Glial cell line-derived neurotrophic factor family ligands and their therapeutic potential

Four glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) have been characterized: GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN). These proteins support and restore multiple neuronal populations such as dopaminergic, sensory, motor, hippocampal, basal forebrain, enteric, sympathetic and parasympathetic neurons. Therefore, GFLs attracted significant attention as a potential cure for the diseases caused by neuronal injury and degeneration. Results of multiple experiments indicate that GFLs can alleviate behavioral symptoms and restore affected neurons in animal models of several neurological disorders including, among others, Parkinson’s disease (PD). During the last decade, GDNF protein and NRTN gene therapy have been tested in several clinical trials in patients with PD. Although the results of phase I clinical trials were positive, phase II clinical trials failed to reach primary end-points. Poor pharmacokinetic properties of GFLs (inability to penetrate tissues barriers, high affinity for extracellular matrix, etc.) could contribute to the absence of clear clinical benefits of these proteins for the patients. The purpose of this paper was to review therapeutic potential of GFLs and discuss possibilities to overcome difficulties associated with pharmacokinetic properties and delivery of GFLs to target neurons.     

Neurotrophic regulation of the development and function of the neuromuscular synapses

Recent studies have established that one of the major functions of neurotrophic factors is to regulate synaptic development and plasticity. This owes a great deal to the studies using the neuromuscular junction (NMJ) as a model system. In this review, we summarize the effects of various neurotrophic factors on the development and function of the neuromuscular synapses. We describe experiments addressing the role of neurotrophins, as well as that of other factors (GFLs, TGF-βs, and Wnts). The synaptic effects of neurotrophic factors are divided into two categories: acute effects on synaptic transmission and plasticity occurring within seconds or minutes after cells are exposed to a particular factor, and long-term regulation of synaptic structure and function that takes days to accomplish. We consider the presynaptic effects on the release of the neurotransmitter ACh, as well as the postsynaptic effects on the clustering of ACh receptors. Further studies of the mechanisms underlying these regulatory effects will help us better understand how neurotrophic factors can achieve diverse and synapse-specific modulation in the brain.     

The GDNF Family: From Neurotrophic Factors to Oncogenesis

The structure and in vivofunctions of the glial cell-derived neurotrophic factor (GDNF) family ligands (GFLs) and their high-affinity receptors are considered. These proteins play an important role in the development of the nervous system, morphogenesis of the kidneys, and regulation of spermatogenesis. Tyrosine kinase Ret is a common receptor component for all GFLs. Its role in multiple endocrine neoplasia type 2 (MEN2) is discussed.     

The GDNF family: from neurotrophic factors to oncogenesis

The structure and in vivo functions of the glial cell-derived neurotrophic factor (GDNF) family ligands (GFLs) and their high-affinity receptors are considered. These proteins play an important role in the development of the nervous system, morphogenesis of the kidneys, and in regulation of spermatogenesis. Tyrosine kinase Ret is a receptor component common for all GFLs. Its role in multiple endocrine neoplasia type 2 (MEN2) is discussed.     

The long and short isoforms of Ret function as independent signaling complexes

Ret, the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family ligands (GFLs), is alternatively spliced to yield at least two isoforms, Ret9 and Ret51, which differ only in their C termini. To identify tyrosines in Ret that are autophosphorylation sites in neurons, we generated antibodies specific to phosphorylated Y905Ret, Y1015Ret, Y1062Ret, and Y1096Ret, all of which are autophosphorylated in cell lines. All four of these tyrosines in Ret became phosphorylated rapidly upon activation by GFLs in sympathetic neurons. These tyrosines remained phosphorylated in sympathetic neurons in the continued presence of GFLs, albeit at a lower level than immediately after GFL treatment. Comparison of GFL activation of Ret9 and Ret51 revealed that phosphorylation of Tyr(905) and Tyr(1062) was greater and more sustained in Ret9 as compared with Ret51. In contrast, Tyr(1015) was more highly phosphorylated over time in Ret51 than in Ret9. Surprisingly, Ret9 and Ret51 did not associate with each other in sympathetic neurons after glial cell line-derived neurotrophic factor stimulation, even though they share identical extracellular domains. Furthermore, the signaling complex associated with Ret9 was markedly different from the Ret51-associated signaling complex. Taken together, these data provide a biochemical basis for the dramatic functional differences between Ret9 and Ret 51 in vivo.     

Structural studies of GDNF family ligands with their receptors—Insights into ligand recognition and activation of receptor tyrosine kinase RET

RET is the receptor for glial cell line-derived neurotrophic factor family of ligands (GFLs). It is different from most other members in the receptor tyrosine kinase (RTK) family with the requirement of a co-receptor, GFRα, for ligand recognition and activation. Through the common signal transducer RET, GFLs are crucial for the development and maintenance of distinct sets of central and peripheral neurons, which has led to a series of studies towards understanding the structure, function and signaling mechanisms of GFLs with GFRα and RET receptors. Here I summarize our current understanding of the molecular basis underlying ligand recognition and activation of RET, focusing on the interactions of GFLs with their respective GFRα receptors, the recently determined crystal structure of RET extracellular region and a proposed GFL–GFRα–RET ternary complex model based on extensive structural, biochemical and functional data. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin

Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson's disease. Soluble GFLs bind to a ligand-specific glycosylphosphatidylinositol-anchored coreceptor (GDNF family receptor α) and signal through the receptor tyrosine kinase RET. In this paper, we show that all immobilized matrix-bound GFLs, except persephin, use a fundamentally different receptor. They interact with syndecan-3, a transmembrane heparan sulfate (HS) proteoglycan, by binding to its HS chains with high affinity. GFL-syndecan-3 interaction mediates both cell spreading and neurite outgrowth with the involvement of Src kinase activation. GDNF promotes migration of cortical neurons in a syndecan-3-dependent manner, and in agreement, mice lacking syndecan-3 or GDNF have a reduced number of cortical γ-aminobutyric acid-releasing neurons, suggesting a central role for the two molecules in cortical development. Collectively, syndecan-3 may directly transduce GFL signals or serve as a coreceptor, presenting GFLs to the signaling receptor RET.     

The role of GDNF family ligand signalling in the differentiation of sympathetic and dorsal root ganglion neurons

The diversity of neurons in sympathetic ganglia and dorsal root ganglia (DRG) provides intriguing systems for the analysis of neuronal differentiation. Cell surface receptors for the GDNF family ligands (GFLs) glial cell-line-derived neurotrophic factor (GDNF), neurturin and artemin, are expressed in subpopulations of these neurons prompting the question regarding their involvement in neuronal subtype specification. Mutational analysis in mice has demonstrated the requirement for GFL signalling during embryonic development of cholinergic sympathetic neurons as shown by the loss of expression from the cholinergic gene locus in ganglia from mice deficient for ret, the signal transducing subunit of the GFL receptor complex. Analysis in mutant animals and transgenic mice overexpressing GFLs demonstrates an effect on sensitivity to thermal and mechanical stimuli in DRG neurons correlating at least partially with the altered expression of transient receptor potential ion channels and acid-sensitive cation channels. Persistence of targeted cells in mutant ganglia suggests that the alterations are caused by differentiation effects and not by cell loss. Because of the massive effect of GFLs on neurite outgrowth, it remains to be determined whether GFL signalling acts directly on neuronal specification or indirectly via altered target innervation and access to other growth factors. The data show that GFL signalling is required for the specification of subpopulations of sensory and autonomic neurons. In order to comprehend this process fully, the role of individual GFLs, the transduction of the GFL signals, and the interplay of GFL signalling with other regulatory pathways need to be deciphered.     

Site-specific gene expression and localization of growth factor ligand receptors RET, GFRα1 and GFRα2 in human adult colon

Two of the glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), namely GDNF and neurturin (NRTN), are essential neurotropic factors for enteric nerve cells. Signal transduction is mediated by a receptor complex composed of GDNF family receptor alpha 1 (GFRα1) for GDNF or GFRα2 for NRTN, together with the tyrosine kinase receptor RET (rearranged during transfection). As both factors and their receptors are crucial for enteric neuron survival, we assess the site-specific gene expression of these GFLs and their corresponding receptors in human adult colon. Full-thickness colonic specimens were obtained after partial colectomy for non-obstructing colorectal carcinoma. Samples were processed for immunohistochemistry and co-localization studies. Site-specific gene expression was determined by real-time quantitative polymerase chain reaction in enteric ganglia and in circular and longitudinal muscle harvested by microdissection. Protein expression of the receptors was mainly localized in the myenteric and submucosal plexus. Dual-label immunohistochemistry with PGP 9.5 as a pan-neuronal marker detected immunoreactivity of the receptors in neuronal somata and ganglionic neuropil. RET immunoreactivity co-localized with neuronal GFRα1 and GFRα2 signals. The dominant source of receptor mRNA expression was in myenteric ganglia, whereas both GFLs showed higher expression in smooth muscle layers. The distribution and expression pattern of GDNF and NRTN and their corresponding receptors in the human adult enteric nervous system indicate a role of both GFLs not only in development but also in the maintenance of neurons in adulthood. The data also provide a basis for the assessment of disturbed signaling components of the GDNF and NRTN system in enteric neuropathies underlying disorders of gastrointestinal motility.     

Functional mapping of receptor specificity domains of glial cell line-derived neurotrophic factor (GDNF) family ligands and production of GFRalpha1 RET-specific agonists

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) (GDNF, neurturin, artemin, and persephin) are critical regulators of neurodevelopment and support the survival of midbrain dopaminergic and spinal motor neurons in vitro and in animal disease models making them attractive therapeutic candidates for treatment of neurodegenerative diseases. The GFLs signal through a multicomponent receptor complex comprised of a high affinity binding component (GDNF-family receptor alpha-component (GFRalpha1-GFRalpha4)) and the receptor tyrosine kinase RET. To begin characterization of GFL receptor specificity at the molecular level, we performed comprehensive homologue-scanning mutagenesis of GDNF, the prototypical member of the GFLs. Replacing short segments of GDNF with the homologous segments from persephin (PSPN) (which cannot bind or activate GFRalpha1.RET or GFRalpha2.RET) identified sites along the second finger of GDNF critical for activating the GFRalpha1.RET and GFRalpha2.RET receptor complexes. Furthermore, introduction of these regions from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient for activating GFRalpha1. RET, but additional determinants are required for interaction with the other GFRalphas. This difference in the molecular basis of GFL-GFRalpha specificity allowed the production of GFRalpha1. RET-specific agonists and provides a foundation for understanding of GFL-GFRalpha.RET signaling at the molecular level.     

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.     

Tyrosine 981, a novel ret autophosphorylation site, binds c-Src to mediate neuronal survival

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are neurotrophic factors that influence several aspects of the developing and injured nervous system. GFLs signal through a common receptor tyrosine kinase (Ret) and one of the four ligand-binding co-receptors (GFRalpha1 to 4). Ligand-induced translocation of Ret to lipid rafts, where it interacts with the nonreceptor tyrosine kinase Src, is a prerequisite for full biological activity of these neurotrophic factors. This interaction and subsequent activation of Src are required for GFL-mediated neuronal survival, neurite outgrowth, or cell proliferation. Here we show by multiple approaches that Ret tyrosine 981 constitutes the major binding site of the Src homology 2 domain of Src and therefore the primary residue responsible for Src activation upon Ret engagement. Other tyrosines such as 1015 and 1029 may contribute to the overall interaction between Ret and Src, as judged by overexpression experiments. By generating a phosphospecific antibody, we demonstrate that tyrosine 981 is a novel autophosphorylation site in Ret. Importantly, we also show that this tyrosine becomes phosphorylated in dissociated sympathetic neurons after ligand stimulation. Mutation of tyrosine 981 to phenylalanine reduces GDNF-mediated survival in a transfected cerebellar granule neuron paradigm.     

Molecular evolution of the neurotrophin family members and their Trk receptors

Neurotrophins are structurally related proteins regulating brain development and function. Molecular evolution studies of neurotrophins and their receptors are essential for understanding the mechanisms underlying the coevolution processes of these gene families and how they correlate with the increased complexity of the vertebrate nervous system. In order to improve our current knowledge of the molecular evolution of neurotrophins and receptors, we have collected all information available in the literature and analyzed the genome database for each of them. Statistical analysis of aminoacid and nucleotide sequences of the neurotrophin and Trk family genes was applied to both complete genes and mature sequences, and different phylogenetic methods were used to compare aminoacid and nucleotide sequences variability among the different species. All collected data favor a model in which several rounds of genome duplications might have facilitated the generation of the many different neurotrophins and the acquisition of specific different functions correlated with the increased complexity of the vertebrate nervous system during evolution. We report findings that refine the structure of the evolutionary trees for neurotrophins and Trk receptors families, indicate different rates of evolution for each member of the two families, and newly demonstrate that the NGF-like genes found in Fowlpox and Canarypox viruses are closely related to reptile NGF.     

Sensitization of recombinant vanilloid receptor-1 by various neurotrophic factors

The vanilloid receptor (VR1) is a central integrator molecule of nociceptive stimuli. In this study, we have measured the effects of various neurotrophins (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and -4) on recombinant rat VR1-mediated intracellular calcium rise in response to capsaicin in VR1/C6 cells. Our results clearly show that all neurotrophins sensitize the VR1 to capsaicin. Furthermore, using K252a, an inhibitor of tyrosine kinases, we present that actions of neurotrophins are mediated by the trk (A, B, C) receptors expressed in these cells. These data argue for the putative roles of neurotrophins in inducing inflammatory (thermal) hyperalgesia via VR1.     

The role of neurotrophins during successive stages of sensory neuron development

Neurotrophins comprise a family of basic homodimeric proteins. The isolation of the first two neurotrophins, nerve growth factor and brain-derived neurotrophic factor, was based on the ability of these proteins to promote the survival of embryonic neurons. However, the identification of additional neurotrophins by homology screening together with recent work on these proteins has shown that neurotrophins do more than just regulate neuronal survival. Neurotrophins influence the proliferation and differentiation of neuron progenitor cells and regulate the expression of several differentiated traits of neurons throughout life. Moreover, the influence of neurotrophins on survival is more complex than originally thought; some neurons switch their survival requirements from one set of neurotrophins to another during development and several neurotrophins may be involved in regulating the survival of a population of neurons at any one time. Most of what is known of the developmental physiology of neurotrophins has come from studying neurons of the peripheral nervous system. Quite apart from the accessibility of these neurons and their progenitor cell populations, investigation of the actions of neurotrophins on several well-characterised populations of sensory neurons has permitted the age-related changes in the effects of neurotrophins to be interpreted in the appropriate developmental context. In this review I provide a chronological account of the action of neurotrophins in neuronal development with special reference to sensory neurons.     

From visual experience to visual function: roles of neurotrophins.

Recently, a role for neurotrophins in regulating cortical developmental plasticity has clearly emerged. We present in this review a summary of the early data on the action of nerve growth factor (NGF) in visual cortical development and plasticity in the rat and of other neurotrophins in the visual cortex of other mammals. In addition, to clarify the differences in the results obtained with the various neurotrophins in different animal preparations, we also report new data on the action of NGF, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)3, and NT4 in the same preparation-namely, the visual cortex of the rat. We discuss old and new results in a physiological model in which different neurotrophins play different roles in regulating visual cortical development and plasticity by acting on different neural targets, such as lateral geniculate nucleus (LGN) afferents, intracortical circuitry, and subcortical afferents, and propose a tentative scheme summarizing these actions.     

Regulation of neuropeptide expression in the brain by neurotrophins

Neurotrophins, which are structurally related to nerve growth factor, have been shown to promote survival of various neurons. Recently, we found a novel activity of a neurotrophin in the brain: Brain-derived neurotrophic factor (BDNF) enhances expression of various neuropeptides. The neuropeptide differentiation activity was then compared among neurotrophins both in vivo and in vitro. In cultured neocortical neurons, BDNF and neurotrophin-5 (NT-5) remarkably increased levels of neuropeptide Y and somatostatin, and neurotrophin-3 (NT-3) also increased these peptides but required higher concentrations. At elevating substance P, however, NT-3 was as potent as BDNF. In contrast, NGF had negligible or no effect. Neurotrophins administered into neonatal brain exhibited slightly different potencies for increasing these neuropeptides: The most marked increase in neuropeptide Y levels was obtained in the neocortex by NT-5, whereas in the striatum and hippocampus by BDNF, although all three neurotrophins increased somatostatin similarly in all the brain regions examined. Overall spatial patterns of the neuropeptide induction were similar among the neurotrophins. Neurons in adult rat brain can also react with the neurotrophins and alter neuropeptide expression in a slightly different fashion. Excitatory neuronal activity and hormones are known to change expression of neurotrophins. Therefore, neurotrophins, neuronal activity, and hormones influence each other and all regulate neurotransmitter/peptide expression in developing and mature brain. Physiological implication of the neurotransmitter/peptide differentiation activities is also discussed.     

Neurotrophins improve synaptic transmission in the adult rodent diaphragm

Neurotrophins are usually viewed as secreted proteins that control long-term survival and differentiation of neurons. However, recent studies have established that among the most important functions of neurotrophins is their capacity to regulate synaptic functions and plasticity. When altering synaptic function, neurotrophins are able to produce two types of outcomes, an immediate effect on synaptic transmission and long-term control of synaptic structure and function. The first effect occurs within seconds or minutes after the neurotrophic factor has been applied and usually involves acute modification of synaptic transmission. The second effect takes hours and days, as protein synthesis is required to complete the structural changes. Neurotrophins and their receptors are expressed within the neuromuscular system, making these agents ideal candidates for the short-and long-term regulation of skeletal muscle function. For instance, neurotrophins can alter neuromuscular function acutely, by modulating the amount of neurotransmitter released with each nerve impulse, or chronically, by changing postsynaptic properties or the content and size of synaptic vesicles. It is obvious that the effects of neurotrophins depend on the specific neurotrophin involved (four neurotrophins have been found in mammals; these are nerve growth factor, brain-derived neurotrophic factor, and neurotrophins-3 and-4) and on the specific synapse being studied. Growing evidence highlights the role of neurotrophins in the development and function of neuromuscular synapses. This review will examine the role of neurotrophins in the regulation of neuromuscular transmission. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 327–337, July–October, 2007.     

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.     

Analysis of neurotrophins in human serum by immunoaffinity capillary electrophoresis (ICE) following traumatic head injury

Neurotrophins, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and β-nerve growth factor (β-NGF), play an active role in the development, maintenance and survival of cells of the central nervous system (CNS). Previous research has indicated that a decrease in concentrations of these neurotrophins is often associated with cell death and ultimately patient demise. However, much of the research conducted analyses of samples taken directly from the CNS, i.e., of samples that are not readily available in clinical trauma centers. In an attempt to obtain a method for evaluating neurotrophins in a more readily accessible matrix, i.e., serum, a precise and accurate immunoaffinity capillary electrophoresis (ICE) method was developed and applied to measure neurotrophins in serum from patients with various degrees of head injury. The five neurotrophins of interest were extracted and concentrated by specific immunochemically immobilized antibodies, bound directly to the capillary wall, and eluted and separated in approximately 10 min. NT-3, BDNF, CNTF and β-NGF showed a marked decrease in concentration as the severity of the head injury increased: mild versus severe: 91% decrease for NT-3; 93 % decrease for BDNF; 93 % decrease for CNTF; and a 87 % decrease for β-NGF. This decrease in concentration is consistent with the neuro-protective roles that neurotrophins play in the maintenance and survival of neuronal cells. The results obtained by the ICE method were closely comparable with those generated by a commercially available ELISA method.