Biological Activity of Nerve Growth Factor Precursor Is Dependent upon Relative Levels of Its Receptors

Department of Biology, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2009; 284(27):18424-33. DOI: 10.1074/jbc.M109.007104
Source: PubMed


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.

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    • "| the final outcome would depend on the relative levels of sortilin , TrkA and p75 NTR receptors ( Nykjaer et al . , 2004 ; Volosin et al . , 2006 , 2008 ; Arnett et al . , 2007 ; Domeniconi et al . , 2007 ; Jansen et al . , 2007 ; Masoudi et al . , 2009 ) . Since the proNGF receptor , p75NTR , acts as a co - receptor for multiple partners , therefore , the presence of different co - receptors would permit the binding of different ligands , which can lead to diverse biological outcomes , such as apoptosis , survival ( Lee et al . , 2001 ; Teng et al . , 2005 ; Schecterson and Bothwell ,"
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    ABSTRACT: Several reports have shown that a sciatic nerve conditioned media (CM) causes neuronal-like differentiation in PC12 cells. This differentiation is featured by neurite outgrowth, which are exclusively dendrites, without axon or sodium current induction. In previous studies, our group reported that the CM supplemented with a generic inhibitor for tyrosine kinase receptors (k252a) enhanced the CM-induced morphological differentiation upregulating neurite outgrowth, axonal formation and sodium current elicitation. Sodium currents were also induced by depletion of endogenous precursor of nerve growth factorr (proNGF) from the CM (pNGFd-CM). Given that sodium currents, neurite outgrowth and axon specification are important features of neuronal differentiation, in the current manuscript, first we investigated if proNGF was hindering the full PC12 cell neuronal-like differentiation. Second, we studied the effects of exogenous wild type (pNGFwt) and mutated (pNGFmut) proNGF isoforms over sodium currents and whether or not their addition to the pNGFd-CM would prevent sodium current elicitation. Third, we investigated if proNGF was exerting its negative regulation through the sortilin receptor, and for this, the proNGF action was blocked with neurotensin (NT), a factor known to compete with proNGF for sortilin. Thereby, here we show that pNGFd-CM enhanced cell differentiation, cell proportion with long neurites, total neurite length, induced axonal formation and sodium current elicitation. Interestingly, treatment of PC12 cells with wild type or mutated proNGF isoforms elicited sodium currents. Supplementing pNGFd-CM with pNGFmut reduced 35% the sodium currents. On the other hand, pNGFd-CM+pNGFwt induced larger sodium currents than pNGFd-CM. Finally, treatments with CM supplemented with NT showed that sortilin was mediating proNGF negative
    Frontiers in Cellular Neuroscience 09/2015; 9(364):11. DOI:10.3389/fncel.2015.00364 · 4.29 Impact Factor
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    • "It has been shown that proNGF can also be 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 for primary sympathetic neurons and for PC12 cells, it is reported to be apoptotic for unprimed PC12 cells [27]. Furthermore, proNGF has been reported to promote apoptosis via its interaction with p75NTR and sortilin receptor [7,28]. "
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    ABSTRACT: Growing evidence shows that, in vivo, the precursor of Nerve Growth Factor (NGF), proNGF, displays biological activities different from those of its mature NGF counterpart, mediated by distinct, and somewhat complementary, receptor binding properties. NGF and proNGF induce distinct transcriptional signatures in target cells, highlighting their different bioactivities. In vivo, proNGF and mature NGF coexist. It was proposed that the relative proNGF/NGF ratio is important for their biological outcomes, especially in pathological conditions, since proNGF, the principal form of NGF in Central Nervous System (CNS), is increased in Alzheimer's disease brains. These observations raise a relevant question: does proNGF, in the presence of NGF, influence the NGF transcriptional response and viceversa? In order to understand the specific proNGF effect on NGF activity, depending on the relative proNGF/NGF concentration, we investigated whether proNGF affects the pattern of well-known NGF-regulated mRNAs. To test any influence of proNGF on pure NGF expression fingerprinting, the expression level of a set of candidate genes was analysed by qReal-Time PCR in rat adrenal pheochromocytoma cell line PC12, treated with a mixture of NGF and proNGF recombinant proteins, in different stoichiometric ratios. These candidates were selected amongst a set of genes well-known as being rapidly induced by NGF treatment. We found that, when PC12 cells are treated with proNGF/NGF mixtures, a unique pattern of gene expression, which does not overlap with that deriving from treatment with either proNGF or NGF alone, is induced. The specific effect is also dependent on the stoichiometric composition of the mixture. The proNGF/NGF equimolar mixture seems to partially neutralize the specific effects of the proNGF or NGF individual treatments, showing a weaker overall response, compared to the individual contributions of NGF and proNGF alone. Using gene expression as a functional read-out, our data demonstrate that the relative availability of NGF and proNGF in vivo might modulate the biological outcome of these ligands.
    BMC Neuroscience 04/2014; 15(1):48. DOI:10.1186/1471-2202-15-48 · 2.67 Impact Factor
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    • "Therefore, it is important to note that overexpression of Praja1 in PC12 cells, in contrast to COS-7 cells, which showed cell rounding, formation of microspikes and pyknotic nuclei (Fig. S3, S5), was not associated with an induction of apoptosis. Praja1, by controlling the level of NRAGE and other intracellular signalling factors [13], may thus contribute to controlling the delicate balance between p75 and TrkA receptor function in differentiating PC12 cells [42]. "
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    ABSTRACT: Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE). We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect.
    PLoS ONE 05/2013; 8(5):e63067. DOI:10.1371/journal.pone.0063067 · 3.23 Impact Factor
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