Not all neurons are destined to survive, and decisions about life and death are therefore aspects of a neuron's fate. The surprising and counterintuitive fact is that cell death is preprogrammed in most animal cells, including neurons.
The Neurotrophic Factor Hypothesis Was Confirmed by the Discovery of Nerve Growth Factor
The target of a neuron is a key source of factors essential for the neuron's survival. The critical role of target cells in neuronal survival was discovered in studies of the dorsal root ganglia.
In the 1930s Samuel Detwiler and Viktor Hamburger discovered that the number of sensory neurons in embryos is increased by transplantation of an additional limb bud into the target field and decreased if the limb target is removed. At the time these findings were thought to reflect an influence of the limb on the proliferation and subsequent differentiation of sensory neuron precursors. In the 1940s, however, Rita Levi-Montalcini made the startling observation that the death of neurons is not simply a consequence of pathology or experimental manipulation, but rather occurs during the normal program of embryonic development. Levi-Montalcini and Hamburger went on to show that removal of a limb leads to the excessive death of sensory neurons rather than a decrease in their production.
These early discoveries on the life and death of sensory neurons were quickly extended to neurons in the central nervous system. Hamburger found that approximately half of all motor neurons generated in the spinal cord die during embryonic development. Moreover, in experiments similar to those performed on sensory ganglia, Hamburger discovered that motor neuron death could be increased by removing a limb and reduced by adding an additional limb (Figure 53–13). These findings indicate that signals from target cells are critical for the survival of neurons within the central as well as peripheral nervous system. We now know that the phenomenon of neuronal overproduction, followed by a phase of neuronal death, occurs in most regions of the vertebrate nervous system. Blocking neuromuscular activity with drugs such as curare also reduces the extent of motor neuron death (Figure 53–13).
The survival of motor neurons depends on signals provided by their muscle targets.
The role of the muscle target in motor neuron survival was demonstrated by Viktor Hamburger in a classic series of experiments performed in the chick embryo. (Adapted, with permission, from Purves and Lichtman 1985.)
A. A limb bud was removed from a 2.5-day-old chick embryo soon after the arrival of motor nerves. A section of the lumbar spinal cord 1 week later reveals few surviving motor neurons on the deprived side of the spinal cord. The number of motor neurons on the contralateral side with an intact limb is normal.
B. An extra limb bud was grafted adjacent to a host limb prior to the normal period of motor neuron death. A section of the lumbar spinal cord 2 weeks later shows an increased number of limb motor neurons on the side with the extra limb.
C. Blockade of nerve-muscle activity with the toxin curare, which blocks acetylcholine receptors, rescues many motor neurons that would otherwise die. Curare may act by enhancing the release of trophic factors from inactive muscle.
The early discoveries of Levi-Montalcini and Hamburger laid the foundations for the neurotrophic factor hypothesis. The core of this hypothesis is that cells at or near the target of a neuron secrete small amounts of an essential nutrient or trophic factor, and that the uptake of this factor by nerve terminals is needed for the survival of the neuron (Figure 53–14). This hypothesis was dramatically confirmed in the 1970s when Levi-Montalcini and Stanley Cohen purified the protein we now know as nerve growth factor (NGF) in the early 1970s and showed that this protein is made by target cells and supports the survival of sensory and sympathetic neurons in vitro. Moreover, neutralizing antibodies directed against NGF were found to cause a profound loss of sympathetic and sensory neurons in vivo.
The neurotrophic factor hypothesis.
(Adapted, with permission, from Reichardt and Farinas 1997.)
A. Neurons extend their axons to target cells, which secrete low levels of neurotrophic factors. (For simplicity only one target cell is shown.) The neurotrophic factor binds to specific receptors and is internalized and transported to the cell body, where it promotes neuronal survival.
B. Neurons that fail to receive adequate amounts of neurotrophic factor die through a program of cell death termed apoptosis.
Neurotrophins Are the Best Studied Neurotrophic Factors
The discovery of NGF prompted a search for additional neurotrophic factors. Today we know of over a dozen secreted factors that promote neuronal survival. The best studied of the neurotrophic factors are related to NGF and are called the neurotrophin family.
There are three main neurotrophins: NGF itself, brain derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). Other classes of proteins that promote neuronal survival include members of the transforming growth factor β family, the interleukin 6-related cytokines, fibroblast growth factors, and even certain inductive signals we encountered earlier (BMPs and hedgehogs). Other neurotrophic factors, notably members of the glial cell line-derived neurotrophic factor (GDNF) family, are responsible for the survival of different types of sensory and sympathetic neurons (Figure 53–15).
Different neurotrophic factors promote the survival of distinct populations of dorsal root ganglion neurons.
Proprioceptive sensory neurons that innervate muscle spindles depend on neurotrophin-3 (NT-3); nociceptive neurons that innervate skin depend on nerve growth factor (NGF); mechanoreceptive neurons that innervate Merkel cells depend on neurotrophin-3; and those that innervate hair follicles depend on neurotrophin-4 and -5 (NT-4/-5). Motor neurons depend on glial cell line-derived neurotrophic factor (GDNF) and other factors. Sympathetic neurons depend on NGF, NT-3, and GDNF. (Adapted, with permission, from Reichardt and Farinas 1997.)
Neurotrophins interact with two major classes of receptors, the Trk receptors and p75. Neurotrophins promote cell survival and promote cell death through activation of Trk receptors, whereas signaling through the p75 receptor promotes cell death. The Trk family comprises three membrane-spanning tyrosine kinases named TrkA, TrkB, and TrkC, each of which exists as a dimer (Figure 53–16).
Neurotrophins and their receptors.
Each of the three main neurotrophins interacts with a different transmembrane tyrosine kinase receptor (Trk). In addition, all three neurotrophins can bind to the low-affinity neurotrophin receptor p75. (BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3.) (Adapted, with permission, from Reichardt and Farinas 1997.)
As with other tyrosine kinase receptors, the binding of neurotrophins to Trk receptors leads to phosphorylation of an intracellular domain of the receptor, resulting in the dimerization of Trk proteins and phosphorylation of specific tyrosine residues in the activation loop of the kinase domain. Phosphorylation of these residues leads to a conformational change in the receptor and to phosphorylation of tyrosine residues that serve as docking sites for adaptor proteins. Activation of Trk receptors promotes the survival of neurons and also triggers their differentiation. These divergent biological responses involve different intracellular signaling pathways: neuronal differentiation largely by the mitogen-activated kinase (MAPK) enzymatic pathways and survival largely by the phosphatidylinositol-3 kinase pathway (Figure 53–17).
Binding of nerve growth factor to the TrkA receptor activates alternative intracellular signaling pathways.
The binding of nerve growth factor (NGF) induces dimerization of the TrkA receptor, which triggers its phosphorylation at many different residues. Phosphorylation of TrkA results in the recruitment of the adaptor proteins SHC, GRB2, and SOS. The additional recruitment of FRS2 to this complex (left) activates a Ras kinase signaling pathway that promotes neuronal differentiation. In the absence of FRS2 (right) the complex activates a phosphatidylinositol-3 kinase (PI3-K) pathway that promotes neuronal survival. (Akt/PKB, protein kinase B; MAPK, microtubule-associated protein kinase; MEK, mitogen-activated/ERK kinase.) (Adapted, with permission, from Reichardt and Farinas 1997.)
In contrast to the specificity of Trk receptor interactions, all neurotrophins bind the receptor p75 (Figure 53–16). The activation of p75 promotes neuronal survival through a pathway that involves activation of the NF-B enzyme. In the absence of exposure to neurotrophins, p75 receptor activation promotes neuronal death. Receptor p75 is a member of the tumor necrosis factor (TNF) receptor family and promotes cell death by activating proteases of the caspase family, which we discuss below.
Neurotrophin signaling is relayed from the axon terminal to the cell body of the neuron through a process that involves internalization of a complex of neurotrophin bound to Trk receptors. The retrograde transport of this complex occurs in a class of endocytotic vesicles called signaling endosomes. The transport of these vesicles brings activated Trk receptors into cellular compartments able to activate signaling pathways and transcriptional programs essential for neuronal survival.
The picture is more complex for neurons in the central nervous system. The survival of motor neurons, for example, is not dependent on a single neurotrophic factor; different classes of motor neurons require neurotrophins, glial cell line-derived neurotrophic factor (GDNF), and interleukin-6-like proteins expressed by muscles or peripheral glial cells. The survival of these neuronal classes depends on the exposure of axons to local neurotrophic factors.
Neurotrophic Factors Suppress a Latent Death Program in Cells
Neurotrophic factors were once believed to promote the survival of neural cells by stimulating their metabolism in beneficial ways, hence their name. It is now evident, however, that neurotrophic factors suppress a latent death program present in all cells of the body, including neurons.
This biochemical pathway can be considered a suicide program. Once it is activated, cells die by apoptosis (Greek, falling away): They round up, form blebs, condense their chromatin, and fragment their nuclei. Apoptotic cell deaths are distinguishable from necrosis, which typically results from acute traumatic injury and involves rapid lysis of cellular membranes without activation of the cell death program.
The first clue that deprivation of neurotrophic factors kills neurons by unleashing an active biochemical program emerged from studies that assessed neuronal survival after inhibition of RNA and protein synthesis. Exposure of sympathetic neurons to protein synthesis inhibitors was found to prevent the death of sympathetic neurons triggered by removal of NGF. These results sparked the idea that neurons are always capable of synthesizing proteins that are lethal and that NGF prevents their synthesis: Neurotrophins suppress an endogenous death program.
Key insights into the biochemical nature of the endogenous cell death program emerged from genetic studies of the nematode Caenorhabditis elegans. During the development of C. elegans a precise number of cells is generated and a fixed number of these cells die—the same number from embryo to embryo. The findings prompted a screen for genes that block or enhance cell death, which led to the identification of the cell death (ced) genes. Two of these genes, ced-3 and ced-4, are needed for the death of neurons; in their absence every one of the cells destined to die instead survives. A third gene, ced-9, is needed for survival, and works by antagonizing the activities of ced-3 and ced-4 (Figure 53–18). Thus, in the absence of ced-9 many additional cells die, even though these deaths still depend on ced-3 and ced-4 activity.
Neurons and other cells express a conserved death program.
Different cellular insults trigger a genetic cascade that involves a series of death effector genes. These death genes and pathways have been conserved in the evolution of species from worms to humans. The target of the core death pathway is the activation of a set of proteolytic enzymes, the caspases. Caspases cleave many downstream and essential protein substrates (see Figure 53–19), resulting in the death of cells by a process termed apoptosis. Genetic analysis of the worm Caenorhabditis elegans indicates that the Ced-9 protein acts upstream and inhibits the activity of Ced-4 and Ced-3, two proteins that promote cell death. Many vertebrate homologs of Ced-9, the Bcl-2 family of proteins, have been identified. Some of these proteins, such as Bcl-2 itself, inhibit cell death, but others promote cell death by antagonizing the actions of the Bcl-2. The Bcl-2 class proteins act upstream of Apaf-1 (a vertebrate homolog of Ced-4) and the caspases (vertebrate homologs of Ced-3).
Neurotrophic factors suppress caspase activation and cell death.
(Adapted, with permission, from Jesenberger and Jentsch 2002.)
A. Two types of pathways trigger cell death: extrinsic activation of surface membrane death receptors and intrinsic activation of a mitochondrial pathway. Both pathways result in activation of caspases such as caspase-8 and caspase-9, which initiate a proteolytic cleavage cascade that converges at the level of caspase-3 activation. Cleavage of the caspase precursor removes the caspase prodomain and produces a proteolytically active enzyme conformation.
The extrinsic pathway involves activation of death receptors by ligands such as tumor-necrosis factor (TNF) receptor 1 or Fas/CD95. The intrinsic pathway involves stress-induced signals such as DNA damage that initiate the release of cytochrome c from the mitochondrial intermembrane space. Cytochrome c binds to Apaf-1 and recruits and activates caspase-9.
B. Binding of neurotrophins to Trk receptors recruits the PI3 kinase pathway and Akt, suppressing the cell death pathway by inhibiting caspase-9. (Akt, protein kinase B [PKB].) This pathway is inhibited in developing neurons by neurotrophic factors explaining why their withdrawal leads to apoptosis.
The cell death pathway in C. elegans has been conserved in mammals. Similar proteins and pathways control the apoptotic death of central and peripheral neurons, indeed of all developing cells. The worm ced-9 gene encodes a protein that is related to members of the mammalian Bcl-2 family, which protect lymphocytes and other cells from apoptotic death. The worm ced-3 gene encodes a protein closely related to a class of mammalian cysteine proteases called caspases. The worm ced-4 gene encodes a protein that is functionally related to a mammalian protein called apoptosis activating factor-1 or Apaf-1.
The mammalian apoptotic cell death pathway works in a way that resembles the worm pathway. The morphological and histochemical changes that accompany the apoptosis of mammalian cells result from the activation of caspases, which cleave specific aspartic acid residues within cellular proteins. Two classes of caspases regulate apoptotic death: the initiator and effector caspases. Initiator caspases (caspase-8, -9 and -10) cleave and activate effector caspases. Effector caspases (caspase-3 and -7) cleave other protein substrates, so triggering the apoptotic process. Perhaps 1% of all proteins in the cell serve as substrates for effector caspases. Their cleavage contributes to neuronal apoptosis through many pathways: by activation of proteolytic cascades, inactivation of repair, DNA cleavage, mitochondrial permeabilization, and initiation of phagocytosis.
The survival of mammalian neurons is determined by the balance between anti-apoptotic and pro-apoptotic members of the Bcl-2 family of proteins. Some Bcl-2- proteins such as BAX and BAK permeabilize mitochondrial outer membranes, causing the release of pro-apoptotic proteins such as cytochrome c into the cytosol. The release of cytochrome c induces Apaf-1 to bind and activate caspase-9, leading to the cleavage and activation of effector caspases. The binding of neurotrophic factors to their tyrosine kinase receptors is thought to lead to the phosphorylation of protein substrates that promote Bcl-2-like activities (Figure 53–19B). Thus withdrawal of neurotrophic factors from neurons changes the balance from anti-apoptotic to pro-apoptotic members of the Bcl-2 family, which triggers the neuron's demise.
The caspase cell death program can also be activated by many cellular insults, including DNA damage and anoxia (Figure 53–19A). The activation of cell-surface death receptors such as Fas by extracellular ligands results in the activation of caspase-8 or -10 as well as the recruitment of death effector proteins such as FADD. Recruitment of an initiator caspase to the Fas-FADD complex then leads to activation of effector caspases. Because many neurodegenerative disorders result in apoptotic death, pharmacological strategies to inhibit caspases are under investigation.