Neurotrophins are a unique family of polypeptide growth factors that influence the proliferation, differentiation, survival and death of neuronal and non-neuronal cells. They are essential for the health and well-being of the nervous system. NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), NT-3 (neurotrophin-3) and NT-4 (neurotrophin-4) also mediate additional higher-order activities, such as learning, memory and behaviour, in addition to their established functions for cell survival. The effects of neurotrophins depend upon their levels of availability, their affinity of binding to transmembrane receptors and the downstream signalling cascades that are stimulated after receptor activation. Alterations in neurotrophin levels have been implicated in neurodegenerative disorders, such as Alzheimer's disease and Huntington's disease, as well as psychiatric disorders, including depression and substance abuse. Difficulties in administering trophic factors have led to the consideration of using small molecules, such as GPCR (G-protein-coupled receptor) ligands, which can participate in transactivation events. In this review, we consider the signalling pathways activated by neurotrophins in both health and disease states.
- nervous system
- neurodegenerative disease
- Trk receptor
The neurotrophins are the best understood trophic factors in the nervous system. Neurotrophins are initially synthesized as precursors or pro-neurotrophins that are cleaved to release the mature active proteins . The mature proteins form stable non-covalent dimers and are normally expressed at very low levels during development. Competition for trophic factors determines the number of surviving neurons during target innervation .
NGF (nerve growth factor) was the first identified neurotrophic factor and displays a restricted target population. In the peripheral nervous system, it acts on sympathetic neurons as well as sensory neurons involved in nociception and temperature sensation. In the CNS (central nervous system), NGF promotes the survival and functioning of cholinergic neurons in the basal forebrain . These neurons project to the hippocampus and are believed to be important for memory processes, which are specifically affected in Alzheimer's disease. The other neurotrophins are more widely expressed in the CNS. BDNF (brain-derived neurotrophic factor) and NT-3 (neurotrophin-3) are highly expressed in cortical and hippocampal structures and have been linked to the survival and functioning of multiple neuronal populations.
Neurotrophins are unique in exerting their cellular effects through the actions of two different receptors, the Trk receptor tyrosine kinase and p75NTR (p75 neurotrophin receptor), a member of the TNF (tumour necrosis factor) receptor superfamily [4,5]. NGF binds most specifically to TrkA, BDNF and NT-4 (neurotrophin-4) to TrkB, and NT-3 to TrkC. p75NTR can bind to each neurotrophin, but has the additional capability of regulating the affinity of a Trk receptor for its cognate ligand. Trk and p75NTR have been referred to as high- and low-affinity receptors respectively; however, this is incorrect nomenclature, since TrkA and TrkB actually bind their ligands with an affinity of 10−9–10−10 M, which is lower than the high-affinity site Kd=10−11 M . In addition, the precursor form of NGF displays high-affinity binding to p75NTR. Trk-mediated responsiveness to low concentrations of NGF is dependent upon the relative levels of p75NTR and TrkA and their combined ability to form high-affinity sites. This is important as the ratio of receptors can determine responsiveness and the number of neurons that survive during development .
Although p75NTR and Trk receptors do not bind to each other directly, there is evidence that complexes form between the two receptors. As a result of these interactions, increased ligand selectivity can be conferred on Trk receptors by p75NTR . One way of generating specificity is by imparting greater discrimination of ligands for Trk receptors. For example, NGF and NT-3 both can bind to TrkA, but p75NTR restricts signalling of TrkA to NGF and not to NT-3. Hence p75NTR and Trk receptors interact in order to provide greater discrimination among different neurotrophins.
Signal transduction through p75 independently gives rise to increases in JNK (c-Jun N-terminal kinase), NF-κB (nuclear factor κB) and ceramide . The p75NTR molecule serves as a pro-apoptotic receptor during developmental cell death and after injury to the nervous system . Cell death triggered by p75 signalling has been observed during conditions of stress, injury or inflammation [9,10]. Pro-neurotrophins are more effective than mature NGF in inducing p75NTR-dependent apoptosis . These results indicate that the biological action of the neurotrophins can be regulated by proteolytic cleavage, with pro-forms preferentially activating p75NTR to mediate apoptosis and mature forms selectively activating Trk receptors to promote survival . Apoptosis by p75NTR is facilitated by binding to pro-neurotrophins and sortilin, a trafficking receptor [11,14].
Neurotrophins may use a death receptor to prune neurons efficiently during periods of developmental cell death. In the event of mis-targeting, neurons may undergo apoptosis if the appropriate trophic factors are not encountered . In this case, a neurotrophin may fail to activate Trk receptors and eliminate cells by an active killing process through p75NTR. Cell death mediated by p75NTR may be important for the refinement of correct target innervation during development. In addition, p75-mediated cell death is associated with inflammation, injury, seizure and nerve lesion.
Neurotrophins ordinarily promote cell survival and differentiation during neural development through engagement of Trk tyrosine kinases . After neurotrophin binding, activated Trk receptors recruit adaptor proteins Shc (Src homology 2-containing protein) and FRS2 (fibroblast growth factor receptor substrate 2), and effectors such as PI3K (phosphoinositide 3-kinase) and PLC-γ (phospolipase C-γ) . The key docking sites on the Trk receptor are Tyr490 (Tyr496 in human TrkA) in the juxtamembrane region and Tyr790 (Tyr791 human TrkA) in the tail of the cytoplasmic domain (Figure 1). PLC-γ binds to Tyr790 and this interaction has been proposed to facilitate interactions with ion channels, such as the VR-1 (vanilloid receptor-1) capsaicin channel. Although Tyr490, Shc or FRS2 become tyrosine phosphorylated and provide a scaffold for other signalling proteins that lead to the activation of the Ras/MAPK (mitogen-activated protein kinase) or PI3K/Akt pathways, this phosphorylation event can have many consequences. During seizure or limbic epileptogenesis, TrkB receptors are induced by specific phosphorylation of the Shc-binding site, Tyr490, in hippocampal neurons .
A distinguishing feature of neurotrophin signalling has been the sustained activation of MAPK activity . Continuous ERK (extracellular-signal-regulated protein kinase) activity occurs for hours after NGF treatment of PC12 cells, whereas EGF (epidermal growth factor) treatment provides a transient activation . The small G-protein Rap1 accounts, at least in part, for the ability of neurotrophins to signal for long time periods by activating the B-Raf and the MEK (MAPK/ERK kinase)/ERK pathway . Recent studies implicate further a critical role of the Rap1/B-Raf pathway in retrograde endosomal signalling [21–23]. The ARMS protein (ankyrin-rich membrane-spanning protein or Kidins 220) serves as a scaffold for Trk-dependent signalling (Figure 1) to promote ERK activity . Association of ARMS protein with Trk receptors generates a complex by which the CrkL–C3G–Rap1 proteins are assembled. Prolonged MAPK activation is facilitated by the formation of this complex. This sustained response is unique to neurotrophin signalling.
NEUROTROPHIN SIGNALLING AND HUMAN DISEASE
In clinical trials for neuropathy and neurodegeneration, neurotrophins have been found to produce acute pain as a side-effect [25,26]. NGF is present at high levels after inflammation and promotes nociceptor sensitization. These responses may well reflect the same process as potentiation of thermal sensitivity by TRPV1 (transient receptor potential vallanoid 1) or related heat-activated ion channels. In NGF-responsive nociceptive sensory neurons, both TrkA and TRPV1 are frequently co-expressed. In other neuronal populations, similar mechanisms may occur to account for the pronounced pain observed when high levels of neurotrophins are administered in animal models or in human clinical trials.
One mechanism to explain the effects of NGF upon hyperalgesia is through the TRPV1 channel, or the capsaicin receptor, a non-selective cation channel that is activated by heat, noxious vanilloid compounds, such as capsaicin, or extracellular protons. . NGF was shown to potentiate the responses of nociceptive sensory neurons to capsaicin . Indeed, NGF produced a profound approx. 30-fold increase in proton (pH 5.5)-evoked currents in Xenopus oocytes co-expressing TrkA and TRPV1. This suggests that cross-talk between capsaicin and NGF exists within sensory neurons. The necessity of TRPV1 channels for NGF-induced thermal hypersensitivity was demonstrated by mice lacking TRPV1. In contrast with NGF-injected normal mice that displayed a marked latency in paw withdrawal in response to a thermal stimulus, injection of NGF in TRPV1-deficient mice did not produce any sensitization .
Signal transduction by TrkA is responsible for the hyperalgesic effects of NGF. Co-immunoprecipitation studies indicated that TRPV1 associated with TrkA and PLC-γ in a complex. Recruitment of PLC-γ to TrkA was essential for NGF-mediated potentiation of channel activity, by decreasing the levels of PIP2 [phosphoinositol (4,5) bisphosphate] that block VR-1 function.
The role of Trk receptors in pain is supported by the expression of TrkA receptors on nociceptive primary afferents and the hyperalgesic effects of exogenously administered NGF in rodents and humans [30–32]. In addition, several mutations have been identified in the TrkA receptor, which are associated in patients suffering from CIPA (congenital insensitivity to pain with anhidrosis). This includes changes in amino acid sequences Gly571→Ala (G571A) [33,34] and Leu93→Pro (L93P), Gly516→Arg (G516R), Arg648→Cys (R648C) and Asp668→Try (D668Y)  (Figure 1). These alterations in Trk structure presumably produce changes in Trk activity, localization or protein interactions that lead to sensitization of ion channels.
Until recently, no genetic associations have been found between neurotrophin genes and human neurological or psychiatric disorders. A recent series of studies, however, have linked a single nucleotide polymorphism in the BDNF gene leading to a Val→Met substitution at position 66 in the pro-domain (BDNFMet) with memory impairments as well as altered susceptibility to neuropsychiatric disorders, such as Alzheimer's disease , Parkinson's disease , depression , eating disorders [39,40] and bipolar disorder [41,42]. This BDNF polymorphism represents the first alteration in a neurotrophin gene that has been linked to clinical pathology. A common clinical symptom among these disorders is varying degrees of impairment of higher cognitive abilities. With the established role of BDNF in mediating processes related to learning and memory [43–45], this susceptibility to cognitive impairment suggests that this alteration may have broad roles in multiple disorders affecting nervous system functioning.
The molecular mechanisms underlying altered BDNFMet function are being studied. Alteration of this site in the prodomain was also found to lead to decreased variant BDNF targeting to secretory granules and subsequent regulated secretion in PC12 cells and primary cultured neurons [46,47]. In addition, when expressed together in the same cell, BDNFMet alters the trafficking of wild-type BDNF (BDNFVal) through the formation of heterodimers that are less efficiently sorted into the regulated secretory pathway . These findings are consistent with previous studies indicating that the prodomain of neurotrophins plays an important role in regulating their intracellular trafficking to secretory pathways . Together, these in vitro processing studies with BDNFMet point to the presence of a specific trafficking signal in the BDNF prodomain region encompassing the methionine substitution that is required for efficient BDNF sorting to the regulated secretory pathway. Perturbations in BDNF trafficking may lead to selective impairments in CNS function. This variant BDNF provides an initial example of how appropriate intracellular trafficking of BDNF may have significant impact on the physiological responses to neurotrophins.
Other variants and mutations have also recently been found in the neurotrophin receptor systems. A case study demonstrated that a de novo missense mutation in the kinase domain of TrkB [Tyr722→Cys (Y722C)] leads to impaired intracellular signalling and obesity and developmental delay . In addition, a polymorphism in the gene encoding p75NTR, resulting in a Ser205→Leu (S205L) substitution in the extracellular domain, leads to increased susceptibility to depressive disorders . Although the molecular mechanisms underlying these genetic alterations needs to be delineated, together they emphasize the role of the neurotrophin system in higher-order CNS functions and how genetic alterations in the neurotrophins or neurotrophin receptors appears to lead to specific sets of neuropsychiatric and neurodegenerative disorders.
THERAPEUTIC POTENTIAL OF NEUROTROPHINS
Neurotrophic factors regulate numerous neuronal functions in development and adult life and in response to neuronal injury . As a result, neurotrophins have been implicated in the pathophysiology of a wide variety of neurodegenerative and psychiatric disorders and have been considered as a therapeutic strategy for neuropsychiatric disorders. However, it should be emphasized that no human disease affecting the nervous system has been shown to be caused directly by a defect in the neurotrophins or their receptors. The finding that neurotrophic factors modulate neuronal survival and axonal growth was the rationale for developing therapeutics for neurodegenerative disorders and neuronal injury, including Alzheimer's disease, Parkinson's disease, Huntington's disease and ALS (amyotrophic lateral sclerosis), as well as spinal cord injury.
The hypothesis underlying these clinical correlations, as well as development of therapeutic strategies using neurotrophic factors, assumes that these disease states result in (i) decreased availability of neurotrophins for the affected neurons, (ii) decreased number of neurotrophin receptors on the affected neurons, and/or (iii) decreased neuronal survival. These deficits can be ameliorated by the addition of neurotrophic factors. In all these conditions, the assumption has been that exogenous neurotrophic factors would provide symptomatic treatment for the disease state rather than a cure for these nervous system disorders.
A link to Alzheimer's disease was made in the 1980s based on studies on aged animals in which cholinergic neurons in the basal forebrain could be rescued with intracerebroventricular NGF. Treatment with NGF led to concomitant improvements in memory function . Subsequent animal studies of impaired motor neuron populations demonstrated that other neurotrophins, BDNF, NT-3, NT-4 and CNTF (ciliary neurotrophic factor), could rescue those neurons in axotomized facial nerve and sciatic nerve. In addition, mutant mouse models of motor neuron disease (progressive motor neuron disease, wobbler), in which there was motor neuron degeneration, demonstrated that BDNF and CNTF could increase the number of motor neurons and improve motor performance. These studies led to the therapeutic strategy to attempt to treat degenerative diseases affecting motor neurons with neurotrophins.
A number of clinical trials involving the use of neurotrophic factors for neurodegenerative disorders have been carried out in the past decade. Studies were carried out with neurotrophic factors as a treatment strategy for ALS, a progressive neurodegenerative disorder affecting motor neurons. However, subcutaneous or intrathecal delivery of BDNF had minimal beneficial effect and produced side-effects such as pain and gastrointestinal symptoms . Similarly, use of another neurotrophic factor, CNTF, also led to even more significant side-effects such as fever, pain and anorexia, which also limited the doses used. Clinical studies using NGF for the treatment of patients with Alzheimer's disease and diabetic neuropathy encountered similar hurdles involving problems of delivery and uncertain pharmacokinetics of the proteins. Recently, efforts to increase levels of NGF by cell therapy in patients with Alzheimer's disease have shown some cognitive improvement . Raising the levels of NGF in Alzheimer's disease patients is likely to result in additional Trk receptor signalling, leading to increased survival or neurotransmission of cholinergic neurons.
It has become apparent that appropriate delivery of sufficient quantities of neurotrophins to target neurons is a major obstacle. It is also clear that with neurotrophins having multiple effects on neuronal activity , indiscriminate ‘flooding’ of the CNS with neurotrophic factors will probably lead to side-effects such as epileptic activity. Development of small molecules that readily cross the blood–brain barrier to activate neurotrophin receptors or potentiate the actions of neurotrophins is an obvious approach. Although some structural data are available for the binding sites of neurotrophin to their receptors, pharmacological studies utilizing peptides and other small molecules that might serve as agonists or antagonists have not been actively pursued.
Activation of the neurotrophin system through other receptor signalling systems offers an alternative strategy. We have shown that GPCRs (G-protein-coupled receptors), the purine adenosine A2A receptor and PACAP (pituitary adenylate cyclase-activating polypeptide) neuropeptide receptor, can transactivate Trk neurotrophin receptors in the absence of neurotrophins in hippocampal neurons in vitro [55,56]. Therefore small molecules can activate Trk receptors in the absence of neurotrophins. These results raise the possibility that small molecules may be used to elicit neurotrophic effects for the treatment of neurodegenerative diseases by selective targeting of neurons that express specific GPCRs and Trk receptors.
TRANSACTIVATION OF Trk RECEPTORS
Trk tyrosine kinase receptors can be activated as a result of transactivation. Treatment of PC12 cells, as well as primary cultures of hippocampal neurons, with adenosine results in the activation of Trk receptors and the phosphorylation of Shc adaptor proteins and PLC-γ, similar to the phosphorylation events induced by NGF. Similar increases were observed for PI3K and Akt.
Hippocampal neuron survival is promoted by BDNF and its withdrawal from hippocampal neurons leads to rapid cell death. We discovered that adenosine maintains the survival of hippocampal neurons grown in the absence of BDNF . Adenosine was able to reverse cell death in hippocampal neurons initiated by withdrawal of trophic support by BDNF. Similar results were observed with PACAP treatment . The action of adenosine required A2A receptor and TrkB receptor activity, as well as Akt activity, but not MAPK activity. Therefore a probable mechanism to account for the neurotrophic effects of adenosine is through activation of specific pathways downstream of Trk receptors.
Adenosine is a neuromodulator, whose levels are increased when ATP is converted into adenosine or during injury, such as hypoxia or ischaemia. Its effects are mediated by specific P1 purinoceptors. Four adenosine receptors have been characterized. A1 and A3 adenosine receptors couple to Gi and Go or Gq, whereas A2A and A2B adenosine receptors couple to Gs. Use of specific adenosine agonists and antagonists established that the A2A receptor functions in PC12 cells to mediate TrkA receptor activation. The effects of adenosine were specifically blocked by K252a, an inhibitor of Trk tyrosine kinase activity.
Transactivation of Trk receptors does not involve increased production of neurotrophins. NGF-blocking antibodies do not affect the ability of adenosine to activate TrkA receptors in PC12 cells at concentrations that block the effects of NGF added exogeneously to PC12 cells. Treatment of PC12 cells with adenosine or adenosine agonists did not result in the production of neurotrophic activities, as assessed by the lack of neurite outgrowth activity from supernatants of PC12 cells subjected to adenosine treatment. Therefore, in these cell systems, adenosine activation of Trk receptors does not result via an autocrine/paracrine mechanism involving the release of neurotrophin.
Small molecules acting through GPCRs can promote trophic activities mediated by receptor tyrosine kinases. Adenosine or PACAP can activate the neurotrophin signalling system in the absence of neurotrophins. This is significant since neurotrophins provide signals to promote neuronal survival, synaptic efficacy and plasticity [51,54]. Depending upon the circumstances, adenosine and PACAP may be neuroprotective against injury initiated by ischaemia, hypoxia or vascular damage. GPCR signalling through Trk neurotrophin receptors leads to selective activation of the PI3K/Akt pathway over a prolonged time course. Intracellular signalling interactions between adenosine and Trk receptors therefore provide a new avenue for developing new approaches to address neurological disorders. Small molecules, such as adenosine, may be used to target populations of neurons that express both adenosine and Trk receptors and, therefore, may be considered as potential treatments for a wide number of nervous system disorders, including cerebral ischaemia, ALS and Parkinson's disease, and other neurodegenerative conditions.
The strategies to apply neurotrophic factors in human neurological diseases are based on an assumption of symptomatic treatment of impaired neurons. This impairment implies not only cell survival, but also proper synaptic functioning of these neurons. With greater understanding of the signal transduction pathways that are activated by neurotrophins, alternative strategies can be devised to manipulate these pathways through new drug development. In addition, further understanding of the core pathophysiological mechanism for neurodegenerative and psychiatric disorders will eventually assist in the development of rational therapies that engage neurotrophin signalling.
This work was supported by National Institutes of Health grants NS21072 and HD23315 (to M. V. C.), and MH068850 and NS052819 (to F. S. L.).
Abbreviations: ALS, amyotrophic lateral sclerosis; ARMS protein, ankyrin-rich membrane spanning protein; BDNF, brain-derived neurotrophic factor; CNS, central nervous system; CNTF, ciliary neurotrophic factor; ERK, extracellular-signal-regulated protein kinase; FRS2, fibroblast growth factor receptor substrate 2; GPCR, G-protein-coupled receptor; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; p75NTR, p75 neurotrophin receptor; PACAP, pituitary adenylate cyclase-activating polypeptide; PI3K, phosphoinositide 3-kinase; PLC-γ, phospolipase C-γ; Shc, Src homology and collagen homology; TRPV1, transient receptor potential vanilloid 1; VR-1, vanilloid receptor 1
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