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Address correspondence to Alun Davies, Department of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, Summerhall Square, Edinburgh, EH9 1QH United Kingdom. Tel.: 44-131-650-6116. Fax: 44-131-650-7962. E-mail alun.davies{at}ed.ac.uk
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Abstract |
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Key Words: phosphoinositide 3-kinase; Akt kinase/protein kinase B; Bax; BcL-xL; signaling
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Introduction |
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Considerable progress has been made in delineating the molecular mechanisms that mediate the survival-enhancing effects of NGF and NT3 on embryonic and neonatal sympathetic neurons. Analysis of the Trk receptor tyrosine kinase isoforms expressed by sympathetic neurons (Wyatt et al., 1997) together with in vitro studies of TrkC-deficient neurons (Davies et al., 1995) and the effects of a mutant NT-3 protein that only signals via TrkA (Belliveau et al., 1997) have shown that both NGF and NT-3 promote sympathetic neuron survival by acting via TrkA. Ligand binding causes TrkA dimerization and autophosphorylation on specific tyrosine residues that form docking sites for several adapter proteins including Shc, PLC, and SHP. These proteins couple Trk to several intracellular signaling pathways that result in Shc/Grb2/Gab1-dependent activation of phosphoinositide (PI)* 3-kinase (Holgado-Madruga et al., 1997) and the production of 3'-phosphorylated phosphoinositides, Shc/Grb2/Sos-dependent activation of Ras, and PLC-
mediated generation of DAG and inositol trisphosphate (Kaplan and Miller, 1997).
Studies using function-blocking Ras antibodies or dominant-negative proteins have shown that Ras plays an important role in mediating the survival response of SCG neurons to NGF (Nobes et al., 1996; Markus et al., 1997; Mazzoni et al., 1999). Activated Ras stimulates several downstream signaling cascades including PI 3-kinase and the mitogen-activated protein kinase kinase (MEK)mitogen-activated protein (MAP) kinase pathways (Shields et al., 2000). Studies using Ras effector mutants suggest that Ras exerts its effects on sympathetic neuron survival principally by activating the catalytic subunit of PI 3-kinase (Rodriguez-Viciana et al., 1994; Mazzoni et al., 1999; Xue et al., 2000), whereas MAP kinase activation plays only a minor role or no role at all (Creedon et al., 1996; Virdee and Tolkovsky, 1996; Klesse and Parada, 1998; Mazzoni et al., 1999; Xue et al., 2000). Although overexpression of P1 3-kinase prevents the death of sympathetic neurons after NGF withdrawal, suggesting that PI 3-kinase is sufficient for survival (Philpott et al., 1997; Crowder and Freeman, 1998), evidence for the involvement of PI 3-kinase in mediating the survival-promoting actions of NGF on embryonic and neonatal sympathetic neurons is conflicting. Whereas several studies have reported that pharmacological or dominant-negative inactivation of PI 3-kinase inhibits the NGF survival response (Crowder and Freeman, 1998; Mazzoni et al., 1999; Vaillant et al., 1999), other studies have reported only modest effects of PI 3-kinase inactivation on the survival of developing sympathetic neurons maintained with NGF (Philpott et al., 1997; Virdee et al., 1999; Tsui-Pierchala et al., 2000). Recently, it has been shown that there is a more critical dependence on PI 3-kinase for survival of sympathetic neurons supported by NGF acting on their distal axons as compared with neurons supported by NGF acting directly on their cell bodies (Kuruvilla et al., 2000).
The serine/threonine protein kinase Akt (protein kinase B), a downstream effector of PI 3-kinase, appears to play a key role in mediating the NGF survival response of developing sympathetic neurons since constitutively active Akt prevents the death of these neurons after NGF deprivation and dominant-negative Akt kills these neurons in the presence of NGF (Crowder and Freeman, 1998; Virdee et al., 1999). Akt influences the activity of several transcription factors implicated in regulating cell survival, including Forkhead 1 (Brunet et al., 1999), NF-B (Kane et al., 1999; Ozes et al., 1999), and CREB (Du and Montminy, 1998). Phosphorylation of Forkhead 1 by Akt prevents it from inducing expression of the cytotoxic factor FasL whose synthesis after growth factor withdrawal plays a role in causing neuronal apoptosis by initiating a caspase cascade (Brunet et al., 1999; Le-Niculescu et al., 1999; Raoul et al., 1999). Induction of NF-
B and CREB transcriptional activity by Akt results in expression of their target genes that include those encoding Bcl-2 and inhibitor of apoptosis proteins, which are required for sympathetic neuron survival in the presence of NGF (Riccio et al., 1999; Wiese et al., 1999). Akt also phosphorylates Bad, a proapoptotic member of the Bcl-2 family, which may prevent it from interacting with and inactivating antiapoptotic members of this family (Datta et al., 1997; del Peso et al., 1997).
Proteins of the Bcl-2 family play a key role in controlling the activation of caspases, which are the proteases that dismantle the cell during apoptosis (Korsmeyer, 1999; Vaux and Korsmeyer, 1999). Bcl-2related proteins fall into two groups that generally either repress apoptosis (e.g., Bcl-2 and Bcl-xL) or promote apoptosis (e.g., Bax, Bcl-xs, Bak, and Bad). These proteins influence caspase activation in part by controlling the release of cytochrome c from mitochondria that interacts with the adapter protein Apaf-1, which in turn activates procaspase-9 (Li et al., 1997; Qin et al., 1999). Proapoptotic members like Bax and Bak increase mitochondrial permeability allowing cytochrome c to pass into the cytosol, whereas antiapoptotic members like Bcl-2 and Bcl-xL prevent cytochrome c release (Kharbanda et al., 1997; Kluck et al., 1997; Yang et al., 1997; Shimizu et al., 1999). In addition, Bcl-2 is also able to regulate activation of membrane-associated procaspase-3 independently of cytochrome c (Krebs et al., 1999).
Although a good deal is known about the molecular mechanisms that mediate the survival effects of neurotrophins and induce apoptosis after their withdrawal in developing neurons, the mechanisms that sustain the survival of adult neurotrophic factorindependent neurons has received very little attention. One experimental paradigm used to address the intriguing issue of neurotrophic factor independence has involved growing embryonic sympathetic or sensory neurons in culture with NGF until they reach a point when they continue to survive after NGF is withdrawn. In sensory neurons grown under these conditions, inhibition of PI 3-kinase did not compromise survival after NGF withdrawal, suggesting that PI 3-kinase activation is not required for sustaining neuronal survival in this model of neurotrophin independence (Vogelbaum et al., 1998). In embryonic sympathetic neurons grown under these conditions, there is evidence for a block in the apoptotic pathway just upstream of caspase activation close to the point at which Bax acts (Greenlund et al., 1995; Easton et al., 1997). To investigate the molecular mechanisms that underlie neurotrophin independence in adult neurons, we purified and cultured sympathetic neurons from the superior cervical ganglion (SCG) of adult rats. We show that PI-3 kinase/Akt activation and the expression of antiapoptotic members of the Bcl-2 family are required for sustaining the survival of these neurons in the absence of added neurotrophic factors.
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Results |
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Akt activation is required for adult SCG neuron survival
Because the downstream effector of PI 3-kinase, Akt, plays a key role in mediating the survival response of neurons to neurotrophins (Crowder and Freeman, 1998; Vaillant et al., 1999), we investigated whether Akt activation is required for the survival of adult SCG neurons. Injection of an expression plasmid that directs the synthesis of a kinase-inactive mutant of Akt that acts as a dominant-negative protein (Virdee et al., 1999) caused the death of a substantial number of adult SCG neurons. Over 70% of these neurons had died 5 d after injection with the dominant-negative Akt expression plasmid compared with 20% of control-injected neurons (Fig. 4). The magnitude of neuronal death effected by expression of dominant-negative Akt was similar to that caused by expression of p85 or overexpression of Rukl. These results suggest that Akt is the main downstream effector of PI 3-kinase in sustaining the survival of adult SCG neurons in the absence of added neurotrophic factors.
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Phospho-Akt is present in adult SCG neurons surviving without neurotrophic factors
To ascertain if the active, phosphorylated form of Akt is present in adult SCG neurons surviving without neurotrophic factors and whether this is dephosphorylated after inhibition of PI 3-kinase, we used immunocytochemistry to visualize Akt protein and phospho-Akt protein in SCG neurons grown with and without the PI 3-kinase inhibitor LY294002. Fig. 5 A shows that SCG neurons were clearly immunoreactive for phospho-Akt protein when grown at low density in defined medium alone. Although phospho-Akt immunoreactivity was clearly reduced by treatment with LY294002 for 45 min (Fig. 5G), expression of total Akt protein was little affected as revealed by the similar level of Akt immunofluorescence in the presence and absence of LY294002 (Fig. 5, B and C). There was no obvious change in the expression of either phospho-Akt immunoreactivity or total Akt protein in adult SCG neurons treated for 45 min with NGF (Fig. 5, A and E). No staining was observed when the primary antibodies were omitted from the staining protocol (Fig. 5 H), indicating that there was no detectable nonspecific staining. Taken together, these results suggest that Akt is constitutively active in adult SCG neurons surviving without neurotrophic factors and that PI 3-kinase is required for sustaining Akt activation.
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Overexpression of Bad or Bax kills adult SCG neurons
In addition to interfering with the synthesis of antiapoptotic members of the Bcl-2 family to ascertain the importance of these proteins in sustaining the survival of adult SCG neurons, we also overexpressed proapoptotic members of this family to see if this would compromise the survival of these neurons. The most marked effect on survival was observed by overexpressing Bad. Injection of a Bad expression plasmid caused the rapid death of the majority of adult SCG neurons, leaving <20% surviving 5 d after injection (Fig. 9). Injection of a Bax expression plasmid caused much less neuronal death, leaving 40% surviving 5 d after injection (Fig. 9).
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Discussion |
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Although the role of PI 3-kinase in mediating the survival-promoting action of NGF on embryonic and neonatal sympathetic neurons is controversial (Philpott et al., 1997; Crowder and Freeman, 1998; Mazzoni et al., 1999; Vaillant et al., 1999; Virdee et al., 1999; Tsui-Pierchala et al., 2000), studies of several other kinds of neurons and many other cell types have shown that PI 3-kinase and serine/threonine kinase Akt/PKB play major roles in mediating the survival response of many different kinds of cells to a variety of growth factors (Datta et al., 1999). However, our study provides the first evidence that activation of PI 3-kinase and Akt is required to prevent the death of cells that survive independently of extracellular growth factors.
Even though adult SCG neurons survive as single cells in serum-free medium, nonetheless, it is possible that they could be sustained by neurotrophic factors that they produce themselves. Autocrine action of neurotrophic factors has been described previously for BDNF acting on embryonic and adult sensory neurons (Wright et al., 1992; Acheson et al., 1995) and for HGF acting on sympathetic neuroblasts (Maina et al., 1998). Although autocrine trophic support of adult sympathetic neurons is entirely possible, preliminary data suggest that several of the likely candidates are unlikely to be involved. Adult SCG neurons are not killed by the inhibitor of Trk receptor tyrosine kinases K252a (Orike et al., 2000) or by function-blocking antibodies to GFR-3, a key component of the artemin receptor (Andres et al., 2001) or HGF (N. Orike, J. Thompson, and A.M. Davies, unpublished data). These findings suggest that neither NGF and NT-3, which promote the survival of developing sympathetic neurons (Levi-Montalcini, 1987; Crowley et al., 1994; Zhou and Rush, 1995; Wyatt et al., 1997; Francis et al., 1999), HGF, which enhances the survival of sympathetic neuroblasts (Maina et al., 1998), nor artemin, which is important for the survival of a SCG neurons during the postnatal period (Nishino et al., 1999; Andres et al., 2001), are required for the survival of adult SCG neurons. Furthermore, the activation of PI 3-kinase by NGF in SCG neurons is thought to occur predominantly by direct activation of the p110 catalytic subunit by Ras (Rodriguez-Viciana et al., 1994; Nobes et al., 1996; Markus et al., 1997; Mazzoni et al., 1999; Xue et al., 2000). Yet we have shown that inhibiting the function of the p85 regulatory subunit of Class IA PI 3-kinase by expressing a dominant-negative protein or overexpressing Rukl kills adult SCG neurons, indicating that this regulatory subunit is crucial for maintaining the activity of the catalytic subunit at the level required to sustain the survival of adult SCG neurons.
Our demonstration that PI 3-kinase activity is required for the survival of adult rat sympathetic neurons in the absence of neurotrophic factors differs from the findings of studies of embryonic sympathetic or sensory neurons that attained neurotrophic factor independence in culture after prolonged incubation with NGF. Dorsal root ganglion neurons from E15 rat embryos that were maintained for 3 wk in the presence of NGF survived after NGF withdrawal and were not killed by inhibiting PI 3-kinase activation with LY294002, but were killed by LY294002 treatment at earlier time points before the acquisition of NGF independence (Vogelbaum et al., 1998). E21 rat SCG neurons maintained for 3 wk with NGF also survived after NGF deprivation, but exhibited a rapid decrease in MAP kinase activation and early changes in gene expression after NGF deprivation that are typical of NGF withdrawal before the acquisition of NGF independence. However, like Bax-deficient embryonic neurons that survive after NGF deprivation, embryonic SCG neurons maintained in long-term culture with NGF do not exhibit c-fos induction after NGF deprivation. These observations, together with the finding that overexpression of Bax in these neurons restored their survival dependence on NGF, suggest that the apoptotic pathway was blocked at a point just upstream of caspase activation close to the point at which Bax acts (Greenlund et al., 1995; Easton et al., 1997). Thus, although embryonic sensory and sympathetic neurons lose dependence on NGF after being cultured with this factor for several weeks, the mechanism of neurotrophin independence appears to be different from that of adult sympathetic neurons that rely on PI 3-kinase activity for survival. However, one potential caveat of our study is that we have studied the role of PI 3-kinase shortly after plating when a stress response to axotomy might make them more susceptible to PI 3-kinase inhibition. In future work, it will be important to address the relevance of our in vitro observations to the role of PI 3-kinase in sustaining the survival of adult sympathetic neurons in vivo.
Another signaling pathway that has been implicated in neuronal survival under certain circumstances is the MEK/MAP kinase pathway. Although this Ras-activated pathway plays either no role or only a minor role in mediating the survival response of developing neurons to NGF (Creedon et al., 1996; Virdee and Tolkovsky, 1996; Klesse and Parada, 1998; Mazzoni et al., 1999; Xue et al., 2000), it plays a major role in BDNF neuroprotection against camptothecin (Hetman et al., 1999) and is capable of reducing p53-dependent apoptosis induced by cytosine arabinoside in sympathetic neurons (Anderson and Tolkovsky, 1999). Our demonstration that the MEK inhibitor PD98059 does not kill adult SCG neurons suggests that the MEK/MAP kinase activation is not required for sustaining the survival of these neurons.
Considerable evidence indicates that members of the Bcl-2 play a key role in regulating neuronal survival during development. Overexpression of Bcl-2 or Bcl-xL promote the survival of developing neurons in vitro and in vivo (Garcia et al., 1992; Allsopp et al., 1993; Martinou et al., 1994; Farlie et al., 1995; Gonzalez-Garcia et al., 1995; Middleton et al., 1996; Parsadanian et al., 1998), whereas reduction or elimination of endogenous Bcl-2 or Bcl-xL reduces neuronal survival (Allsopp et al., 1995; Motoyama et al., 1995; Michaelidis et al., 1996; Piñón et al., 1997). In contrast, Bax overexpression accelerates neuronal death after neurotrophin withdrawal (Vekrellis et al., 1997; Martinou et al., 1998), and reduction or elimination of endogenous Bax promotes the survival of neurons without neurotrophic factors in culture and prevents their death in vivo (Deckwerth et al., 1996; Gillardon et al., 1996; Miller et al., 1997; White et al., 1998; Bar-Peled et al., 1999). Our studies of manipulating the expression of Bcl-2 family proteins in adult sympathetic neurons have likewise shown that these proteins also play an important role in regulating the survival of mature sympathetic neurons. Expression of the proapoptotic proteins Bad, Bax, and Bcl-xS killed these neurons as did antisense RNA to the antiapoptotic proteins Bcl-xL and Bcl-2. A link between PI 3-kinase/Akt signaling and the function of these proteins has come from the demonstration that activated Akt phosphorylates Bad, resulting in its sequestration by cytosolic 14-3-3 proteins (Datta et al., 1997; del Peso et al., 1997). Because Bad brings about apoptosis by binding to and inhibiting the antiapoptotic actions of Bcl-xL, the sequestration of Bad in the cytosol by 14-3-3 proteins results in enhanced survival.
In summary, we have shown that the survival of adult sympathetic neurons is dependent on PI 3-kinase/Akt signaling and the expression of the antiapoptotic proteins Bcl-xL and Bcl-2. In future work, it will be important to ascertain which proteins interact with the p85 regulatory subunit of Class IA PI 3-kinase in adult sympathetic neurons to maintain the activity of the holoenzyme and understand what brings about the change in PI 3-kinase regulation between postnatal animals and the adult.
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Materials and methods |
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The neurons were initially grown overnight and were microinjected intranuclearly with the aid of a manually operated Narishige micromanipulator (Allsopp et al., 1993) with expression plasmids encoding wild-type or mutated signaling proteins or members of the Bcl-2 family in the sense or antisense orientation. Expression plasmids without a cDNA insert were used to control for any nonspecific effects of the injection procedure. The expression plasmids were diluted in 100 mM potassium phosphate buffer, pH 7, to a concentration of 100 ng/ml and filtered through a 0.22-µm filter before injection. All neurons within a specific grid square marked on the inside of the culture dish were pressure-injected. Intranuclear injection resulted in >95% viability assessed 1 h after injection. The number of surviving neurons was counted at 12 or 24 hourly intervals after injection, and is expressed as a percentage of the number injected in the grid square. About 150 neurons were injected for each experimental condition and followed longitudinally in each experiment.
Live and dead neurons were clearly distinguished by the morphology when examined by phase-contrast microscopy. Live neurons had large, smooth, intact cell bodies and smooth, unbroken neurites. Dead neurons rapidly disintegrated, leaving cell body ghosts comprised of cellular debris and fragmented neurites, which are appearances that are typical of neuronal apoptosis in culture (Allsopp et al., 1993). In addition to these characteristic appearances, we also used DAPI to stain nuclear chromatin to determine if the chromatin condensation, which is typical of apoptosis, occurs in adult sympathetic neurons dying as a consequence of inhibiting PI-3 kinase.
Plasmids and pharmacological reagents
A variety of expression plasmids were used to express wild-type or mutated proteins or antisense RNA in cultured sympathetic neurons. cDNAs encoding members of the Bcl-2 family (Bcl-2, Bcl-xL, Bcl-xS, Bad, and Bax) were cloned into the pSFFV vector in the sense and antisense orientations. The pCMV vector was used to express the kinase-inactive mutant of PKB/Akt, Rukl and Rukm, and the pSG5 vector was used to express the p85 dominant-negative form of PI 3-kinase. The PI 3-kinase inhibitors LY294002 and Wortmannin, the MEK inhibitor PD98059, and the caspase inhibitor Z-VAD-FMK were obtained from Calbiochem.
Immunocytochemistry
Cultures of adult SCG neurons were fixed with fresh 4% paraformaldehyde for 15 min, washed in TBS with .1% Triton X-100 (TBT) and blocked with TBT plus 10% goat serum. They were incubated with primary antibody (rabbit polyclonals from Santa Cruz Biotechnology, Inc. and New England Biolabs, Inc.) at a dilution of 1:100 overnight at 4°C, washed in TBT, and incubated with FITC-labeled goat antirabbit antiserum (Sigma-Aldrich) at a dilution of 1:500 at room temperature for 1 h. The cultures were washed, mounted beneath coverslips, and viewed with an Axioskop fluorescence microscope.
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Footnotes |
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The present address of N. Orike is Montreal Neurological Institute, Montreal, Quebec, H3A 2B4, Canada.
* Abbreviations used in this paper: MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase kinase; PI, phosphoinositide; SCG, superior cervical ganglion.
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Acknowledgments |
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This work was supported by grants from the Wellcome Trust (to V. Buchman, T. Cowen, and A.M. Davies).
Submitted: 22 January 2001
Revised: 27 July 2001
Accepted: 31 August 2001
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References |
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---|
Acheson, A., J.C. Conover, J.P. Fandl, T.M. DeChlara, M. Russell, A. Thadani, S.P. Squinto, G.D. Yancopoulos, and R.M. Lindsay. 1995. A BDNF autocrine loop in adult sensory neurons prevents cell death. Nature. 374:450453.[Medline]
Alessi, D.R., A. Cuenda, P. Cohen, D.T. Dudley, and A.R. Saltiel. 1995. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270:2748927494.
Allsopp, T.E., S. Wyatt, H.F. Paterson, and A.M. Davies. 1993. The proto-oncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis. Cell. 73:295307.[Medline]
Allsopp, T.E., S. Kiselev, S. Wyatt, and A.M. Davies. 1995. Role of Bcl-2 expression in the BDNF survival response. Eur. J. Neurosci. 7:12661272.[Medline]
Anderson, C.N.G., and A.M. Tolkovsky. 1999. A role for MAPK/ERK in sympathetic neuron survival: protection against a p53-dependent, JNK-independent induction of apoptosis by cytosine arabinoside. J. Neurosci. 19:664673.
Andres, R., A. Forgie, S. Wyatt, Q. Chen, F.J. de Sauvage, and A.M. Davies. 2001. Multiple effects of artemin on sympathetic neuron generation, survival and growth. Development. In press.
Bar-Peled, O., M. Knudson, S.J. Korsmeyer, and J.D. Rothstein. 1999. Motor neuron degeneration is attenuated in bax-deficient neurons in vitro. J. Neurosci. Res. 55:542556.[Medline]
Belliveau, D.J., I. Krivko, J. Kohn, C. Lachance, C. Pozniak, D. Rusakov, D. Kaplan, and F.D. Miller. 1997. NGF and neurotrophin-3 both activate TrkA on sympathetic neurons but differentially regulate survival and neuritogenesis. J. Cell Biol. 136:375388.
Boise, L.H., G.M. Gonzalez, C.E. Postema, L. Ding, T. Lindsten, L.A. Turka, X. Mao, G. Nunez, and C.B. Thompson. 1993. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 74:597608.[Medline]
Brunet, A., A. Bonni, M.J. Zigmond, M.Z. Lin, P. Juo, L.S. Hu, M.J. Anderson, K.C. Arden, J. Blenis, and M.E. Greenberg. 1999. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell. 96:857868.[Medline]
Burgering, B.M., and P.J. Coffer. 1995. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 376:599602.[Medline]
Creedon, D.J., E.M. Johnson, and J.C. Lawrence. 1996. Mitogen-activated protein kinase-independent pathways mediate the effects of nerve growth factor and cAMP on neuronal survival. J. Biol. Chem. 271:2071320718.
Crowder, R.J., and R.S. Freeman. 1998. Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons. J. Neurosci. 18:29332943.
Crowley, C., S.D. Spencer, M.C. Nishimura, K.S. Chen, S. Pitts-Meek, M.P. Armanini, L.H. Ling, S.B. McMahon, D.L. Shelton, A.D. Levinson, and H.S. Phillips. 1994. Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell. 76:10011011.[Medline]
Datta, S.R., A. Brunet, and M.E. Greenberg. 1999. Cellular survival: a play in three Akts. Genes Dev. 13:29052927.
Datta, S.R., H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, and M.E. Greenberg. 1997. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 91:231241.[Medline]
Davies, A.M. 1986. The survival and growth of embryonic proprioceptive neurons is promoted by a factor present in skeletal muscle. Dev. Biol. 115:5667.[Medline]
Davies, A.M., L. Minichiello, and R. Klein. 1995. Developmental changes in NT3 signalling via TrkA and TrkB in embryonic neurons. EMBO J. 14:44824489.[Abstract]
Deckwerth, T.L., J.L. Elliott, C.M. Knudson, E.M. Johnson, Jr., W.D. Snider, and S.J. Korsmeyer. 1996. BAX is required for neuronal death after trophic factor deprivation and during development. Neuron. 17:401411.[Medline]
del Peso, L., M. Gonzalez-Garcia, C. Page, R. Herrera, and G. Nunez. 1997. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science. 278:687689.
Dhand, R., K. Hara, I. Hiles, B. Bax, I. Gout, G. Panayotou, M.J. Fry, K. Yonezawa, M. Kasuga, and M.D. Waterfield. 1994. PI 3-kinase: structural and functional analysis of intersubunit interactions. EMBO J. 13:511521.[Abstract]
Du, K., and M. Montminy. 1998. CREB is a regulatory target for the protein kinase Akt/PKB. J. Biol. Chem. 273:3237732379.
Easton, R.M., T.L. Deckwerth, A.S. Parsadanian, and E.M. Johnson, Jr. 1997. Analysis of the mechanism of loss of trophic factor dependence associated with neuronal maturation: a phenotype indistinguishable from Bax deletion. J. Neurosci. 17:96569666.
Farlie, P.G., R. Dringen, S.M. Rees, G. Kannourakis, and O. Bernard. 1995. bcl-2 transgene expression can protect neurons against developmental and induced cell death. Proc. Natl. Acad. Sci. USA. 92:43974401.[Abstract]
Fearnhead, H.O., D. Dinsdale, and G.M. Cohen. 1995. An interleukin-1 beta-converting enzyme-like protease is a common mediator of apoptosis in thymocytes. FEBS Lett. 375:283288.[Medline]
Francis, N., I. Farinas, C. Brennan, K. Rivas-Plata, C. Backus, L. Reichardt, and S. Landis. 1999. NT-3, like NGF, is required for survival of sympathetic neurons, but not their precursors. Dev. Biol. 210:411427.[Medline]
Garcia, I., I. Martinou, Y. Tsujimoto, and J.C. Martinou. 1992. Prevention of programmed cell death of sympathetic neurons by the bcl-2 proto-oncogene. Science. 258:302304.[Medline]
Gillardon, F., M. Zimmermann, E. Uhlmann, S. Krajewski, J.C. Reed, and L. Klimaschewski. 1996. Antisense oligodeoxynucleotides to bax mRNA promote survival of rat sympathetic neurons in culture. J. Neurosci. Res. 43:726734.[Medline]
Gonzalez-Garcia, M., I. Garcia, L. Ding, S. O'Shea, L.H. Boise, C.B. Thompson, and G. Nunez. 1995. bcl-x is expressed in embryonic and postnatal neural tissues and functions to prevent neuronal cell death. Proc. Natl. Acad. Sci. USA. 92:43044308.[Abstract]
Gout, I., G. Middleton, J. Adu, N.N. Ninkina, L.B. Drobot, V. Filonenko, G. Matsuka, A.M. Davies, M. Waterfield, and V.L. Buchman. 2000. Negative regulation of PI 3-kinase by Ruk, a novel adaptor protein. EMBO J. 19:40154025.
Greenlund, L.J., S.J. Korsmeyer, and E.M. Johnson. 1995. Role of BCL-2 in the survival and function of developing and mature sympathetic neurons. Neuron. 15:649661.[Medline]
Hetman, M., K. Kanning, J.E. Cavanaugh, and Z. Xia. 1999. Neuroprotection by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. J. Biol. Chem. 274:2256922580.
Holgado-Madruga, M., D.K. Moscatello, D.R. Emlet, R. Dieterich, and A.J. Wong. 1997. Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor. Proc. Natl. Acad. Sci. USA. 94:1241912424.
Kane, L.P., V.S. Shapiro, D. Stokoe, and A. Weiss. 1999. Induction of NF-kappaB by the Akt/PKB kinase. Curr. Biol. 9:601604.[Medline]
Kaplan, D.R., and F.D. Miller. 1997. Signal transduction by the neurotrophin receptors. Curr. Opin. Cell Biol. 9:213221.[Medline]
Kharbanda, S., P. Pandey, L. Schofield, S. Israels, R. Roncinske, K. Yoshida, A. Bharti, Z.M. Yuan, S. Saxena, R. Weichselbaum, C. Nalin, and D. Kufe. 1997. Role for Bcl-xL as an inhibitor of cytosolic cytochrome c accumulation in DNA damage-induced apoptosis. Proc. Natl. Acad. Sci. USA. 94:69396942.
Klesse, L.J., and L.F. Parada. 1998. p21 ras and phosphatidylinositol-3 kinase are required for survival of wild-type and NF1 mutant sensory neurons. J. Neurosci. 18:1042010428.
Kluck, R.M., E. Bossy-Wetzel, D.R. Green, and D.D. Newmeyer. 1997. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science. 275:11321136.
Korsmeyer, S.J. 1999. BCL-2 gene family and the regulation of programmed cell death. Cancer Res. 59:1693s1700s.[Medline]
Krebs, J.F., R.C. Armstrong, A. Srinivasan, T. Aja, A.M. Wong, A. Aboy, R. Sayers, B. Pham, T. Vu, K. Hoang, et al. 1999. Activation of membrane-associated procaspase-3 is regulated by Bcl-2. J. Cell Biol. 144:915926.
Kuruvilla, R., H. Ye, and D.D. Ginty. 2000. Spatially and functionally distinct roles of the PI3-K effector pathway during NGF signaling in sympathetic neurons. Neuron. 27:499512[Medline]
Le-Niculescu, H., E. Bonfoco, Y. Kasuya, F.X. Claret, D.R. Green, and M. Karin. 1999. Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to Fas ligand induction and cell death. Mol. Cell. Biol. 19:751763.
Levi-Montalcini, R. 1987. The nerve growth factor 35 years later. Science. 237:11541162.[Medline]
Li, P., D. Nijhawan, I. Budihardjo, S.M. Srinivasula, M. Ahmad, E.S. Alnemri, and X. Wang. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91:479489.[Medline]
Maina, F., M.C. Hilton, R. Andres, S. Wyatt, R. Klein, and A.M. Davies. 1998. Multiple roles for hepatocyte growth factor in sympathetic neuron development. Neuron. 20:835846.[Medline]
Markus, A., A. von Holst, H. Rohrer, and R. Heumann. 1997. NGF-mediated survival depends on p21ras in chick sympathetic neurons from the superior cervical but not from lumbosacral ganglia. Dev. Biol. 191:306310.[Medline]
Martinou, I., M. Missotten, P.A. Fernandez, R. Sadoul, and J.C. Martinou. 1998. Bax and Bak proteins require caspase activity to trigger apoptosis in sympathetic neurons. Neuroreport. 9:1519.[Medline]
Martinou, J.C., M. Dubois-Dauphin, J.K. Staple, I. Rodriguez, H. Frankowsky, M. Missotten, P. Albertini, D. Talabot, S. Catsicas, C. Pietra, and J. Huarte. 1994. Overexpression of bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischaemia. Neuron. 13:10171030.[Medline]
Mazzoni, I.E., F.A. Said, R. Aloyz, F.D. Miller, and D. Kaplan. 1999. Ras regulates sympathetic neuron survival by suppressing the p53-mediated cell death pathway. J. Neurosci. 19:97169727.
Michaelidis, T.M., M. Sendtner, J.D. Cooper, M.S. Airaksinen, B. Holtmann, M. Meyer, and H. Thoenen. 1996. Inactivation of bcl-2 results in progressive degeneration of motoneurons, sympathetic neurons and sensory neurons during the early postnatal development. Neuron. 17:7589.[Medline]
Middleton, G., G. Nunez, and A.M. Davies. 1996. Bax promotes neuronal survival and antogonises the survival effects of trophic factors. Development. 122:695701.
Miller, T.M., K.L. Moulder, C.M. Knudson, D.J. Creedon, M. Deshmukh, S.J. Korsmeyer, and E.M. Johnson, Jr. 1997. Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death. J. Cell Biol. 139:205217.
Motoyama, N., F. Wang, K.A. Roth, H. Sawa, K. Nakayama, K. Nakayama, I. Negishi, S. Senju, Q. Zhang, S. Fujii, and D.Y. Loh. 1995. Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science. 267:15061510.[Medline]
Nishino, J., K. Mochida, Y. Ohfuji, T. Shimazaki, C. Meno, S. Ohishi, Y. Matsuda, H. Fujii, Y. Saijoh, and H. Hamada. 1999. GFR alpha3, a component of the artemin receptor, is required for migration and survival of the superior cervical ganglion. Neuron. 23:725736.[Medline]
Nobes, C.D., J.B. Reppas, A. Markus, and A.M. Tolkovsky. 1996. Active p21Ras is sufficient for rescue of NGF-dependent rat sympathetic neurons. Neuroscience. 70:10671079.[Medline]
Okada, T., Y. Kawano, T. Sakakibara, O. Hazeki, and M. Ui. 1994. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J. Biol. Chem. 269:35683573.
Orike, N., C. Thrasivoulou, A. Wrigley, and T. Cowen. 2000. Differential regulation of survival and growth in adult sympathetic neurons: an in vitro study of neurotrophin responsiveness. J. Neurobiol. 47:295305.
Ozes, O.N., L.D. Mayo, J.A. Gustin, S.R. Pfeffer, L.M. Pfeffer, and D.B. Donner. 1999. NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature. 401:8285.[Medline]
Parsadanian, A.S., Y. Cheng, C.R. Keller-Peck, D.M. Holtzman, and W.D. Snider. 1998. Bcl-xL is an antiapoptotic regulator for postnatal CNS neurons. J. Neurosci. 18:10091019.
Philpott, K.L., M.J. McCarthy, A. Klippel, and L.L. Rubin. 1997. Activated phosphatidylinositol 3-kinase and Akt kinase promote survival of superior cervical neurons. J. Cell Biol. 139:809815.
Piñón, L.G.P., G. Middleton, and A.M. Davies. 1997. Bcl-2 is required for cranial sensory neuron survival at defined stages of embryonic development. Development. 124:41734178.
Qin, H., S.M. Srinivasula, G. Wu, T. Fernandes-Alnemri, E.S. Alnemri, and Y. Shi. 1999. Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1. Nature. 399:549557.[Medline]
Raoul, C., C.E. Henderson, and B. Pettmann. 1999. Programmed cell death of embryonic motoneurons triggered through the Fas death receptor. J. Cell Biol. 147:10491062.
Riccio, A., S. Ahn, C.M. Davenport, J.A. Blendy, and D.D. Ginty. 1999. Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons. Science. 286:23582361.
Rodriguez-Viciana, P., P.H. Warne, R. Dhand, B. Vanhaesebroeck, I. Gout, M.J. Fry, M.D. Waterfield, and J. Downward. 1994. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 370:527532.[Medline]
Shields, J.M., K. Pruitt, A. McFall, A. Shaub, and C.J. Der. 2000. Understanding Ras: "it ain't over 'til it's over." Trends Cell Biol. 10:147154.[Medline]
Shimizu, S., M. Narita, and Y. Tsujimoto. 1999. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature. 399:483487.[Medline]
Tsui-Pierchala, B.A., G.V. Putcha, E.M. Johnson, Jr. 2000. Phosphatidylinositol 3-kinase is required for the trophic, but not the survival-promoting, actions of NGF on sympathetic neurons. J. Neurosci. 20:72287237.
Vaillant, A.R., I. Mazzoni, C. Tudan, M. Boudreau, D.R. Kaplan, and F.D. Miller. 1999. Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinase-Akt pathway to synergistically regulate neuronal survival. J. Cell Biol. 146:955966.
Vaux, D.L., and S.J. Korsmeyer. 1999. Cell death in development. Cell. 96:245254.[Medline]
Vekrellis, K., M.J. McCarthy, A. Watson, J. Whitfield, L.L. Rubin, and J. Ham. 1997. Bax promotes neuronal cell death and is downregulated during the development of the nervous system. Development. 124:12391249.
Virdee, K., and A.M. Tolkovsky. 1996. Inhibition of p42 and p44 mitogen-activated protein kinase activity by PD98059 does not suppress nerve growth factor-induced survival of sympathetic neurones. J. Neurochem. 67:18011805.[Medline]
Virdee, K., L. Xue, B.A. Hemmings, C. Goemans, R. Heumann, and A.M. Tolkovsky. 1999. Nerve growth factor-induced PKB/Akt activity is sustained by phosphoinositide 3-kinase dependent and independent signals in sympathetic neurons. Brain Res. 837:127142.[Medline]
Vlahos, C.J., W.F. Matter, K.Y. Hui, and R.F. Brown. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269:52415248.
Vogelbaum, M.A., J.X. Tong, and K.M. Rich. 1998. Developmental regulation of apoptosis in dorsal root ganglion neurons. J. Neurosci. 18:89288935.
White, F.A., C.R. Keller-Peck, C.M. Knudson, S.J. Korsmeyer, and W.D. Snider. 1998. Widespread elimination of naturally occurring neuronal death in Bax-deficient mice. J. Neurosci. 18:14281439.
Wiese, S., M.R. Digby, J.M. Gunnersen, R. Gotz, G. Pei, B. Holtmann, J. Lowenthal, and M. Sendtner. 1999. The anti-apoptotic protein ITA is essential for NGF-mediated survival of embryonic chick neurons. Nat. Neurosci. 2:978983.[Medline]
Wright, E.M., K.S. Vogel, and A.M. Davies. 1992. Neurotrophic factors promote the maturation of developing sensory neurons before they become dependent on these factors for survival. Neuron. 9:139150.[Medline]
Wyatt, S., L.G.P. Piñón, P. Ernfors, and A.M. Davies. 1997. Sympathetic neuron survival and TrkA expression in NT3-deficient mouse embryos. EMBO J. 16:31153123.
Xue, L., J.H. Murray, and A.M. Tolkovsky. 2000. The Ras/phosphatidylinositol 3-kinase and Ras/ERK pathways function as independent survival modules each of which inhibits a distinct apoptotic signaling pathway in sympathetic neurons. J. Biol. Chem. 275:88178824.
Yang, J., X. Liu, K. Bhalla, C.N. Kim, A.M. Ibrado, J. Cai, T.I. Peng, D.P. Jones, and X. Wang. 1997. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 275:11291132.
Zhou, X., and R.A. Rush. 1995. Sympathetic neurons in neonatal rats require endogenous neurotrophin-3 for survival. J. Neurosci. 15:65216530.[Medline]