin receptor (p75NTR). However, some reports have indicated that proneurotrophins bind and activate Trk receptors. In this study, we have examined proneurotrophin receptor binding and activation properties in an attempt to reconcile these findings. We show that proNGF readily binds p75NTR expressed in HEK293T cells but does not interact with TrkA expressed under similar circumstances. Importantly, proNGF activates TrkA tyrosine phosphorylation, induces Erk and Akt activation, and causes PC12 cell differentiation. We show that inhibiting endocytosis or furin activity reduced TrkA activation induced by proNGF but not that induced by mature NGF and that proNGF123, a mutant form of NGF lacking dibasic cleavage sites in the prodomain, does not induce TrkA phosphorylation in PC12 cells. Therefore, endocytosis and cleavage appear to be prerequisites for proNGF-induced TrkA activity. We also found that proBDNF induces activation of TrkB in cerebellar granule neurons and that proBDNF cleavage by furin and metalloproteases facilitates this effect. Taken together, these data indicate that under physiological conditions, proneurotrophins do not directly bind or activate Trk receptors. However, endocytosis and cleavage of proneurotrophins produce processed forms of neurotrophins that are capable of inducing Trk activation.

TrkA receptor "hot spots" for binding of NT-3 as a heterologous ligand.

Neurotrophins signal via Trk tyrosine kinase receptors. Nerve growth factor (NGF) is the cognate ligand for TrkA, the brain-derived neurotrophic factor for TrkB, and NT-3 for TrkC. NT-3 also binds TrkA as a lower affinity heterologous ligand. Because neurotrophin-3 (NT-3) interactions with TrkA are biologically relevant, we aimed to define the TrkA "hot spot" functional docking sites of NT-3. The Trk extracellular domain consists of two cysteine-rich subdomains (D1 and D3), flanking a leucine-rich subdomain (D2), and two immunoglobulin-like subdomains IgC1(D4) and IgC2(D5). Previously, the D5 subdomain was defined as the primary ligand-binding site of neurotrophins for their cognate receptors (e.g. NGF binds and activates through TRKA-D5 hot spots). Here binding studies with truncated and chimeric extracellular subdomains show that TRKA-D5 also includes an NT-3 docking and activation hot spot (site 1), and competition studies show that the NGF and NT-3 hot spots on TRKA-D5 are distinct but partially overlapping. In addition, ligand binding studies provide evidence for an NT-3-binding/allosteric site on TRKA-D4 (site 2). NT-3 docking on sites 1 and/or 2 partially blocks NGF binding. Functional survival studies showed that sites 1 and 2 regulate TrkA activation. NT-3 docking on both sites 1 and 2 affords full agonism, which can be additive with NGF activation of Trk. However, NT-3 docking solely on site 1 is partially agonistic but noncompetitively antagonizes NGF binding and activation of Trk. This study demonstrates that Trk signaling is more complex than previously thought because it involves several receptor subdomains and hot spots.

Construction of a mutated pro-nerve growth factor resistant to degradation and suitable for biophysical and cellular utilization.

Precursor of nerve growth factor (proNGF) has been found to be proapoptotic in several cell types and mediates its effects by binding to p75 neurotrophin receptor (p75NTR) and sortilin. The proNGF molecule is processed by proteases at three dibasic sites found in the pro domain to form mature NGF (termed herein as sites 1, 2, and 3 from the proNGF N terminus). Of these processing sites, site 3, adjacent to the N terminus of mature NGF, was thought to be the major site responsible for processing of proNGF to mature NGF. We found that mutating this major processing site (site 3) resulted in a form of proNGF that was only partially stable. On introducing additional mutations in the pro domain at the other two dibasic sites, we found the stability of proNGF to increase significantly. Here we describe the construction, expression, and purification of this more stable proNGF molecule. The two consecutive basic residues at each of the three sites were mutated to neutral alanine residues. Expression was performed in stably transfected Sf21 insect cells. Purification involved strong cation-exchange chromatography and N60 immunoaffinity column chromatography. The construct with all three sites mutated (termed proNGF123) gave all proNGF with no mature NGF and was not cleaved by three proconvertases (furin, PACE-4, and PC-2) known to proteolyze proneurotrophins in vivo. This stable proNGF molecule demonstrated proapoptotic activity on rat pheocytochroma PC12 cells, PC12nnr cells, C6 glioblastoma cells, and RN22 schwannoma cells.

Activation of the mitogen-activated protein kinase pathway through p75NTR: a common mechanism for the neurotrophin family.

Neurotrophins interact with two distinct classes of cell-surface receptors, the Trk receptor tyrosine kinase family and the common neurotrophin receptor p75(NTR). For many years, the biological role of p75(NTR) remained obscure, being relegated to modulating Trk binding of neurotrophins. Recently, the importance of p75(NTR) as a signaling receptor in itself has become increasingly clear. The signals initiated by p75(NTR) are likely to be as complex as those for the Trk family and probably depend on the cell system in which such signaling is being studied. In this study, all members of the neurotrophin family were demonstrated to be capable of stimulating p75(NTR)-mediated activation of the mitogen-activated protein kinase (MAPK) family (ERK1,2). This activation is rapid and transient, peaking at 5-15 min, depending on the cell system. The classical MAPK cascade consists of the reaction series Ras-Raf-MEK-MAPK. The p75(NTR)-induced MAPK activation is MEK dependent but Raf independent. This result implies that neurotrophin activation of p75(NTR) results in some cascade (as yet unknown) that bypasses Raf and converges on MEK to result in activation of MAPK. This activated MAPK is then able to translocate to the nucleus. The effect of this MAPK activation on cell survival is dependent on cell type. These results support the concept that signaling from the p75(NTR) receptor is more diverse and extensive than previously believed.

Biophysical characterization of proteins in the post-genomic era of proteomics.

Proteomics focuses on the high throughput study of the expression, structure, interactions, and, to some extent, function of large numbers of proteins. A true understanding of the functioning of a living cell also requires a quantitative description of the stoichiometry, kinetics, and energetics of each protein complex in a cellular pathway. Classical molecular biophysical studies contribute to understanding of these detailed properties of proteins on a smaller scale than does proteomics in that individual proteins are usually studied. This perspective article deals with the role of biophysical methods in the study of proteins in the proteomic era. Several important physical biochemical methods are discussed briefly and critiqued from the standpoint of information content and data acquisition. The focus is on conformational changes and macromolecular assembly, the utility of dynamic and static structural data, and the necessity to combine experimental approaches to obtain a full functional description. The conclusions are that biophysical information on proteins is a useful adjunct to "standard" proteomic methods, that data can be obtained by high throughput technology in some instances, but that hypothesis-driven experimentation may frequently be required.

Nerve growth factor withdrawal-mediated apoptosis in naive and differentiated PC12 cells through p53/caspase-3-dependent and -independent pathways.

Programmed cell death is regulated in response to a variety of stimuli, including the tumor suppressor protein p53, that can mediate cell cycle arrest through p21/Waf1 and apoptosis through the Bcl-2/Bax equilibrium and caspases. Neuronal cell apoptosis has been reported to require p53, whereas other data suggest that neuronal cell death may be independent of p53. Comparison of wild type PC12 to a temperature-sensitive PC12 cell line that depresses the normal function of p53 has permitted investigation of the importance of p53 in a variety of cell functions. This study examined the role of p53 in trophic factor withdrawal-mediated apoptosis in both naïve and differentiated PC12 cells. Our data show that as PC12 cells differentiate they are more poised to undergo apoptosis than their undifferentiated counterparts. Survival assays with XTT (sodium 3'-1-(phenylaminocarbonyl)-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene sulfonic acid) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) demonstrated that lack of p53 is initially protective against apoptosis. The window of protection is about 20 h for naïve and 36 h for differentiated cells. Apoptosis involved caspases 3, 6, and 9. However, caspase 3 activation was absent in cells lacking p53, concomitant with the delayed apoptosis. When the expression of caspase 3 was silenced with interference RNA, wild type PC12 cells revealed a morphology and biochemistry similar to PC12[p53ts] cells, indicating that caspase 3 accounts for the observed delay in apoptosis in p53 dysfunction. These results suggest that p53 is important, but not essential, in factor withdrawal-mediated apoptosis. Parallel pathways of caspase-mediated apoptosis are activated later in the absence of functional p53.

Individual and combined effects of TrkA and p75NTR nerve growth factor receptors. A role for the high affinity receptor site.

A long-standing question in neurotrophin signal transduction is whether heteromeric TrkA-p75NTR complexes possess signaling capabilities that are significantly different from homo-oligomeric TrkA or p75NTR alone. To address this issue, various combinations of transfected PC12 cells expressing a platelet-derived growth factor receptor-TrkA chimera and the p75NTR-selective nerve growth factor mutant (Delta9/13 NGF) were utilized to selectively stimulate TrkA or p75NTR signaling, respectively. The contribution of individual and combined receptor effects was analyzed in terms of downstream signaling and certain end points. The results suggest two unique functions for the high affinity heteromeric NGF receptor site: (a) integration of both the MAPK and Akt pathways in the production of NGF-induced neurite outgrowth, and (b) rapid and sustained activation of the Akt pathway, with consequent long term cellular survival. Whereas activation of TrkA signaling is sufficient for eliciting neurite outgrowth in PC12 cells, signaling through p75NTR plays a modulatory role, especially in the increased formation of fine, synaptic "bouton-like" structures, in which both TrkA and p75NTR appear to co-localize. In addition, a new interaction in the TrkA/p75NTR heteromeric receptor signal transduction network was revealed, namely that NGF-induced activation of the MAPK pathway appears to inhibit the parallel NGF-induced Akt pathway.