++
The belief that extracellular signals can promote the growth and differentiation of nerve cells is almost a century old, yet the molecular diversity of neurotrophic factors and of their intracellular signaling cascades did not become apparent until the past two decades. Knowledge of neurotrophic factor signaling has dramatically enhanced our understanding of the ways in which the nervous system evolves during development and adapts throughout the adult life of an organism. Such knowledge also has provided insight into mechanisms responsible for neuronal survival, whose failure may underlie neurodegenerative disorders such as Alzheimer disease, Parkinson disease, Huntington disease, and amyotropic lateral sclerosis (ALS) (Chapter 18). Moreover, neurotrophic factors and their signaling proteins represent a large number of potential targets for the pharmacotherapeutic treatment of these and other neuropsychiatric disorders.
++
A discussion of neurotrophic factors requires the definition of several terms. Neurotrophic factors themselves are peptide growth factors for nerve cells: they influence the cell cycle, growth, differentiation, and survival of neurons. Due to overlap among growth factors for neurons and glia, we also apply the term neurotrophic factor to any molecule that produces trophic effects on the nervous system by influencing glia. To add to the confusion, the term neurotrophin applies to only one family of neurotrophic factors. A broader term is cytokine, which is borrowed from the field of immunology and refers to molecules released by one cell to modulate the activity of other cells. The term cytokine therefore applies to all growth factors, including neurotrophic factors. The term neurotrophic factor is restricted to proteins and excludes the many nonpeptide molecules—for example, steroid hormones, retinoic acid, and many small-molecule neurotransmitters—that also can influence the growth and integrity of the nervous system.
++
Although neurotrophic factors originally were distinguished from neurotransmitters based on their role in nervous system development, as opposed to synaptic transmission in the adult, we now know that there is considerable overlap in the actions of these molecules. Like neurotransmitters, many neurotrophic factors are synthesized by neurons and alter the functioning of other neurons; under some circumstances, they may even be released as a result of neuronal activity. Moreover, some neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), may produce rapid changes in target neurons that are indistinguishable from those elicited by conventional neurotransmitters. Likewise, many small-molecule neurotransmitters not only elicit the rapid changes associated with synaptic transmission but also affect the growth, differentiation, and survival of neurons during development and in adulthood.
++
Certain key features of neurotrophic factors distinguish them from peptide neurotransmitters. Notably, neurotrophic factors tend to be larger proteins, whereas peptide neurotransmitters typically are very small peptides (Chapter 7). In addition, most neurotrophic factors produce their biologic effects through the regulation of protein tyrosine kinases, whereas most peptide neurotransmitters signal through G protein–coupled receptors and classic second messenger cascades.
+++
Functional Characteristics of Neurotrophic Factors
++
Neurotrophic factors are synthesized by transcription and translation in the cell bodies of particular neurons and glia. Some are stored in these cells, perhaps in large dense core vesicles (Chapter 3), and are transported either to nerve terminals or to dendrites. Many factors, such as interleukin-1 (IL-1), BDNF, and glial cell line–derived neurotrophic factor (GDNF), are encoded by immediate early genes (Chapter 4); consequently, activity-dependent transcription may be the major determinant of release of these factors. The release of many neurotrophic factors can be triggered by depolarization, and there is evidence that such activity-dependent release of a neurotrophic factor requires more extended periods of neuronal activation compared with the release of small-molecule transmitters. The major mechanism responsible for the termination of neurotrophic factor signals is proteolytic degradation. However, some factors, such as BDNF, are sequestered by a functionally inactive, truncated receptor that limits their diffusion and thereby perhaps the duration of their action.
++
According to the neurotrophic hypothesis, neurotrophic factors are indispensable in the establishment of suitable nerve–target connections during development. While the sites of action of many neurotrophic factors are unclear, the case of nerve growth factor (NGF) in the peripheral nervous system is well understood. The first growth factor to be identified, NGF was discovered in the 1950s when certain sympathetic and sensory neurons were shown to require this protein for their survival 8–9. NGF was subsequently demonstrated to be synthesized by the target organs of these nerve cells and required to maintain the sympathetic and sensory neurons that innervate the organs. Because the supply of NGF is limited, competition for the factor exists among growing nerve fibers. Growing neurons that do not receive the NGF signal from their target do not survive. Likewise, only neurons that successfully respond to NGF survive and make appropriate connections with their targets. These findings demonstrate that NGF is required for proper nerve–target connections during development 8–10.
++
++
++
Although this pattern of NGF synthesis and action predominates in the peripheral nervous system, different patterns emerge in the brain and spinal cord (see 8–10). In the CNS, a target neuron may supply neurotrophic factor for an innervating neuron, but may also synthesize many other neurotrophic factors and receptors. Thus, factors in the brain and spinal cord may serve additional autoregulatory, or autocrine, functions. Furthermore, certain neurotrophic factors can be transported in an anterograde fashion to axon terminals where, after release, they act on the cell bodies or nerve terminals of other nerve cells. As well, neurotrophic factor–receptor complexes, formed on the plasma membrane of nerve terminals, can be retrogradely transported back to cell bodies, where they exert some of their biologic effects.
++
Glia further complicate our understanding of the production of neurotrophic factors. Some factors are synthesized by both neurons and glia and act on receptors expressed by both cell types. Such patterns of synthesis and activity result in highly complex forms of intercellular communication among neurons and glia that investigators have yet to disentangle.
+++
Families of Neurotrophic Factors
++
The classification of neurotrophic factors is complicated by the history of their discovery: the names of many factors were based on the actions with which they were originally associated. Interleukins were given their name because they were identified as proteins that mediate communication among white blood cells, even though they are produced by glia and perhaps certain neurons as well. GDNF was named for its original source, even though it is made by many types of neurons and other cells in the body. Fibroblast growth factor (FGF) was regarded as a growth factor for fibroblasts, even though it is also produced by glia. Ciliary neurotrophic factor (CNTF) was named for its contribution to the growth and maintenance of ciliary ganglion neurons in the eye, even though it is generated by glia and neurons and is important for the survival of many types of neurons, notably motor neurons.
++
Currently, neurotrophic factors can be categorized based on their homologies and on the shared signal transduction mechanisms through which they produce their biologic effects 8–2. This chapter focuses primarily on the neurotrophins, which are among the best characterized in the nervous system. The chapter also provides brief discussions of GDNF, CNTF, some neurotrophic factors better known for their role as immune-response cytokines or chemokines, as well as vascular endothelial growth factor (VEGF), neuregulin, and nonacronymic VGF (which is not an abbreviation).
++
++
The neurotrophin family comprises NGF and subsequently identified factors that employ similar signaling mechanisms, including BDNF, neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4; also known as neurotrophin-4/5). All neurotrophins are small proteins. BDNF, for example, has a molecular mass of approximately 14 kDa. Another common feature of neurotrophins is that they produce their physiologic effects by means of the tropomyosin receptor kinase (Trk) receptor family (also known as the tyrosine receptor kinase family).
++
Neurotrophins produce effects on a wide range of neurons. As previously mentioned in connection with NGF, their role in ensuring the survival of neurons in the peripheral nervous system is well established. NGF, for example, is present in target fields of small sympathetic and sensory neurons that have nociceptive and temperature-sensing functions, and the NGF receptor is expressed in these neurons. BDNF is produced in skeletal muscle (innervated by motor neurons). BDNF, NT-4, and NT-3 each supports the survival of a subset of peripheral sensory neurons.
++
Neurotrophins similarly affect the survival and maintenance of some neurons in the CNS, although their precise roles are less clear. In different circumstances their primary function might be target-derived support of afferent neurons, the support of efferent neurons, or the generation and maintenance of differentiated neurons. For example, NGF promotes the survival of cholinergic neurons in the septal nuclei of the basal forebrain, and BDNF, NT-3, and NT-4 promote the survival of some cortical motor and hippocampal neurons, and of noradrenergic, dopaminergic, and serotoninergic neurons located in the brainstem. Because cholinergic neurons are affected early in Alzheimer disease (Chapter 18), NGF has been proposed as a potential treatment; the use of other neurotrophins has been proposed for the treatment of Parkinson disease. Unfortunately, clinical trials have not shown significant efficacy as shown later in this chapter. To complicate matters further, recent work has shown that precursors of mature neurotrophins (eg, pro-NGF or pro-BDNF) are themselves released from neurons and antagonize the actions of mature neurotrophins. These proneurotrophins may, therefore, also be important for regulating cell death during development and after injury.
++
We now know that neurotrophins continue to play a role in the adult CNS, where they have been shown to support the survival and plasticity of fully differentiated neurons and in some instances to contribute to synaptic transmission. BDNF, in particular, is important for some forms of long-term potentiation (LTP) as well as certain types of learning and memory. BDNF also has been implicated in regulation of adult hippocampal neurogenesis, epilepsies, and animal models of several psychiatric disorders, in particular, depression, as will be discussed in later chapters of this book. NGF, which acts by activating the TrkA receptor (next paragraph), has been implicated in neuropathic pain, as well (Chapter 11). This has prompted the evaluation of anti-NGF antibodies or small-molecule antagonists of TrkA as potential treatments of chronic pain syndromes.
++
All neurotrophins bind to a class of highly homologous receptor tyrosine kinases known as Trk receptors, of which three types are known: TrkA, TrkB, and TrkC. These transmembrane receptors are glycoproteins whose molecular masses range from 140 to 145 kDa. Each type of Trk receptor binds specific neurotrophins: TrkA is the receptor for NGF, TrkB the receptor for BDNF and NT-4, and TrkC the receptor for NT-3. However, some overlap in the specificity of these receptors has been noted 8–11.
++
++
The characteristic structural domains of the Trk receptor are represented in 8–12. The binding of a neurotrophin to its Trk receptor causes activation of the receptor’s catalytic domain. Neurotrophins bind as dimers, and in turn cause the dimerization of Trk molecules, which results in the autophosphorylation of Trk on several key cytoplasmic tyrosine residues. Such autophosphorylation initiates intracellular signaling cascades that lead to many of the biologic effects of receptor activation (Chapter 4). Intracellular signaling occurs by way of phosphorylated tyrosine residues that form a recognition sequence for the SH2 domains that are present on several types of cellular proteins. SH2 domains on Shc and Grb2, for example, eventually link Trk receptors to the activation of the small G protein Ras, which in turn triggers the activation of the microtubule-associated protein (MAP)-kinase cascade. Genetic abnormalities in Ras-related proteins cause neurofibromatosis in humans, which involves excessive growth and occasionally cancerous degeneration of Schwann cells (Chapter 4).
++
++
Other biologic effects of Trk receptor activation result from the phosphorylation of different signaling proteins on tyrosine residues. The most important of these are phospholipase Cγ (PLCγ), which triggers the phosphatidylinositol cascade, and insulin receptor substrate (IRS), which leads to activation of the phosphatidylinositol-3-kinase cascade. Mutations in the ATM gene, which encodes one subtype of phosphatidylinositol-3-kinase, have been shown to cause ataxia telangiectasia, a disease characterized by progressive degeneration and atrophy of several brain regions, particularly that of the cerebellum. The various intracellular signaling cascades activated by the neurotrophins and their Trk receptors are discussed in greater detail in Chapter 4.
++
Trk receptors display multiple splice variants. The best understood are the TrkB and TrkC isoforms that contain normal extracellular ligand-binding domains but lack the catalytic tyrosine kinase domain. As previously discussed, truncated Trk receptors limit the scope and duration of neurotrophin activity, either by heterodimerizing with full-length Trk receptors and preventing their phosphorylation or by sequestering extracellular neurotrophins and preventing their activation of full-length Trk receptors.
++
The first neurotrophin receptor to be cloned was not a Trk receptor, but p75, a 75-kDa protein that is currently described as the low-affinity neurotrophin receptor. The p75 receptor binds to all neurotrophins with roughly equal affinity. It also appears to modulate Trk signaling, perhaps by allowing Trk receptors to respond to lower concentrations of NGF and other neurotrophins—a highly desirable trait in a receptor that competes for limited quantities of growth factor. In addition, p75 may make TrkA and TrkB more selective for their respective primary ligands, NGF and BDNF. While the mechanism by which p75 exerts these effects is not well understood, it is hypothesized that p75, Trk, and a neurotrophin form a stable trimolecular complex. Importantly, however, Trk receptors are fully capable of mediating functional responses to neurotrophins in the absence of p75.
++
Despite evidence that p75 can enhance neurotrophin and Trk function and promote cell survival under some conditions, p75 has been implicated in cell death pathways, which are described in greater detail in Chapter 18. p75 has a so-called “death domain,” analogous to similar domains in the tumor necrosis factor-α (TNF-α) receptor (see below), through which it activates programmed cell death or apoptosis (Chapter 18). Recent discoveries suggest that the opposing actions of p75 may depend on the presence of the coreceptor sortilin, a member of the Vps10p-receptor family. The p75–sortilin complex has high affinity for the proneurotrophins (eg, pro-BDNF and pro-NGF), which have strong proapoptotic activity. Studies to determine the functional implications of proneurotrophin action as well as that of p75 and sortilin are ongoing. Interestingly, genetic variations in certain sortilin family genes have been implicated in risk for Alzheimer disease (Chapter 18).
+++
Neurotrophins and synaptic plasticity
++
Studies of developing visual cortex illustrate that neurotrophins play a role in some forms of synaptic plasticity. As axons from the lateral geniculate nucleus (LGN) of the thalamus grow into the primary visual cortex and form synapses, they segregate into eye-specific, alternating patches known as ocular dominance columns. Monocular deprivation experiments have revealed that LGN neurons that receive inputs from the deprived eye exhibit weakened synaptic connections. NT-4 can rescue these neurons from such plastic changes. It is possible that LGN neurons might compete for TrkB ligands and that this competition might be crucial for the formation of ocular dominance columns. In support of this hypothesis, both infusion of excess BDNF or NT-4 and infusion of a neurotrophin antagonist into the visual cortex are sufficient to block formation of ocular dominance columns.
++
Evidence suggests that neurotrophins also are involved in regulating synaptic plasticity in the adult brain. Neuronal activity dramatically and rapidly regulates the expression of certain neurotrophins and their Trk receptors in adult neurons. The induction of BDNF and TrkB, for example, has been observed in neurons of the hippocampus in response to trains of synaptic stimuli (including those associated with the formation of LTP; Chapter 5). The rapidity of BDNF induction is consistent with that of other immediate early genes, such as c-Fos (Chapter 4), and induction of BDNF is mediated by the activation of preexisting transcription factors such as CREB.
++
Increasing evidence suggests that neurotrophins can modulate synaptic transmission and regulate the formation and strengthening of synapses. Such actions may be mediated by cross-talk between neurotrophin and neurotransmitter signaling pathways. For instance, NT-3, via its Trk receptor, rapidly enhances synaptic transmission at the neuromuscular junction by increasing the probability of acetylcholine release from the presynaptic neuron. BDNF and NT-3 are reported to increase the size of excitatory postsynaptic potentials at the Schaffer collateral–CA1 synapse in the hippocampus. Accordingly, BDNF knockout mice exhibit reduced basal synaptic transmission at this synapse, and hippocampal slices from these mice are reportedly deficient in LTP. These data clearly implicate the involvement of BDNF in hippocampal function in the adult, although further research is needed to better understand the underlying mechanisms involved.
++
GDNF, a glycosylated protein of approximately 18 kDa, was first isolated from a glial cell line that supports the survival of dopaminergic neurons from the midbrain. Because Parkinson disease is caused by the degeneration of dopaminergic neurons in the midbrain (Chapter 18), GDNF has received considerable attention as a potential therapeutic agent for this disease, although obstacles remain as will be discussed toward the end of this chapter.
++
More recently, GDNF has been proven to support the survival of many other neuronal cells, including those of the myenteric plexus within the gut. However, the most profound abnormality observed in GDNF knockout mice is maldevelopment of the kidney, which causes death shortly after birth. This finding demonstrates that GDNF plays a critical role outside of the nervous system.
++
Like the neurotrophins, GDNF produces its biologic effects through the activation of a protein tyrosine kinase, but such activation is achieved indirectly through an intervening receptor protein 8–13. This receptor, termed GFRα1, binds to and activates Ret, a transmembrane protein tyrosine kinase of about 150 kDa, which then mediates GDNF action. Loss-of-function mutations in Ret are associated with Hirschsprung disease in humans, a disorder characterized by abnormal gut motility, consistent with Hirschsprung-like abnormalities in GDNF knockout mice. In contrast, gain-of-function mutations in Ret are associated with neural crest malignancies in humans, such as multiple endocrine neoplasias and medullary thyroid carcinoma.
++
++
Other members of the GDNF family of neurotrophic factors include neurturin, artemin, and persephin, which also signal by means of Ret. Like GDNF, each binds to a specific receptor-α (Rα) subunit, which subsequently converges on Ret. Also like GDNF, neurturin can support the survival of dopaminergic neurons in the midbrain, which raises its potential utility for Parkinson disease.
++
As previously mentioned, CNTF initially was studied as a survival factor for chick ciliary ganglion neurons where it upregulates choline acetyltransferase (Chapter 6). More recently, CNTF, a protein of 24 kDa in size, has been proven to regulate the survival or differentiation of many other neuronal cells, including motor neurons, hippocampal neurons, and midbrain dopaminergic neurons. Its effects on motor neurons are perhaps the most dramatic: CNTF prevents their degeneration after axotomy and improves some motor defects in murine models of motor neuron disease. Consequently, CNTF has been tested as a therapeutic agent in the treatment of ALS, which is characterized by the degeneration of motor neurons. However, clinical trials were abandoned due to toxic side effects. Attempts to overcome these side effects have been unsuccessful (see below), but there is still interest in CNTF in the treatment of Huntington disease as well as in recovery from spinal cord injury.
++
CNTF, along with several cytokines (see next section), exerts its effects through a characteristic glycoprotein of 130-kDa (gp130) signaling pathway 8–14. Each ligand binds a unique Rα subunit, which is tethered to the plasma membrane by glycophosphatidylinositol (GPI). On ligand binding, the receptor complexes with gp130 and in some cases with other proteins. The formation of this receptor complex triggers activation of Janus kinase (JAK) and of related protein tyrosine kinases, such as Tyk, which subsequently mediate biologic effects such as activation of the signal transducer and activator of transcription (STAT) family of transcription factors (Chapter 4).
++
++
Surprisingly, CNTF knockout mice develop normally and exhibit only mild motor neuron defects during adulthood. Also surprising is a related finding in humans: approximately 2.5% of the Japanese population are homozygous for inactivating mutations of CNTF and thus are, in a sense, human CNTF “knockouts”; these individuals develop without obvious deficits. In contrast, mice lacking the CNTF receptor CNTFRα lose almost all of their motor neurons and die within 24 hours of birth. The striking discrepancies between CNTF and CNTFRα knockout mice suggest the existence of additional endogenous ligands for CNTFRα that have yet to be discovered.
++
CNTF and its receptor complex are expressed by neurons and glial cells in the CNS. However, much remains to be learned about the details of how CNTF contributes to cell–cell communication and its functional consequences in health and disease.
+++
Immune-Response Cytokines and the CNS
++
As alluded to previously, the best studied effects of many cytokines are their actions on the immune system. Only recently has it emerged that many of these factors, particularly IL-1 and interleukin-6 (IL-6), TNF-α, and transforming growth factor-β (TGF-β), also mediate CNS responses to immunologic challenges. In addition, increasing evidence supports their involvement in the regulation of neural function and plasticity far more broadly.
++
Several cytokine signaling pathways are known. Many cytokines exert their actions at least partly via the activation of the JAK–STAT pathway. In some instances, such as for IL-6, leukemia inhibitory factor, and oncostatin M, such activation is achieved via the gp130 signaling pathway mentioned above for CNTF 8–14. However, many other cytokines (eg, several interferons) activate JAK–STAT via other receptor mechanisms. Interesting, several peripheral hormones—prolactin, growth hormone, leptin, and erythropoietin, among others—also signal through JAK–STAT (Chapter 10).
++
IL-1 activates Toll-like receptors, which act via nuclear factor κB (NF-κB) and MAP-kinase pathways, among others (Chapter 4). Toll-like receptors, numerous subtypes of which are known, are crucial for mammalian immunity and the ability to fight infections (Chapter 12). TGF-β activates the TGF-β receptor, which is a serine–threonine protein kinase that phosphorylates a family of transcription factors known as SMADs. Bone morphogenetic proteins (BMPs), classified as part of the TGF-β family, also signal through serine–threonine protein kinase receptors and SMADs. Finally, TNF-α signals through a complex receptor mechanism, involving the TNF-α receptor (which binds TNF-α) and its subsequent interaction with any of several effector proteins that determine the specific functional response elicited, which includes activation of NF-κB, MAP-kinases, and caspases (proteolytic enzymes involved in cell death; Chapter 18). One key adaptor is tumor necrosis factor receptor type 1–associated death domain protein (TRADD). Antibodies directed against several of these cytokines or their receptors are being used increasingly to treat a range of inflammatory and autoimmune diseases, including those that affect the nervous system (Chapter 12).
++
Cytokines implicated in immune function are critical for systemic homeostasis. Serious illness or stressors elicit a defense response that aids in the body’s recovery. This response involves processes that are mediated in part by the CNS, such as fever, reduced appetite, cardiovascular changes, sleep disturbances, and malaise. A more detailed discussion of these processes is provided in Chapters 10 and 11; the section that follows is devoted to the general schemes by which cytokines affect brain function, whether they are generated in the CNS or periphery.
++
The impact that immune-response cytokines can have on the brain is exemplified by the fever—which is mediated by the brain—that occurs in animals and humans in response to the peripheral injection of IL-1. Although the blood–brain barrier is believed to limit cytokine entry to most of the brain, some cytokines enter at sites where the blood–brain barrier is incomplete, such as certain periventricular areas. Cytokines also may act by inducing lipophilic signals, such as prostaglandins, within endothelial cells, which can diffuse into the brain parenchyma.
++
Interestingly, the brain itself can synthesize many of these immune-response cytokines. Such cytokines are believed to be synthesized primarily in glia—particularly in microglia, the CNS equivalent of macrophages—and in astrocytes. Expression by certain types of neurons now appears likely as well. Likewise, while receptors for these cytokines are expressed primarily in glia, increasingly such expression is being demonstrated in neurons. After almost any dramatic perturbation, such as brain infection or injury, activated microglia and astrocytes produce cytokines, including IL-1, IL-6, TNF-α, and TGF-β. These factors may further activate glial cells and in turn stimulate gliosis—the generation of new astrocytes and microglia. The activated glia presumably help the brain recover by restoring normal homeostasis.
++
However, excessive levels of these cytokines can contribute to neural injury. This phenomenon is demonstrated dramatically in mutant mice that overexpress specific factors such as IL-1 or IL-6; such mice exhibit significant neurodegeneration in the vicinities of cytokine overexpression. Moreover, elevated levels of immune-response cytokines have been associated with several neurodegenerative disorders, including Alzheimer disease. Elevated cytokine levels are also observed in individuals with multiple sclerosis, an autoimmune disease that results in the degeneration of myelin sheaths around axons. Treatment of multiple sclerosis now centers on the systemic delivery of antibodies that neutralize certain immune-response cytokines or of certain cytokines themselves that suppress immune responses (eg, interferon-β) (Chapter 12). In addition, there is increasing evidence for a role of immune-response cytokines in psychiatric disorders, in particular, depression (Chapter 15).
++
In some cases, immune-response cytokines produce effects similar to those produced by CNTF and related proteins. IL-1, like IL-6, enhances survival of several types of central neurons. TGF-β, like CNTF, is important for neural crest cell differentiation during development. On the other hand, TNF-α released from macrophages contributes to the waves of programmed cell death of motor neurons required for proper development. Like other neurotrophic factors, these cytokines may also directly affect neurons in the adult brain. Receptors for IL-1, TNF-α, and TGF-β are most densely concentrated in the hippocampus and hypothalamus, where some of their corresponding factors have been proven to influence synaptic plasticity. For example, IL-1 is reported to attenuate the generation of hippocampal LTP while TNF-α may directly influence synaptic properties in developing neural circuits. Immune-response cytokines also may influence the rate of neurogenesis and the survival of newly formed neurons in the dentate gyrus of the adult hippocampus. More research will be required to better characterize the direct effects of these cytokines on neurons, and to explore their possible role in the regulation of nervous system function under normal and pathologic conditions.