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Signalling for death and survival in neurons

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Bax is a proapoptotic member of the Bcl-2 family of proteins. The Bax protein is dormant in the cytosol of normal cells and is activated upon induction of apoptosis. In apoptotic cells, Bax gets translocated to mitochondria, inserts into the outer membrane, oligomerizes and triggers the release of cytochrome c, possibly by channel formation. The BH3 domain-only protein Bid induces a conformational change in Bax before its insertion into the outer membrane. The mechanism by which Bid promotes Bax activation is not understood, and whether Bid is the only protein required for Bax activation is unclear. Here we report that recombinant full-length Bax (Bax(FL)) does not form channels in lipid bilayers when purified as a monomer. In contrast, in the presence of Bid cut with caspase 8 (cut Bid), Bax forms ionic channels in liposomes and planar bilayers. This channel-forming activity requires an interaction between cut Bid and Bax, and is inhibited by Bcl-x(L). Moreover, in the absence of the putative transmembrane C-terminal domain, Bax does not form ionic channels in the presence of cut Bid. Cut Bid does not induce Bax oligomerization in liposomes and the Bax channels formed in the presence of cut Bid are not large enough to permeabilize vesicles to cytochrome c. In conclusion, our results suggest that monomeric Bax(FL) can form channels only in the presence of cut Bid. Cut Bid by itself is unable to induce Bax oligomerization in lipid membranes. It is suggested that another factor that might be present in mitochondria is required for Bax oligomerization.
Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In these cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Here we report that Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerated Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delayed apoptosis. When phosphorylated, Bid was insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis.
The main results were described in a publication (Maina et al., Molecular Cell 7:1293, 2001)Receptor tyrosine kinases (RTKs) mediate distinct biological responses by stimulating similar intracellular signalling pathways. Whether the specificity of the response is determined by qualitative or quantitative differences in signalling output is not known. We addressed this question in vivo by replacing the multifunctional docking sites of Met, the receptor for hepatocyte growth factor, with specific binding motifs for phosphatidylinositol-3 kinase, Src tyrosine kinase, or Grb2 (Met(2P), Met(2S), and Met(2G), respectively). All three mutants retained normal signalling through the multiadaptor Gab1, but differentially recruited specific effectors. While Met(2G) mice developed normally, Met(2P) and Met(2S) mice were loss-of-function mutants displaying different phenotypes and rescue of distinct tissues. These data indicate that RTK-mediated activation of specific signalling pathways is required to fulfil cell-specific functions in vivo
It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). We analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells. We also demonstrated that calmodulin is able to precipitate TrkA from PC12 cell lysates. Using recombinant GST-fusion proteins containing the complete intracellular domain of TrkA, or fragments of this region, we show that calmodulin binds directly to the C-terminal domain of TrkA in a Ca2+-dependent manner. We have also co-immunoprecipitated endogenous Trk and calmodulin in primary cultures of cortical neurones. Moreover, we provided evidence that calmodulin is involved in the regulation of TrkA processing in PC12 cells. Calmodulin inhibition results in the generation of a TrkA-derived p41 fragment from the cytosolic portion of the protein. This fragment is autophosphorylated in tyrosines and can recruit PLCgamma and Shc adaptor proteins. These results suggest that calmodulin binding to Trk may be important for the regulation of Trk intracellular localization and cleavage.
Cholinergic neurons innervating cortical structures are among the most affected neuronal populations in Alzheimer's disease. In rodents, they express high levels of the neurotrophin receptor p75NTR. We analyzed cholinergic septohippocampal neurons of the medial septal nucleus in p75exonIII (partial p75NTR knock-out) and p75exonIV (complete p75NTR knock-out) mice, in their original genetic background and in congenic strains. At postnatal day 15, the p75exonIII mutation leads to a moderate increase (+13%) in these neurons among littermates only after back-crossing in a C57BL/6 background. In contrast, the null p75exonIV mutation, which prevents expression of both the full-length and the shorter p75NTR isoforms, results in a 28% neuronal increase, independent of genetic background. The incomplete nature of the p75NTR mutation used previously, coupled with difficulties in delineating the mouse medial septum and the impact of the genetic background on cell numbers, all contribute to explain previous difficulties in establishing the role of p75NTR in regulating cholinergic neuron numbers in the mouse forebrain.
Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and neublastin/artemin (ART) are distant members of the transforming growth factor beta family, and have been shown to elicit neurotrophic effects upon several classes of peripheral and central neurons. Limited information from in vitro and expression studies has also substantiated a role for GDNF family ligands in mammalian somatosensory neuron development. Here, we show that although dorsal root ganglion (DRG) sensory neurons express GDNF family receptors embryonically, they do not survive in response to their ligands. The regulation of survival emerges postnatally for all GDNF family ligands. GDNF and NTN support distinct subpopulations that can be separated with respect to their expression of GDNF family receptors, whereas ART supports neurons in populations that are also responsive to GDNF or NTN. Sensory neurons that co-express GDNF family receptors are medium sized, whereas small-caliber nociceptive cells preferentially express a single receptor. In contrast to brain-derived neurotrophic factor (BDNF)-dependent neurons, embryonic nerve growth factor (NGF)-dependent nociceptive neurons switch dependency to GDNF, NTN and ART postnatally. Neurons that survive in the presence of neurotrophin 3 (NT3) or neurotrophin 4 (NT4), including proprioceptive afferents, Merkel end organs and D-hair afferents, are also supported by GDNF family ligands neonatally, although at postnatal stages they lose their dependency on GDNF and NTN. At late postnatal stages, ART prevents survival elicited by GDNF and NTN. These data provide new insights on the roles of GDNF family ligands in sensory neuron development.
The TrkB receptor tyrosine kinase and its ligand, BDNF, have an essential role in certain forms of synaptic plasticity. However, the downstream pathways required to mediate these functions are unknown. We studied mice with a targeted mutation in either the Shc or the phospholipase Cgamma (PLCgamma) docking sites of TrkB (trkB(SHC/SHC) and trkB(PLC/PLC) mice). We found that hippocampal long-term potentiation was impaired in trkB(PLC/PLC) mice, but not trkB(SHC/SHC) mice. BDNF stimulation of primary neurons derived from trkB (PLC/PLC) mice fully retained their ability to activate MAP kinases, whereas induction of CREB and CaMKIV phosphorylation was strongly impaired. The opposite effect was observed in trkB (SHC/SHC) neurons, suggesting that MAPKs and CREB act in parallel pathways. Our results provide genetic evidence that TrkB mediates hippocampal plasticity via recruitment of PLCgamma, and by subsequent phosphorylation of CaMKIV and CREB. Signalling by brain-derived neurotrophic factor (BDNF) via the TrkB receptor, or by neurotrophin-3 (NT3) through the TrkC receptor support distinct populations of sensory neurons. The intracellular signalling pathways activated by Trk (tyrosine kinase) receptors, which in vivo promote neuronal survival and target innervation, are not well understood. Using mice with TrkB or TrkC receptors lacking the docking site for Shc adaptors (trkB(shc/shc) and trkC(shc/shc) mice), we show that TrkB and TrkC promote survival of sensory neurons mainly through Shc site-independent pathways, suggesting that these receptors use similar pathways to prevent apoptosis. In contrast, the regulation of target innervation appears different: in trkB(shc/shc) mice neurons lose target innervation, whereas in trkC(shc/shc) mice the surviving TrkC-dependent neurons maintain target innervation and function. Biochemical analysis indicates that phosphorylation at the Shc site positively regulates autophosphorylation of TrkB, but not of TrkC. Our findings show that although TrkB and TrkC signals mediating survival are largely similar, TrkB and TrkC signals required for maintenance of target innervation in vivo are regulated by distinct mechanisms.
Although the requirement of neurotrophins for the prevention of cell death in the peripheral nervous system is well established, their physiological involvement in nerve growth is still unclear. To address this question, we generated a mouse that expresses the green fluorescent protein in post-mitotic neurons, allowing the repeated visualization of all motor and sensory axons during development. We imaged the growth of these axons into the limb bud of day 10.5 embryos. Sensory axons, but rarely motor axons, were targeted to ectopically placed beads containing any of the neurotrophins NGF, BDNF, NT-3 or NT-4/5. Conversely, a combination of function-blocking monoclonal antibodies to NGF, BDNF and NT-3 dramatically inhibited elongation of both sensory and motor axons in the limb bud, indicating that the growth of mixed nerves is dependent upon neurotrophins during development. To understand the mode of action of another group of factors on growth and survival, we have performed an oligonucleotide microarray experiment. We found that several hundred genes were regulated between embryonic and postnatal stages, and that several important classes of genes were differentially regulated by GDNF treatment, including genes related to translation and to phenotypic specification and maturation. Interestingly, a set of genes related to cell adhesion, cytoskeleton and cellular morphology were consistently down regulated by GDNF, suggesting a previously uncharacterized role for GDNF in repressing neurite growth and/or branching. This nuclear program initiated by GDNF was functionally confirmed in cultures of embryonic wild-type neurons sustained with nerve growth factor and in bax(-/-) neurons that survive in the absence of trophic support. Target innervation by specific neuronal populations involves still incompletely understood interactions between central and peripheral factors. We showed that glial cell line-derived neurotrophic factor (GDNF), initially characterized for its role as a survival factor, is present early in the plexus of the developing forelimb. In the absence of GDNF signalling, motor neurons show dramatic reductions in axonal invasion of target muscles. The ETS transcription factor PEA3 is normally expressed by these motor neurons and fails to be induced in most of them in GDNF signalling mutants. Thus, GDNF acts as a peripheral signal to induce PEA3 expression in specific motor neuron pools thereby regulating growth into the target area.
Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. We showed that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-kappaB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival-signalling pathway of CNTF-related cytokines.
If neuron-specific cell death pathways existed, this would be important for explaining the coordinated elimination of neurons during development and perhaps even more so for understanding the selective loss of specific classes in human patients. We showed that motoneuron death could be triggered by activation of the Fas death receptor. In motoneurons, we found a novel Fas signalling pathway, which involves transcription of nNOS and production of NO. Unexpectedly, only motoneurons of the cell types tested activate this pathway in response to Fas. We therefore tested its possible implication in motoneuron disease. Motoneurons purified from mice expressing mutant SOD are less than 100-fold more sensitive to the activation of this pathway than those from mice overexpressing wildtype SOD. This sensitization is restricted to motoneurons. Although they require further in vivo confirmation, these data support the idea that a novel pathway downstream of the Fas death receptor might be a useful therapeutic target in patients with ALS.
The main results were described in three publications (Livet et al., Neuron 35:877, 2002; Haase et al., Neuron 35:893, 2002; Helmbacher et al., Neuron 39:767, 2003). This series of studies used as a model system a particular group of motor neurons in the spinal cord, which innervate two flank muscles called cutaneus maximus (CM) and latissimus dorsi (LD). Based on project result 12225 (above), we knew that HGF signalling is required for the proper innervation of these muscles and analysed the signalling pathways required downstream of the Met receptor. These three papers concerned the coordinated signalling within motor neurons required to achieve muscle innervation. One key aspect of motor neuron positioning is physical segregation into motor pools within the cord, each innervating a single muscle. Neither the mechanism by which these pools are formed, nor the link between cell body grouping and target innervation, were known. The CM and LD pools specifically express the PEA3 transcription factor. In collaboration with S. Arber (Basel) and T. Jessell (NY), we showed that in the absence of PEA3 these motoneurons are mispositioned within the spinal cord and do not correctly innervate their target muscles. Downstream of PEA3, we identified potential effectors (cadherin 8, semaphorin 3E). Upstream, we showed that the neurotrophic factor GDNF, expressed specifically in the brachial plexus and then in the CM and LD muscles, is necessary to trigger expression of PEA3. Consequently, GDNF and PEA3 knockout mice are phenocopies at this level, and this new signalling pathway provides an unexpected link between the peripheral and central factors that contribute to the formation of a motor unit. Lastly, we discovered an additional level of regulation of the formation of the same pools. After initial induction by GDNF, the recruitment of the complete set of PEA3 motoneurons is brought about by a non-cell-autonomous action of the HGF/Met system, thus explaining the deficits first observed in Met mutant mice. These results show that interactions between different neurotrophic factors and signalling pathways are remarkably complex, and can be specific to very limited neuronal ensembles. This undoubtedly has consequences for strategies involving neurotrophic treatments of neurodegenerative disease.
Morphine is a powerful analgesic for severe pain, and this effect is thought to be mediated by inhibitory G-protein coupled receptors (GPCRs). We showed that morphine activates TrkB in a NT-4�dependent manner and provide evidence from transgenic mice that such activation partially mediates morphine-induced analgesia. These findings show that the anti-nociceptive effect of morphine is partially mediated by NT-4�induced TrkB receptor activation.
This work was described in two articles (Schmidt et al., EJN 15:101, 2002; Forgie et al., Development 130:995, 2003). Macrophage stimulating protein (MSP) is a pleiotropic growth factor. MSP is structurally related to hepatocyte growth factor (HGF), another pleiotropic factor whose many functions include promoting neuronal survival and growth. To investigate whether MSP is also capable of acting as a neurotrophic factor, we purified hypoglossal motoneurons from the embryonic chicken hindbrain because these neurons are known to express the MSP receptor tyrosine kinase RON. MSP promoted the in vitro survival of these neurons during the period of naturally occurring neuronal death and enhanced the growth of neurites from these neurons. MSP mRNA was detected in the developing tongue whose musculature is innervated by hypoglossal neurons. Our study demonstrates that MSP is a neurotrophic factor for a population of developing motoneurons. We reported that Ron mRNA is expressed by NGF-dependent sensory and sympathetic neurons and that these neurons survive and grow with MSP at different stages of development. Whereas NGF-dependent sensory neurons become increasingly responsive to MSP with age, sympathetic neurons exhibit an early response to MSP that is lost by birth. MSP mRNA expression increases with age in sensory neuron targets and decreases in sympathetic targets. After the phase of naturally occurring neuronal death, significant numbers of NGF-dependent sensory neurons, but not sensory neurons, dependent on other neurotrophins, are lost in mice lacking a functional Ron receptor. These results show that MSP is a target-derived neurotrophic factor for subsets of sensory and sympathetic neurons at different times during their development.

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