Two conserved domains in the NGF propeptide are necessary and sufficient for the biosynthesis of correctly processed and biologically active NGF.

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.     

The nerve growth factor family

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are small, basic, secretory proteins that allow the survival of specific neuronal populations. In their biologically active form, after cleavage from their biosynthetic precursors, these three neurotrophic proteins, or neurotrophins, show about 50% amino acid identities. The genes coding for the neurotrophins are not only expressed during development, but also in the adult, in a variety of tissues including the central nervous system. In the adult brain, the hippocampal formation is the site of highest expression of the three neurotrophin genes. These genes are expressed in neurons, and the mRNA levels of two of them (NGF and BDNF) have been shown to be regulated by neurotransmitters. There are also convincing indications that the administration of NGF prevents the atrophy and death of axotomized cholinergic neurons in the adult central nervous system, and improves the performance of rats selected for their poor memory retention in simple behavioral tasks.     

Development of trophic interactions in the vertebrate peripheral nervous system

During embryogenesis, the neurons of vertebrate sympathetic and sensory ganglia become dependent on neurotrophic factors, derived from their targets, for survival and maintenance of differentiated functions. Many of these interactions are mediated by the neurotrophins NGF, BDNF, and NT3 and the receptor tyrosine kinases encoded by genes of thetrk family. Both sympathetic and sensory neurons undergo developmental changes in their responsiveness to NGF, the first neurotrophin to be identified and characterized. Subpopulations of sensory neurons do not require NGF for survival, but respond instead to BDNF or NT3 with enhanced survival. In addition to their classic effects on neuron survival, neurotrophins influence the differentiation and proliferation of neural crest-derived neuronal precursors. In both sympathetic and sensory systems, production of neurotrophins by target cells and expression of neurotrophin receptors by neurons are correlated temporally and spatially with innervation patterns. In vitro, embryonic sympathetic neurons require exposure to environmental cues, such as basic FGF and retinoic acid to acquire neurotrophin-responsiveness; in contrast, embryonic sensory neurons acquire neurotrophin-responsiveness on schedule in the absence of these molecules.     

Activin A Binds to Perlecan through Its Pro-region That Has Heparin/Heparan Sulfate Binding Activity

Activin A, a member of the transforming growth factor-β family, plays important roles in hormonal homeostasis and embryogenesis. In this study, we produced recombinant human activin A and examined its abilities to bind to extracellular matrix proteins. Recombinant activin A expressed in 293-F cells was purified as complexes of mature dimeric activin A with its pro-region. Among a panel of extracellular matrix proteins tested, recombinant activin A bound to perlecan and agrin, but not to laminins, nidogens, collagens I and IV, fibronectin, and nephronectin. The binding of recombinant activin A to perlecan was inhibited by heparin and high concentrations of NaCl and abolished by heparitinase treatment of perlecan, suggesting that activin A binds to the heparan sulfate chains of perlecan. In support of this possibility, recombinant activin A was capable of directly binding to heparin and heparan sulfate chains. Site-directed mutagenesis of recombinant activin A revealed that clusters of basic amino acid residues, Lys²⁵⁹-Lys²⁶³ and Lys²⁷⁰-Lys²⁷², in the pro-region were required for binding to perlecan. Interestingly, deletion of the peptide segment Lys²⁵⁹-Gly²⁷⁷ containing both basic amino acid clusters from the pro-region did not impair the activity of activin A to stimulate Smad-dependent gene expressions, although it completely ablated the perlecan-binding activity. The binding of activin A to basement membrane heparan sulfate proteoglycans through the basic residues in the pro-region was further confirmed by in situ activin A overlay assays using frozen tissue sections. Taken together, the present results indicate that activin A binds to heparan sulfate proteoglycans through its pro-region and thereby regulates its localization within tissues.     

The expression of neurotrophins and their receptors in the prenatal and adult human testis: evidence for functions in Leydig cells

Previous studies have demonstrated local functions for neurotrophins in the developing and mature testis of rodents. To examine whether these signaling molecules are present and also potentially active in the human testis, we characterized immunohistochemically the expression and cellular localization of the known neurotrophins and their receptors during prenatal testicular development as well as in the adult human testis. Results obtained revealed the presence of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 and 4, as well as neurotrophin receptors p75^NTR, TrkA, TrkB, and TrkC during testis morphogenesis. These proteins were also detectable in the adult human testis, and their local expression could be confirmed largely by immunoblot and RT-PCR analyses. Remarkably, the Leydig cells were found to represent the predominant neurotrophin/receptor expression sites within both fetal and adult human testes. Functional assays performed with a mouse tumor Leydig cell line revealed that NGF exposure increases cellular steroid production, indicating a role in differentiation processes. These findings support previously-recognized neuronal characteristics of Leydig cells, provide additional evidence for potential roles of neurotrophins during testis morphogenesis and in the mature testis, and demonstrate for the first time a neurotrophin-induced functional activity in Leydig cells.     

Site-Specific Labeling of Neurotrophins and Their Receptors via Short and Versatile Peptide Tags

We present a toolbox for the study of molecular interactions occurring between NGF and its receptors. By means of a suitable insertional mutagenesis method we show the insertion of an 8 amino acid tag (A4) into the sequence of NGF and of 12 amino acid tags (A1 and S6) into the sequence of TrkA and P75NTR NGF-receptors. These tags are shortened versions of the acyl and peptidyl carrier proteins; they are here covalently conjugated to the biotin-substituted arm of a coenzyme A (coA) substrate by phosphopantetheinyl transferase enzymes (PPTases). We demonstrate site-specific biotinylation of the purified recombinant tagged neurotrophin, in both the immature proNGF and mature NGF forms. The resulting tagged NGF is fully functional: it can signal and promote PC12 cells differentiation similarly to recombinant wild-type NGF. Furthermore, we show that the insertion of A1 and S6 tags into human TrkA and P75NTR sequences leads to the site-specific biotinylation of these receptors at the cell surface of living cells. Crucially, the two tags are labeled selectively by two different PPTases: this is exploited to reach orthogonal fluorolabeling of the two receptors co-expressed at low density in living cells. We describe the protocols to obtain the enzymatic, site-specific biotinylation of neurotrophins and their receptors as an alternative to their chemical, nonspecific biotinylation. The present strategy has three main advantages: i) it yields precise control of stoichiometry and site of biotin conjugation; ii) the tags used can be functionalized with virtually any small probe that can be carried by coA substrates, besides (and in addition to) biotin; iii) above all it makes possible to image and track interacting molecules at the single-molecule level in living systems.     

The autocatalytic processing of the subtilisin Carlsberg pro-region is independent of the primary structure of the cleavage site

Subtilisins are extracellular seryl-proteases produced by bacilli (Markland and Emil, 1971). In addition to signal sequences, these proteases have N-terminal extensions (pro-regions) which have also been identified in several other proteases (Silen et al., 1988; Vasantha et al., 1984; Polhner et al., 1987; Henderson et al., 1987; Yanagida et al., 1986; Takagi et al., 1985). The pro-region holds the pro-protease associated with the membrane and release of the protease takes place as a result of pro-region removal by autocatalytic processing (Egnell and Flock, 1991). In this report we describe the construction of four deletion-mutations in the gene encoding subtilisin Carlsberg at the junction between the pro-region and mature subtilisin Carlsberg. We found that the introduction of different deletions abolished the ability of subtilisin to undergo autocatalytic cleavage of the pro-region in cis, whereas cleavage by exogenous subtilisin could still occur in trans. Point mutations were also introduced in positions -5 to +4 around the pro-region and native subtilisin cleavage site. Processing of pro-subtilisin with the point mutations showed that the autocatalytic cleavage and recognition of this junction of the subtilisin Carlsberg pro-region is independent of the amino acid sequence around the cleavage site.     

Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.

In eukaryotes small secreted peptides are often proteolytically cleaved from larger precursors. In Saccharomyces cerevisiae multiple proteolytic processing steps are required for production of mature 12-amino-acid a-factor from its 36-amino-acid precursor. This study provides additional genetic data supporting a direct role for Afc1p in cleavage of the carboxyl-terminal tripeptide from the CAAX motif of the prenylated a-factor precursor. In addition, Afc1p had a second role in a-factor processing that was independent of, and in addition to, its role in the carboxyl-terminal processing in vivo. Using ubiquitin-a-factor fusions we confirmed that the pro-region of the a-factor precursor was not required for production of the mature pheromone. However, the pro-region of the a-factor precursor contributed quantitatively to a-factor production.     

Distribution and localization of pro-brain-derived neurotrophic factor-like immunoreactivity in the peripheral and central nervous system of the adult rat

The precursors for neurotrophins are proteolytically cleaved to form biologically active mature molecules which activate their receptors p75NTR and trks. A recent study showed that the precursor for nerve growth factor (NGF) can bind to p75NTR with a high affinity and induces apoptosis of neurons in vitro. Mutation in Val66Met of brain-derived neurotrophic factor (BDNF) results in reduction in hippocampal function in learning and in the dysfunction of intracellular BDNF sorting and secretion. To examine the functions of pro-neurotrophins in vivo, it is essential to know where they are expressed in the nervous system. In the present study, we have raised and characterized rabbit polyclonal antibodies against a peptide coding for the precursor region of the BDNF gene. The antibody specifically recognizes the precursor for BDNF by western blot. With the affinity purified precursor antibody, we have mapped the distribution and localization of the precursor for BDNF. The results showed that, like mature BDNF, pro-BDNF is localized to nerve terminals in the superficial layers of dorsal horn, trigeminal nuclei, nuclei tractus solitarius, amygdaloid complex, hippocampus, hypothalamus and some peripheral tissues. These results suggest that pro-BDNF, like mature BDNF, is anterogradely transported to nerve terminals and may have important functions in synaptic transmission in the spinal cord and brain.     

The unprocessed C-terminal dipeptide of recombinant beta-nerve growth factor determines three stable forms with distinct biological activities.

The processing of polypeptide neurotrophins in the nervous system is poorly understood. In this paper, we provide information on the effects of C-terminal processing of nerve growth factor. Three forms of recombinant mouse beta-nerve growth factor (rNGF) were produced and isolated from insect cells infected with a recombinant baculovirus. The three purified forms of rNGF exhibited distinct biological activities and differed in their abilities to compete with high affinity binding of mouse beta-nerve growth factor (mNGF). However, they were chemically and structurally indistinguishable from each other. All three forms of rNGF differed from mature mNGF from mouse submaxillary gland in that the C-terminal Arg-Gly dipeptide had not been proteolytically removed. Removal of the C-terminal dipeptide by gamma-NGF peptidase treatment converted the three forms into a single form identical with mature mNGF. The above results demonstrate that a single polypeptide of rNGF, due to the presence of a C-terminal dipeptide, exhibits three stable dimeric protein conformations with distinct biological activities. The apparent lack of gamma-NGF peptidase in the nervous system raises the possibility that the biologically significant form of NGF may differ from mature mNGF; such a difference may be of physiological relevance.     

Monocytes of allergics and non-allergics produce, store and release the neurotrophins NGF, BDNF and NT-3

INTRODUCTION: Recent studies have shown that neurotrophins (NTs) are involved in inflammatory processes. Elevated plasma levels of NTs were found allergic diseases with the highest levels in allergic asthma. However, the exact cellular sources involved in the regulation and release of neurotrophins in allergic inflammation are still not well defined. OBJECTIVE: The aim of this study was to assess whether monocytes of allergic and non-allergic subjects produce, store and release the neurotrophins NGF, BDNF and NT-3. METHODS: Monocytes of allergic and non-allergic donors were purified by immunomagnetic selection. APAAP-staining for the presence of NTs and their receptors was performed. RT-PCR and Western blot evaluated the production and storage of NTs. Monocytes were incubated and supernatants were collected for measurement of neurotrophic factors after stimulation with lipopolysaccharide (LPS) as inflammatory stimulus. The neurotrophin content in lysates and cell culture supernatants was determined by ELISA. RESULTS: Human monocytes express the neurotrophins NGF, BDNF and NT-3 but also their specific receptors TrkA, TrkB and TrkC. RT-PCR amplification of isolated mRNA demonstrated expression of the examined neurotrophins. Proteins were detectable by Western blot. NTs were found in the monocyte lysates and supernatants at different levels in allergic and non-allergic donors. Cell stimulation with LPS leads to release of NGF and NT3. CONCLUSIONS: Monocytes, produce, store and release NGF, BDNF and NT-3. They are a possible source of elevated neurotrophin levels found in allergy and asthma.     

Activity-dependent and hormonal regulation of neurotrophin mRNA levels in brain–implications for neuronal plasticity

The neurotrophins exhibit neurotrophic effects on specific, partially overlapping populations of neurons both in the peripheral and the central nervous system (CNS). In the periphery, they are synthesized by a variety of nonneuronal cells, and their synthesis seems to be independent of the neuronal input. In contrast, in the CNS all neurotrophins are expressed under physiological conditions primarily by neurons. The production of NGF and BDNF is controlled by neuronal activity: up-regulation by glutamate and acetylcholine, down-regulation by gamma-aminobutyric acid. In contrast, NT-3 regulation is independent of neuronal activity, but it is up-regulated by thyroid hormones and BDNF. The latter observation suggests that NT-3 might be controlled indirectly by neuronal activity via BDNF. In peripheral nonneuronal tissues, glucocorticoid hormones down-regulate NGF mRNA levels both in vitro and in vivo. In contrast, in the CNS, neuronal production of NGF is enhanced by glucocorticoids. The rapid regulation of NGF and BDNF by subtle physiological stimuli together with the recent demonstration that the neurotrophin release neurotransmitters such as acetylcholine opens up interesting perspectives for the function of neurotrophins as mediators of neuronal plasticity. 1994 John Wiley & Sons, Inc.     

An immunoglobulin-like domain determines the specificity of neurotrophin receptors.

The neurotrophins influence survival and maintenance of vertebrate neurons in the embryonic, early post-natal and post-developmental stages of the nervous system. Binding of neurotrophins to receptors encoded by the gene family trk initiates signal transduction into the cell. trkA interacts preferably with nerve growth factor (NGF), trkB with brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) and trkC with neurotrophin-3 (NT-3). By constructing 17 different chimeras and domain deletions of the human trk receptors and analyzing their binding affinities to the neurotrophins we have shown that an immunoglobulin-like domain located adjacent to the transmembrane domain is the structural element that determines the interaction of neurotrophins with their receptors. Chimeras of trkC where this domain was exchanged for the homologous sequences from trkB or trkA gained high affinity binding to BDNF or NGF respectively, while deletion of this domain in trkC or trkA abolished binding to NT-3 or NGF respectively. This domain alone retained affinities to neurotrophins similar to the full-length receptors and when expressed on NIH 3T3 cells in fusion with the kinase domain showed neurotrophin-dependent activation.     

Potential role of NGF,BDNF, and their receptors in oligodendrocytes differentiation from neural stem cell: An in vitro study

Neural stem cells (NSCs) or neuronal progenitor cells are cells capable of differentiating into oligodendrocytes, myelin-forming cells that have the potential of remyelination. Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are two neurotrophic factors that have been studied to stimulate NSC differentiation thus playing a role in multiple sclerosis pathogenesis and several other demyelinating disorders. While several studies have demonstrated the proliferative and protective capabilities of these neurotrophic factors, their cellular and molecular functions are still not well understood. Thus, in the present study, we focus on understanding the role of these neurotrophins (BDNF and NGF) in oligodendrogenesis from NSCs. Both neurotrophic factors have been shown to promote NSC proliferation and NSC differentiation particularly into oligodendroglial lineage in a dose-dependent fashion. Further, to establish the role of these neurotrophins in NSC differentiation, we have employed pharmacological inhibitors for TrkA and TrkB receptors in NSCs. The use of these inhibitors suppressed NSC differentiation into oligodendrocytes along with the downregulation of phosphorylated ERK suggesting active involvement of ERK in the functioning of these neurotrophins. The morphometric analysis also revealed the important role of both neurotrophins in oligodendrocytes development. These findings highlight the importance of neurotrophic factors in stimulating NSC differentiation and may pave a role for future studies to develop neurotrophic factor replacement therapies to achieve remyelination.     

Pro-region of neurotrophins: role in synaptic modulation

Neurotrophins are synthesized first as precursors, followed by maturation through proteolytic removal of the "pro" region. Since pro- and mature neurotrophins elicit opposite functional effects by differential interactions with Trks and p75 receptors, extracellular cleavage represents a new way to control the synaptic functions of neurotrophins. A single nucleotide mutation in the pro-region appears to affect synaptic targeting and activity-dependent secretion of BDNF in hippocampal neurons. These results demonstrate new mechanisms by which neurotrophins regulate synaptic plasticity and memory function.     

Intraperitoneal injection of neuropeptide Y (NPY) alters neurotrophin rat hypothalamic levels: Implications for NPY potential role in stress-related disorders

Neuropeptide Y (NPY) is a 36-amino acid peptide which exerts several regulatory actions within peripheral and central nervous systems. Among NPY actions preclinical and clinical data have suggested that the anxiolytic and antidepressant actions of NPY may be related to its antagonist action on the hypothalamic-pituitary-adrenal (HPA) axis. The neurotrophins brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are proteins involved in the growth, survival and function of neurons. In addition to this, a possible role of neurotrophins, particularly BDNF, in HPA axis hyperactivation has been proposed. To characterize the effect of NPY on the production of neurotrophins in the hypothalamus we exposed young adult rats to NPY intraperitoneal administration for three consecutive days and then evaluated BDNF and NGF synthesis in this brain region. We found that NPY treatment decreased BDNF and increased NGF production in the hypothalamus. Given the role of neurotrophins in the hypothalamus, these findings, although preliminary, provide evidence for a role of NPY as inhibitor of HPA axis and support the idea that NPY might be involved in pathologies characterized by HPA axis dysfunctions.     

Molecular cloning of the gene encoding Vibrio metalloproteinase vimelysin and isolation of a mutant with high stability in organic solvents

Vimelysin is a unique metalloproteinase from Vibrio sp. T1800 exhibiting high activity at low temperature and high stability in organic solvents such as ethanol. A 1,821 bp open reading frame of the vimelysin gene encoded 607 amino acid residues consisting of an N-terminal pro-region, a mature enzyme, and a C-terminal pro-region. The mature enzyme region showed 80%, 57% and 35% sequence identity with the mature forms of vibriolysin from V. vulnificus, pseudolysin from Pseudomonas aeruginosa, and thermolysin from Bacillus thermoproteolyticus, respectively. The catalytic residues and zinc-binding motifs of metalloproteinases are well conserved in vimelysin. The vimelysin gene was expressed in E. coli JM109 cells and the recombinant enzyme was purified as a 38-kDa mature form from cell-free extracts. The purified recombinant enzyme is indistinguishable from the enzyme purified directly from Vibrio. To obtain mutants exhibiting higher stability in organic solvents, random mutations were introduced by error-prone PCR and 600 transformants were screened. The N123D mutant exhibits two times higher stability in organic solvents than the wild-type enzyme. A plausible mechanism for the stability of the N123D mutant in organic solvents was discussed based on homology models of vimelysin and the N123D mutant.     

Effect of NRG1, GDNF,EGF and NGF in the Migration of a Schwann Cell Precursor Line

The Schwann cells are the myelinating glia of the peripheral nervous system that originated during development from the highly motile neural crest. However, we do not know what the guidance signals for the Schwann cell precursors are. Therefore, we set to test some of the known neurotrophins that are expressed early in developing embryos and have been shown to be critical for the survival and patterning of developing glia and neurons. The goal of this study was to determine more specifically if GDNF, NRG1 and NGF are chemoattractants and/or chemokinetic molecules for a Schwann cell precursor line, the Spl201. We performed live chemoattraction assays, with imaging and also presented these molecules as part of their growing substrate. Our results show for the first time that GDNF and NRG1 are potent chemoattractive and chemokinetic molecules for these cells while NGF is a chemokinetic molecule stimulating their motility.