Peptides other than the neurotrophins that can be cleaved from proneurotrophins: a neglected story

The members of the family of neurotrophic factors known as neurotrophins, NGF, BDNF, NT-3 and NT4/5 are known to be cleaved intracellularly from immature precursors, the proneurotrophins. NGF and the other neurotrophins regulate neurite outgrowth and neuronal survival during development via binding to Trk receptor tyrosine kinases and the p75 neurotrophin receptor. Surprisingly, the proneurotrophins were shown to be also biologically active ligands. ProNGF and proBDNF induce neuronal apoptosis via binding to a complex of p75 and sortilin. Therefore, life and death seems to be a delicate interplay between 'cleavage' or 'not cleavage' of the proneurotrophins. However, there is a third aspect to this story. In general, peptide-hormone precursors are known to give rise to several biologically active peptides from one precursor molecule. The paradox with the proneurotrophins is that although they have several additional potential cleavage sites that would necessarily give rise to other peptides besides the neurotrophins and thus new members in the neurotrophin family, this aspect has been largely neglected. This article aims to review evidence for biologically active peptides other than the NGF and NT-3 that can be generated from the proNGF and proNT-3.     

Decrease in the levels of NGF and BDNF in brains of mice fed a tryptophan-deficient diet

The roles of dietary tryptophan (Trp) were evaluated in regulation of production of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 in the various brain regions in ddY mice. Feeding the mice a Trp-deficient diet for 2 weeks significantly decreased in the hippocampal level of NGF but not those of BDNF and NT-3, as compared with feeding an adequate Trp diet. The mice fed excess Trp did not have different levels of any of these neurotrophins than in the mice fed an adequate Trp diet. The levels of BDNF in the cerebral cortex were also significantly lower in the mice fed on a Trp-deficient diet, while the levels of NGF and NT-3 in the region were not modulated upon feeding of the diet. The dietary Trp level had no significant effect on the levels of NGF, BDNF, or NT-3 in the entorhinal cortex nor septum of the mice. These results demonstrate that the brain levels of NGF and BDNF are dependent on the dietary content of tryptophan.     

NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression.

To obtain insight into the site and stage specificity of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) action in vivo, we compared the expression patterns of the genes for these three related neurotrophic factors as well as for the NGF receptor in developing and adult rats. Initial embryonic expression of these related neurotrophic factors approximately coincides with the onset of neurogenesis. However, the levels at which the three factors are expressed at this time and throughout the developing nervous system are dramatically different. NT-3 is by far the most highly expressed in immature regions of the CNS in which proliferation, migration, and differentiation of neuronal precursors is ongoing. NT-3 expression dramatically decreases with maturation of these regions. By contrast, BDNF expression is low in developing regions of the CNS and increases as these regions mature. NGF expression varies during the development of discrete CNS regions, but not in any consistent manner compared with NT-3 and BDNF. Despite the dramatic variations, NT-3, BDNF, and NGF do share one striking similarity--high level expression in the adult hippocampus. Our observations are consistent with the idea that NT-3, BDNF, and NGF have paralleled as well as reciprocal roles in vivo.     

Immunohistochemical distribution of NGF, BDNF, NT-3, and NT-4 in adult rhesus monkey brains.

Immunohistochemical distribution and cellular localization of neurotrophins was investigated in adult monkey brains using antisera against nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Western blot analysis showed that each antibody specifically recognized appropriate bands of approximately 14.7 kDa, 14.2 kDa, 13.6 kDa, and 14.5 kDa, for NGF, BDNF, NT-3, and NT-4, respectively. These positions coincided with the molecular masses of the neurotrophins studied. Furthermore, sections exposed to primary antiserum preadsorbed with full-length NGF, BDNF, NT-3, and NT-4 exhibited no detectable immunoreactivity, demonstrating specificities of the antibodies against the tissues prepared from rhesus monkeys. The study provided a systematic report on the distribution of NGF, BDNF, NT-3, and NT-4 in the monkey brain. Varying intensity of immunostaining was observed in the somata and processes of a wide variety of neurons and glial cells in the cerebrum, cerebellum, hippocampus, and other regions of the brain. Neurons in some regions such as the cerebral cortex and the hippocampus, which stained for neurotrophins, also expressed neurotrophic factor mRNA. In some other brain regions, there was discrepancy of protein distribution and mRNA expression reported previously, indicating a retrograde or anterograde action mode of neurotrophins. Results of this study provide a morphological basis for the elucidation of the roles of NGF, BDNF, NT-3, and NT-4 in adult primate brains.     

A common mechanism for recombinant human NGF, BDNF, NT-3, and murine NGF slow unfolding

The recombinant human nerve growth factor (hNGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF) dimers all undergo rapid unfolding and dissociation to monomer in GdnHCl. Fluorescence spectroscopy, reversed-phase high-performance liquid chromatography, and size-exclusion chromatography were used to show that this monomer M1 converts slowly to a more fully unfolded monomer, M2, by a first order process with half-lives of 22, 2.5, 1.6, and 0.73 h for hNGF, mNGF, NT-3, and BDNF, respectively, at 25 degrees C. Linear Arrhenius plots for the conversion of M1 to M2 yielded activation energies of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT-3, and BDNF, respectively. The refolding of these neurotrophins from 5 M GdnHCl was also first order with NT-3 the slowest to refold and BDNF the fastest. Threading of the N-terminus out through the cystine-knot loop present in each of these proteins is proposed as the slow step in unfolding. The number of amino acids in the cystine-knot loop (14 for hNGF, mNGF, NT-3, and BDNF; 21 for NT4/5), and the number and position of the proline residues in this loop (2 for hNGF; 1 for mNGF, NT-3, BDNF, and NT4/5) correlate with the relative rates of unfolding. The smaller the loop and the greater the number of prolines, the more hindered and slower the unfolding.     

The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons.

The pattern of retrograde axonal transport of the target-derived neurotrophic molecule, nerve growth factor (NGF), correlates with its trophic actions in adult neurons. We have determined that the NGF-related neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are also retrogradely transported by distinct populations of peripheral and central nervous system neurons in the adult. All three 125I-labeled neurotrophins are retrogradely transported to sites previously shown to contain neurotrophin-responsive neurons as assessed in vitro, such as dorsal root ganglion and basal forebrain neurons. The patterns of transport also indicate the existence of neuronal populations that selectively transport NT-3 and/or BDNF, but not NGF, such as spinal cord motor neurons, neurons in the entorhinal cortex, thalamus, and neurons within the hippocampus itself. Our observations suggest that neurotrophins are transported by overlapping as well as distinct populations of neurons when injected into a given target field. Retrograde transport may thus be predictive of neuronal types selectively responsive to either BDNF or NT-3 in the adult, as first demonstrated for NGF.     

Spatiotemporal Changes of NGF,BDNF and NT-3 in the Developing Spinal Cords of Embryonic Chicken

Spatiotemporal changes of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the spinal cords of chick embryonic stage day 7 (E7) and day 14 (E14) were examined by using immunohistochemistry and Western blot. Intensive NGF immunoreaction (IR) was detected in the white matter of the spinal cords, while BDNF-IR in perikaryon and neurite, and NT-3-IR in the nucleus and cytoplasm were seen in the neurons of the ventral horn in the gray matter. Comparatively, the expressions for three growth factors have expanded largely into the dorsal horn at E14, and the level of proteins for these growth factors increased significantly in the spinal cords from E7 to E14. Morphological observation showed that the lumbar spinal cords of E7 appeared rectangular, whereas it gave a butterfly shape in the gray matter consisting of the typical ventral horn, dorsal horn and intermediate zone at E14. The present findings indicated that the spatiotemporal changes of NGF, BDNF and NT-3 could be associated to the morphological changes of developing spinal cords, suggesting the possible roles of three growth factors in the development of spinal cords.     

Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brain-derived neurotrophic factor.

Neurotrophin-3 (NT-3) has low-affinity (Kd = 8 x 10(-10) M), as well as high-affinity receptors (Kd = 1.8 x 10(-11) M) on embryonic chick sensory neurons, the latter in surprisingly high numbers. Like the structurally related proteins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), NT-3 also binds to the low-affinity NGF receptor, a molecule that we suggest to designate low-affinity neurotrophin receptor (LANR). NT-3 dissociates from the LANR much more rapidly than BDNF, and more slowly than NGF. The binding of labelled NT-3 to the LANR can be reduced by half using a concentration of BDNF corresponding to the Kd of BDNF to the LANR. In contrast, the binding of NT-3 to its high-affinity neuronal receptors can only be prevented by BDNF or NGF when used at concentrations several thousand-fold higher than those corresponding to their Kd to their high-affinity neuronal receptors. Thus, specific high-affinity NT-3 receptors exist on sensory neurons that can readily discriminate between three structurally related ligands. These findings, including the remarkable property of the LANR to bind three related ligands with similar affinity, but different rate constants, are discussed.     

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.     

Molecular Evolution of Three Avian Neurotrophin Genes: Implications for Proregion Functional Constraints

Neurotrophin proteins are essential for the survival, differentiation, and maintenance of neurons in the peripheral and central nervous systems. Recent studies have shown that the unprocessed proforms of the neurotrophins are preferential high-affinity ligands for p75^NTR and potent inducers of p75^NTR-mediated cell death. Here, we explore differences in the selective constraints acting on the proregions of the three avian neurotrophin genes—NT-3, BDNF, and NGF—in an explicit phylogenetic context. We found a 50-fold difference in levels of constraint as estimated by d _N/d _S ratios, with the NGF proregion showing the lowest degree of constraint and BDNF the highest. These patterns suggest that the high conservation exhibited by the BDNF proregion results from intense functional constraints that are relaxed in NGF and somewhat relaxed in NT-3. The proregion of BDNF is likely to have a function that differentiates it from the corresponding regions of the NGF and NT-3 genes, suggesting that BDNF is the avian neurotrophin most likely to be used both in its precursor and mature forms in vivo.     

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.     

神经营养因子与神经干细胞

生长因子在神经干细胞的增殖,分化和存活过程中有重要作用。神经营养因子是其中的一类,它包括神经生长因子（NGF）家族,胶质源性神经营养因子（GDNF）家族和其它神经营养因子。NGF家族包括NGF,BDNF,NT－3,NT－4／5和NT－6。这一家族可促进epidermic growth facter(EGF)反应 海马及前脑室管膜下区神经干细胞的存活和分化。GDNF家族包括GDNF,NTN,PSP和ART。GDNF家族促神经发育的作用主要在外周,它促进肠神经嵴前体细胞的存活和增殖,且对外周感觉神经的发育至关重要。其它生长因子如bFGF和EGF,它们能促进神经干细胞增殖和存活;CNTF和LIF等在神经干细胞的分化中也有重要作用。     

NT-3 modulates BDNF and proBDNF levels in naïve and kindled rat hippocampus

Both mature and precursor forms of neurotrophins regulate nerve development, survival and plasticity. Brain-derived neurotrophic factor (BDNF) synthesis and secretion in turn are regulated by neuronal activity, such as epilepsy. Further, neurotrophins themselves are regulated by neurotrophin levels. Neurotrophin-3 (NT-3) and BDNF in particular can be co-expressed and each can regulate the levels of the other. This regulation is thought to be mediated through receptor tyrosine kinase (Trk) activity. It is not known whether this neurotrophin-neurotrophin interaction occurs in hippocampal tissue in vivo, or how it is influenced by neuronal activation. In this study, we explored the reciprocal influences of intraventricular infusions of NT-3 and BDNF in na?ve and kindled hippocampi of rats using Western blotting. We confirm that hippocampal kindling resulted in a significant increase in levels of BDNF both in cytochrome C (control) infused and NT-3 infused kindled rats. However, NT-3 infusion significantly reduced BDNF levels in both kindled and non-kindled hippocampi compared to their cytochrome C infused counterparts. These results are consistent with our earlier studies demonstrating lowered levels of TrkA and TrkC (NGF modulates BDNF levels via TrkA) following chronic NT-3 infusion. Although kindling led to an increase in BDNF, this was not accompanied by any detectable change in the levels of proBDNF. However, there was a significant increase in proBDNF following NT-3 infusions, suggesting NT-3 may reduce proBDNF processing. In contrast, neither NT-3 nor proNT-3 levels were affected by kindling or chronic BDNF infusions, consistent with down-regulation of TrkB by chronic BDNF infusion. Thus, modulation of BDNF by NT-3, likely mediated by Trk receptors, occurs in na?ve and kindled adult rat hippocampus.     

Expression of mRNAs for Neurotrophins (NGF,BDNF, and NT-3) and their Receptors (p75NGFR,Trk, TrkB,and TrkC) in Human Peripheral Neuropathies

The steady-state mRNA levels of NGF, BDNF and NT-3, and the mRNA levels of their receptors p75^NGFR, trk, trk,B and trkC were examined in various human peripheral neuropathies, to determine the correlation with myelinated fiber pathology and T cell and macrophage invasions in the diseased nerves. Steady state levels of p75^NGFR mRNAs were significantly elevated in nerves with axonal pathology. In contrast, steady state levels of trkB and trkC mRNA levels were diminished, trk mRNA was not detected in the human nerves. The NGF, BDNF, and NT-3 mRNA levels were elevated in the diseased nerves. The increase in BDNF and NT-3 mRNA levels were proportional to the extent of invasion of the nerves by T cells and macrophages, but did not directly correlate with axonal nor demyelinating pathology, thus suggesting that inflammatory cell invasions are involved in the regulation of BDNF and NT-3 mRNA expressions. These neurotrophin and their receptor gene expressions in the diseased human nerves would be regulated by an underlying pathology-related process, and could play a role in peripheral nerve repair.     

The interaction of neurotrophins with the p75NTR common neurotrophin receptor: a comprehensive molecular modeling study

Neurotrophins are a family of proteins with pleiotropic effects mediated by two distinct receptor types, namely the Trk family, and the common neurotrophin receptor p75NTR. Binding of four mammalian neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), to p75NTR is studied by molecular modeling based on X-ray structures of the neurotrophins and the extracellular domain of p55TNFR, a homologue of p75NTR. The model of neurotrophin/receptor interactions suggests that the receptor binding domains of neurotrophins (loops I and IV) are geometrically and electrostatically complementary to a putative binding site of p75NTR, formed by the second and part of the third cysteine-rich domains. Geometric match of neurotrophin/receptor binding domains in the complexes, as characterized by shape complementarity statistic Sc, is comparable to known protein/protein complexes. All charged residues within the loops I and IV of the neurotrophins, previously determined as being critical for p75NTR binding, directly participate in receptor binding in the framework of the model. Principal residues of the binding site of p75NTR include Asp47, Lys56, Asp75, Asp76, Asp88, and Glu89. The additional involvement of Arg80 and Glu53 is specific for NGF and BDNF, respectively, and Glu73 participates in binding with NT-3 and NT-4/5. Neurotrophins are likely to induce similar, but not identical, conformational changes within the p75NTR binding site.     

Brain-derived neurotrophic factor is an anterograde survival factor in the rat visual system

BACKGROUND: The neurotrophins, which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5 and NT-6, are a family of proteins that play fundamental roles in the differentiation, survival and maintenance of peripheral and central neurons. Much research has focused on the role of neurotrophins as target-derived, retrogradely transported trophic molecules. Although there is recent evidence that BDNF and NT-3 can be transported in an anterograde direction along peripheral and central axons, there is as yet no conclusive evidence that these anterograde factors have direct post-synaptic actions. RESULTS: We report that BDNF travels in an anterograde direction along the optic nerve. The anterogradely transported BDNF had rapid effects on retinal target neurons in the superior colliculus and lateral geniculate nucleus of the brain. When endogenous BDNF within the developing superior colliculus was neutralised, the rate of programmed neuronal death increased. Conversely, provision of an afferent supply of BDNF prevented the degeneration of geniculate neurons after removal of their cortical target. CONCLUSIONS: BDNF released from retinal ganglion cells acts as a survival factor for post-synaptic neurons in retinal target fields.     

Nerve growth factor antibodies recognize neurotrophin-3

The immunological properties of the neurotrophins NGF, BDNF, and NT-3 were compared using polyclonal and monoclonal antibodies against the subunit of mouse NGF. Affinity-purified anti-NGF IgG consistently recognized NGF and NT-3 on Western blots, and inhibited the trophic activity of NGF and NT-3 but not BDNF. In contrast, anti-NGF monoclonal antibodies did not block the trophic activity of either NT-3 or BDNF. These results are consistent with the greater structural overlap between NGF and NT-3 than between NGF and BDNF.     

The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3.

trkB is a tyrosine protein kinase gene highly related to trk, a proto-oncogene that encodes a receptor for nerve growth factor (NGF) and neurotrophin-3 (NT-3). trkB expression is confined to structures of the central and peripheral nervous systems, suggesting it also encodes a receptor for neurotrophic factors. Here we show that brain-derived neurotrophic factor (BDNF) and NT-3, but not NGF, can induce rapid phosphorylation on tyrosine of gp145trkB, one of the receptors encoded by trkB. BDNF and NT-3 can induce DNA synthesis in quiescent NIH 3T3 cells that express gp145trkB. Cotransfection of plasmids encoding gp145trkB and BDNF or NT-3 leads to transformation of recipient NIH 3T3 cells. In these assays, BDNF elicits a response at least two orders of magnitude higher than NT-3. Finally, 125I-NT-3 binds to NIH 3T3 cells expressing gp145trkB; binding can be competed by NT-3 and BDNF but not by NGF. These findings indicate that gp145trkB may function as a neurotrophic receptor for BDNF and NT-3.     

Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve

The neurotrophin family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Previous studies have demonstrated that expression of NGF and its low-affinity receptor is induced in nonneuronal cells of the distal segment of the transected sciatic nerve suggesting a role for NGF during axonal regeneration (Johnson, E. M., M. Taniuchi, and P. S. DeStefano. 1988. Trends Neurosci. 11:299-304). To assess the role of the other neurotrophins and the members of the family of Trk signaling neurotrophin receptors, we have here quantified the levels of mRNAs for BDNF, NT-3, and NT-4 as well as mRNAs for trkA, trkB, and trkC at different times after transection of the sciatic nerve in adult rats. A marked increase of BDNF and NT-4 mRNAs in the distal segment of the sciatic nerve was seen 2 wk after the lesion. The increase in BDNF mRNA was mediated by a selective activation of the BDNF exon IV promoter and adrenalectomy attenuated this increase by 50%. NT-3 mRNA, on the other hand, decreased shortly after the transection but returned to control levels 2 wk later. In Schwann cells ensheathing the sciatic nerve, only trkB mRNA encoding truncated TrkB receptors was detected with reduced levels in the distal part of the lesioned nerve. Similar results were seen using a probe that detects all forms of trkC mRNA. In the denervated gastrocnemius muscle, the level of BDNF mRNA increased, NT-3 mRNA did not change, while NT-4 mRNA decreased. In the spinal cord, only small changes were seen in the levels of neutrophin and trk mRNAs. These results show that expression of mRNAs for neurotrophins and their Trk receptors is differentially regulated after a peripheral nerve injury. Based on these results a model is presented for how the different neurotrophins could cooperate to promote regeneration of injured peripheral nerves.