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fibroblast growth factor receptor signalling cascade
www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 26 (2004) 17 – 23
A dimeric version of the short N-cadherin binding motif HAVDI promotes neuronal cell survival by activating an N-cadherin/ fibroblast growth factor receptor signalling cascade Stephen D. Skaper, a,* Laura Facci, a Gareth Williams, b Emma-Jane Williams, b Frank S. Walsh, 1 and Patrick Doherty b a
Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research & Development Limited, New Frontiers Science Park, Harlow CM19 5AW, Essex, UK b Molecular Neurobiology Group, Medical Research Council Centre for Developmental Neurobiology, King’s College London, London SE1 1UL, UK Received 21 July 2003; revised 11 December 2003; accepted 19 December 2003 Available online 20 March 2004 The HAVDI and INPISGQ sequences have been identified as functional binding motifs in extracellular domain 1 (ECD1) of N-cadherin. Cyclic peptides containing a tandem repeat of the individual motifs function as N-cadherin agonists and stimulate neurite outgrowth. We now show that the cyclic peptide N-Ac-CHAVDINGHAVDIC-NH2 (SW4) containing the HAVDI sequence in tandem is efficacious also in promoting the in vitro survival of several populations of central nervous system neurons in paradigms where fibroblast growth factor-2 (FGF-2) is active. SW4 supported the survival of rat postnatal cerebellar granule neurons plated in serum-free medium and limited the death of differentiated granule neurons induced to die by switch to low K+ medium. In addition, SW4 rescued embryonic hippocampal and cortical neurons from injury caused by glutamic acid excitotoxicity. The neuroprotective effects of SW4 displayed a concentration dependence similar to those inducing neuritogenesis, were inhibited by a monomeric version of the same motif and by a specific FGF receptor antagonist (PD173074), and were not mimicked by the linear peptide. Inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase), MAP kinase, and p38 kinase signalling pathways did not interfere with SW4 function. These data suggest that SW4 functions by binding to and clustering N-cadherin in neurons and thereby activating and Ncadherin/FGF receptor signalling cascade, and propose that such agonists may represent a starting point for the development of therapeutic agents promoting neuronal cell survival and regeneration. D 2004 Elsevier Inc. All rights reserved.
Introduction Cell – cell interactions mediated by cell adhesion molecules (CAMs) are fundamental to many developmental processes. In the * Corresponding author. Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research & Development Limited, New Frontiers Science Park, Third Avenue, Harlow CM19 5AW, Essex, UK. Fax: +44-1279-622660. E-mail address: Stephen [email protected] (S.D. Skaper). 1 Present address: Discovery Research, Wyeth Research, Collegeville, PA 19426, USA. Available online on ScienceDirect (www.sciencedirect.com.) 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2003.12.015
nervous system, the ability of neurons to extend axons and innervate their targets in an appropriate manner is governed to a large extent by the binding of CAMs on the neuronal growth cone to CAMs on the surface of other axons or nonneuronal cells (Dodd and Jessell, 1988; Goodman and Shatz, 1993). NCAM, N-cadherin, and L1 are CAMs that promote axonal growth during development (Walsh and Doherty, 1997). N-cadherin is a member of the classical cadherin family of transmembrane glycoproteins that mediate cellular recognition via a homophilic binding mechanism (Takeichi, 1995). In the nervous system, N-cadherin function has been implicated in several key events encompassing cell migration (Barami et al., 1994), axonal growth and guidance (Matsunaga et al., 1988; Riehl et al., 1996), and synapse formation and synaptic plasticity (Bozdagi et al., 2000; Inoue and Sanes, 1997; Tang et al., 1998). In addition to homophilic binding, cadherins have been shown to interact with many adaptor or signalling molecules. Many groups have implicated the fibroblast growth factor receptor (FGFR) in N-cadherin function. For example, neurite outgrowth stimulated by N-cadherin is inhibited by a variety of agents that inhibit FGFR function in neurons (Lom et al., 1998; Saffell et al., 1997; Williams et al., 1994a, 2001). N-cadherin can promote ‘‘contact-dependent’’ survival of ovarian granulosa cells in an FGFR-dependent fashion (Trolice et al., 1997). The FGFR requirement may reflect a more direct interaction among these molecules (Doherty and Walsh, 1996), as the antibody clustering of Ncadherin in cells is associated with the co-clustering of the FGFR (Utton et al., 2001) and FGFR and N-cadherin will co-precipitate from cells (Cavallaro et al., 2001). Soluble forms of some adhesion molecules (Doherty et al., 1995), including N-cadherin (Utton et al., 2001), are effective also in promoting axonal growth. The homophilic binding site resides in extracellular domain 1 (ECD1) (Koch et al., 1999; Shan et al., 1999), and peptide mimetics of two linear sequences from ECD1 (HAVDI and INPISGQ) function as highly specific N-cadherin antagonists in a physiologically relevant assay (Williams et al., 2000a,b). More recently, Williams et al. (2002) have shown that cyclic peptides containing a tandem repeat of the individual motifs
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function as N-cadherin agonists that stimulate neurite outgrowth in a similar manner to native N-cadherin. We now demonstrate that these dimeric peptides are capable of promoting the survival of several populations of central nervous system (CNS) neurons in vitro. Moreover, we show that, as with native N-cadherin, the response to the peptide agonists requires the function of both Ncadherin and the FGFR in the target neuron.
Results A dimeric version of the HAVDI motif displays neuron survivalpromoting and neuroprotective properties Peptide mimetics of the HAVDI motif in ECD1 of N-cadherin function as specific N-cadherin antagonists (Williams et al., 2000a), suggesting this sequence to contain a functional N-cadherin binding motif. A cyclic peptide (SW4) containing the HAVDI sequence in tandem (N-Ac-CHAVDINGHAVDIC-NH2), with the potential to promote cis-dimerisation of N-cadherin in the cell membrane, stimulated neurite outgrowth from rat cerebellar granule neurons cultured on monolayers of 3T3 fibroblasts in an FGFRdependent manner (Williams et al., 2002). As FGF-2 is capable of supporting survival of cerebellar granule neurons plated directly in serum-free (Sato’s) medium without insulin (Williams and Doherty, 1999), the dimeric HAVDI peptide SW4 was tested for its ability to promote survival of granule neurons under these conditions. Fig. 1 shows that SW4 increased the survival of granule neurons cultured in serum-free medium. The effect of SW4 was concentration-dependent, being already significant at 10 Ag/ml and maximal at 30 – 50 Ag/ml peptide (5 – 20 AM). When the dimeric HAVDI motif (peptide SW5) was presented to the granule neurons as a linear rather than as a cyclic peptide (N-Ac-HAVDINGHAVDI-NH2), it had no effect on survival (Table 1). Apoptotic death of differentiated cerebellar granule neurons can be induced by removing serum and lowering the extracellular K+ concentration from 25 to 5 mM (D’Mello et al., 1993; Miller and Johnson, 1996). Pretreatment of granule neurons with FGF-2 for
Fig. 1. The dimeric HAVDI peptide (SW4) promotes survival of cerebellar granule neurons plated in serum-free medium. Granule neurons were plated in Sato’s medium without insulin; SW4 peptide was added at this time. Survival was quantified after 48 h by MTT. Data are means F SD (three experiments) expressed relative to 10% FCS (100%). Under these conditions, survival promoted by FGF-2 (50 ng/ml) was 87.6 F 5.2% (n = 3). *P < 0.05 or #P < 0.01 vs. no FCS.
Table 1 Pharmacology of the dimeric HAVDI peptide in promoting neuronal cell survival Treatment
None SW4 (50 Ag/ml) SW4 (50 Ag/ml) + PD173074 (1 AM) SW4 (30 Ag/ml) + Ad126 (250 Ag/ml) SW5 (50 Ag/ml)
Neuronal cell survival (% control) Granule neurons (serum-free)
Granule neurons (low K+)
Hippocampal neurons (+glutamate)
41.9 F 6.6 70.9 F 5.8* 46.7 F 4.0
42.5 F 2.1 71.4 F 5.1* 47.9 F 6.5
45.0 F 5.4 67.5 F 2.6* 42.7 F 6.7
41.4 F 0.5
47.4 F 0.8
42.4 F 5.3
44.1 F 4.3
51.7 F 6.2
39.1 F 1.7
Cerebellar granule neurons were plated in Sato’s medium (‘serum-free’) for 48 h with the indicated test compound. Survival was quantified after 48 h by MTT. Data are means F SD (three experiments) expressed relative to 10% FCS (100%). Granule neurons at DIV 7 were incubated with the indicated test compound for 24 h and then shifted to serum-free/low-K+ medium. Survival was quantified after 48 h by MTT. Data are means F SD (three experiments) expressed relative to cells switched to serum-free/high (25 mM)-K+ medium (100%). Hippocampal cell cultures (DIV 7 – 9) were pretreated with the indicated test compound for 48 h and then exposed to 50 AM glutamate, and neuronal cell survival was quantified 24 h later by MTT. Data are means F SD (three experiments) expressed relative to no glutamate (100%). * P < 0.01 vs. all other conditions.
24 h rescues the majority of cells from death caused by K+ deprivation (Skaper et al., 2000). Addition of the dimeric HAVDI peptide SW4 to granule neurons at DIV 7, followed by switch to low-K+/serum-free medium 24 h later led to a significant increase in the numbers of surviving cells (Fig. 2), in comparison to cultures not pretreated with SW4. The effect of SW4 was concentration-
Fig. 2. The dimeric HAVDI peptide (SW4) rescues cerebellar granule neurons from death induced by switch to serum-free/low-K+ medium. Granule neurons at DIV 7 were incubated with the indicated concentrations of SW4 peptide for 24 h, and then shifted to serum-free/low-K+ medium. Survival was quantified after 48 h by MTT. Data are means F SD (three experiments) expressed relative to cells switched to serum-free/high (25 mM)-K+ medium (100%). In some culture wells, FGF-2 (50 ng/ml) was added at DIV 7, instead of SW4. Under these conditions, survival promoted by FGF-2 was 87.0 F 5.5% (n = 3). *P < 0.01 vs. serum-free/low-K+ medium.
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dependent, again being maximal at approximately 30 – 50 Ag/ml peptide. As with granule neurons plated under serum-free conditions, the dimeric HAVDI motif (SW5) presented as a linear rather than as a cyclic peptide failed to rescue K+-deprived cells (Table 1). Fibroblast growth factor-2 (FGF-2) is capable of protecting cultured rat hippocampal neurons against glutamate excitotoxicity (Mattson et al., 1989, 1995), and these neurons in vitro express high-afinity receptors for FGF-2 (Walicke et al., 1989). The survival of hippocampal neurons approximately doubled when cells were first incubated with FGF-2 for 24 h before exposure to 50 AM glutamate (Fig. 3, legend). Hippocampal neurons were then incubated with the dimeric HAVDI peptide SW4 for 24 h. When subsequently challenged with glutamate for a further 24 h, a concentration-dependent protection was observed, becoming significant already at 10 Ag/ml and maximal with 30 – 50 Ag/ml peptide (Fig. 3). Increasing the glutamate concentration to 100 AM reduced hippocampal neuronal cell survival to 18.8 F 4.2%, which improved to 36.1 F 4.1% (n = 3, P < 0.05) in cultures pretreated for 24 h with 50 Ag/ml SW4 peptide. The dimeric HAVDI motif (SW5) presented as a linear rather than as a cyclic peptide was inactive in rescuing hippocampal neurons against glutamate toxicity (Table 1). Pharmacology of dimeric HAVDI peptide survival-promoting effects As described above, the dimeric HAVDI motif, when presented as a linear cyclic peptide, failed to exhibit a neuroprotective action (Table 1). Moreover, the monomeric cyclic mimetic of the HAVDI sequence (N-Ac-CHAVDIC-NH 2 ) (Ad126) inhibited the survival-promoting function of the agonist peptide SW4 when evaluated in all three neuronal cell culture models (Table 1). Under the same conditions, the monomeric peptide failed to affect the survival response elicited by FGF-2 (data not shown). Similar experiments using function-blocking antibodies that act as well-established cadherin agonists were not
Fig. 3. The dimeric HAVDI peptide (SW4) attenuates glutamate-induced toxicity in cultured hippocampal neurons. Cultures (DIV 7 – 9) were pretreated with the indicated concentrations of SW4 peptide for 48 h and then exposed to 50 AM glutamate, and neuronal survival was quantified 24 h later by MTT. Data are means F SD (three experiments) expressed relative to no glutamate (100%). In some culture wells, FGF-2 (25 ng/ml) was added instead of SW4. Under these conditions, survival promoted by FGF-2 was 82.2 F 1.5% (n = 3). *P < 0.05 or #P < 0.01 vs. glutamate only.
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Fig. 4. Time dependence of neuroprotection by the dimeric HAVDI peptide. Cerebellar granule neurons at DIV 7 were incubated with the dimeric peptide agonist (SW4, 30 Ag/ml) for 1 or 24 h and then shifted to serum-free/low-K+ medium. Survival was quantified after 48 h by MTT. Data are means F SD (three experiments) expressed relative to cells switched to serum-free/high (25 mM)-K+ medium (100%). Hippocampal and cortical cell cultures (DIV 7 – 9) were pretreated with SW4 (30 Ag/ml) for 1 or 48 h and then exposed to 50 AM glutamate. Neuronal cell survival was quantified 24 h later by MTT. Data are means F SD (three experiments) expressed relative to no glutamate (100%). *P < 0.01 vs. all other conditions.
possible, as such reagents are not available. One may predict that in the assay described here, bivalent antibodies would cluster and internalise N-cadherin in the neurons, complicating analysis of the results. The dimeric peptide agonist appeared to promote neuronal cell survival via an FGFR-dependent mechanism, as a highly specific antagonist of the FGFR (PD173074) (Mohammadi et al., 1998; Skaper et al., 2000) fully inhibited the survival responses (Table 1). The ability of FGF-2 to protect hippocampal neurons from excitotoxicity (Mattson et al., 1989, 1995) and cerebellar granule neurons from death by K+ withdrawal (Skaper et al., 2000) requires a pretreatment of at least 24 h. When tested under similar conditions, neurons required a 24-h exposure to the dimeric HAVDI peptide agonist to observe a protective action (Fig. 4). Addition of the agonist peptide 1 h before, or only at the time of glutamate addition (or K+ withdrawal) did not provide any neuroprotective benefit (Fig. 4). The dimeric peptide agonist was efficacious also in limiting the excitotoxic death of cortical neurons, but only when added 24 h before glutamate exposure (Fig. 4). Tyrosine kinase growth factor receptors activate several intracellular signalling pathways. Of these, the MAP kinase and phosphatidylinositol 3-kinase (PI 3-kinase) pathways have been implicated in survival (Kaplan and Miller, 2000). In a previous study (Williams and Doherty, 1999), we established that the PI 3kinase pathway is not required for FGFR-dependent survival responses in cerebellar granule neurons, and that activation of the FGFR does not result in a robust activation of the PI 3-kinase pathway. The structurally distinct PI 3-kinase inhibitors LY294002 (Vlahos et al., 1994) and wortmannin (Okada et al., 1994), which act via distinct mechanisms, clearly failed to block the dimeric HAVDI peptide agonist promotion of survival of granule neurons under serum-free conditions and excitotoxic injury to hippocampal neurons (Fig. 5). In addition, the selective and potent MAP kinase inhibitor U0126 (Favata et al., 1998), and
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Fig. 5. Neuronal cell survival promoted by the dimeric HAVDI peptide is independent of the PI 3-kinase and MAPK kinase pathways. Hippocampal cell cultures (DIV 7) were pretreated 30 min with one of the following inhibitors: LY294002 (5 AM), wortmannin (0.1 AM), U0126 (10 AM), SB-203580 (10 AM) (optimal published concentrations; cf. Vlahos et al., 1994; D’Mello et al., 1997; Williams and Doherty, 1999; Davies et al., 2000). Medium was then replaced with fresh medium containing inhibitor and SW4 (30 Ag/ml). In the case of wortmannin, inhibitor was re-added after 8 h. After 48 h, cells were exposed to 50 AM glutamate. Neuronal cell survival was quantified 24 h later by MTT. Data are means F SD (two experiments) expressed relative to control survival (no glutamate; 100%). Alternatively, granule neurons were plated in Sato’s medium and, after allowing 1 h for cell attachment, were treated 30 min with inhibitor as above. Medium was then replaced with fresh medium containing inhibitor and SW4 (30 Ag/ml). Wortmannin was re-added after 8 h. Survival was quantified after 48 h by MTT. Data are means F SD (two experiments) expressed relative to control survival with 10% FCS (100%). Inhibitors at the concentrations tested were not cytotoxic on their own. P < 0.01 for all drug treatment conditions vs. insult only (glutamate or serum-free culture for hippocampal and cerebellar granule neurons, respectively).
the p38 inhibitor SB-203580 (Cuenda et al., 1995), were ineffective, as well (Fig. 5).
Discussion A principal finding of this study is that a dimeric version of a short N-cadherin binding motif (HAVDI) is capable of promoting the survival of several populations of CNS neurons, under distinct paradigms of injury, in an FGFR-dependent manner. The responses to the dimeric agonist peptide were inhibited by a monomeric version of the same motif (itself a highly specific N-cadherin antagonist; Williams et al., 2000a), and by a specific FGFR antagonist. These data suggest that the dimeric agonist peptide competes for the same binding site on cells as the monomeric antagonist peptide and that the agonist peptide stimulates neuronal cell survival via and N-cadherin-dependent mechanism. Much of the function of the classical cadherins depends on their ability to promote the stable adhesion of cells to each other. However, it is becoming increasingly evident that simple adhesion models cannot readily explain the diverse functions of some cadherins. Rather, some cadherin functions might be explained by their ability to activate signal transduction cascades in cells rather than by adhesion per se. This idea is supported by the known ability of N-cadherin to stimulate neurite outgrowth, where a soluble dimeric form of N-cadherin can stimulate neuritogenesis as effectively as N-cadherin expressed in a transfected cell line (Utton et al., 2001). Rational design of cyclic peptides that contain tandem mimetics of the functional binding motifs in ECD1 domain of N-cadherin has yielded molecules capable of promoting neurite
outgrowth; in the case of the HAVDI agonist peptide, the response was almost as good as that stimulated by native N-cadherin (Williams et al., 2002). Peptides were designed that contained the motifs in an antiparallel manner, based upon molecular modeling studies, which suggested that this orientation would be required for the simultaneous engagement of two cadherin units (Williams et al., 2002). The present findings clearly show that such interaction is critical also for prevention of neuronal cell death. The neural cell adhesion molecules L1 and CHL1 (Chen et al., 1999) and the synthetic NCAM peptide ligand, C3d (Ditlevsen et al., 2003), have been reported to display survival-promoting effects (albeit modest) on cultured CNS neurons. The neurotropic and neurotrophic effects of CAMs strengthens the emerging roles for adhesion molecules such as N-cadherin in synaptic plasticity (for review, see Goda, 2002; Togashi et al., 2002). The molecular basis for the observed neurosurvival effects of the N-cadherin dimeric peptide agonist are not known. The ability of CAMs to activate an FGFR-signalling cascade in growth cones is required for, and sufficient to explain, their positive effects on growth cone motility (Saffell et al., 1997; Williams et al., 1994a). The initial description of the dimeric peptide demonstrated that the effects of the peptide on neurite outgrowth were absolutely dependent on N-cadherin in the neurons; we can therefore conclude that the peptide cannot directly interact and activate the FGFR in neurons (see Williams et al., 2002). The micromolar concentrations of dimeric peptide agonist needed for neuronal survival (present study) and neurite outgrowth (Williams et al., 2002) further support the notion of N-cadherin activation of an FGFR signalling cascade rather than a direct interaction with FGFR, in contrast to FGF engagement
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of its cognate receptor which operates at nanomolar concentrations (cf. Figs. 1 – 3, legends). A role for DAG lipase activity in the control of axonal growth and guidance in vitro and in vivo has been established (Brittis et al., 1996; Lom et al., 1998) and appears to be a key element in FGF (and CAM)-stimulated calcium influx into neuronal growth cones, which in turn is both necessary and sufficient for an axonal growth response (Doherty and Walsh, 1996; Lom et al., 1998; Williams et al., 1994b). Recent evidence proposes that signalling via the CB1 cannabinoid receptor is not only required for, but can also mediate, the neurite outgrowth response stimulated by N-cadherin and FGF-2, and that it does so by coupling DAG hydrolysis to a signalling cascade that depends upon calcium influx into neurons via both N- and L-type calcium channels (Williams et al., 2003). Although CB1 cannabinoid receptor antagonists inhibited axonal growth responses stimulated by N-cadherin and FGF-2 (Williams et al., 2003), the former failed to show any consistent inhibition of neuronal survival responses by the N-cadherin agonist peptide in the cell culture paradigms utilised here (our unpublished observations). Tyrosine kinase growth factor receptors activate several intracellular signalling pathways. Of these, the MAP kinase and phosphatidylinositol 3-kinase (PI 3-kinase) pathways have been implicated in survival (Kaplan and Miller, 2000). However, selective small molecule inhibitors of PI 3-kinase, MAP kinase, and p38 kinase were unable to neutralise neuroprotection by the HAVDI agonist peptide. In a previous study (Williams and Doherty, 1999), we established that the PI 3kinase pathway is not required for FGFR-dependent survival responses in these neurons, and that activation of the FGFR does not result in a robust activation of the PI 3-kinase pathway. It is therefore not surprising that PI 3-kinase inhibitors do not block the peptide. The pathway that couples the activated FGFR to the neuronal survival response remains to be established and might not be the same for all neurons in each of the experimental paradigms. The neuronal cell survival and axonal regeneration signalling pathways linked to N-cadherin function likely comprise, at least in part, nonoverlapping elements. Neuroprotection afforded by the dimeric agonist peptide required, in several cases, a lengthy exposure to be effective. A similar behaviour has been described for neuronal cell survival promotion by FGF-2 (Mattson et al., 1989, 1995; Skaper et al., 2000), suggesting that a transcriptional response is likely to be important for the Ncadherin/FGFR signalling cascade. A comprehensive study on gene regulation by the peptide and FGF-2 will be required to fully understand the signalling mechanisms and is beyond the scope of the present work. FGF-2 can protect cultured central neurons against injury by a variety of insults, including hypoxia/ischemia and hypoglycemia (Mattson and Barger, 1995) and h-amyloid peptide (Mark et al., 1997). Expression of FGFR is induced after trauma or cerebral ischemia (Logan et al., 1992; Masumura et al., 1996), especially in the penumbral regions of the cortex after focal ischemia (Masumura et al., 1996). Exogenous FGF-2 administration is neuroprotective in experimental ischemia models and enhances functional recovery (Ay et al., 1999) and prevents the death of lesioned basal forebrain cholinergic neurons (Anderson et al., 1988), the latter being of relevance to Alzheimer’s disease (Hefti, 1997). Although exogenously given neurotrophic proteins are efficacious in animal models of nervous system injury, their general clinical usefulness remains in doubt, given their lack of blood – brain barrier penetration and side effects.
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Small molecule nonpeptide mimetics that maintain the neurotrophic and neurotropic activities of the protein while having improved pharmacokinetic and disposition characteristics may minimise these problems (Skaper and Walsh, 1998; Swain et al., 1999). The synthetic peptides described here and elsewhere (Williams et al., 2002) can be viewed as a starting point for the development of such non-peptide mimetics with the potential as therapeutic agents for the promotion of cell survival and axonal regeneration.
Experimental methods Materials Neurobasal medium, B27 supplement, and fetal calf serum (FCS) were obtained from Life Technologies (Paisley, UK); all other cell culture media, supplements, and 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma (St. Louis, MO, USA); U0126 was from Promega (Southampton, UK); LY294002, wortmannin, and SB-203580 were from Calbiochem (Nottingham, UK); recombinant human FGF-2 was from R & D Systems (Abingdon, UK); the dimeric HAVDI peptide N-AcCHAVDINGHAVDIC-NH2 (SW4), the linear form of the dimeric HAVDI motif (N-Ac-HAVDINGHAVDI-NH2) (SW5), and the monomeric cyclic mimetic of the HAVDI sequence (N-Ac-CHAVDIC-NH2) (Ad126) have been described previously (Williams et al., 2000a, 2002). Neuronal cell cultures Cerebellar granule neurons Cultures of cerebellar granule neurons were prepared from 8day-old Sprague – Dawley rats (Charles River, Margate, UK) as described previously (Skaper et al., 1990). Dissociated cells were seeded in poly-D-lysine (35,000 – 70,000 Mr)-coated 48-well plates (Nunclon) (3.5 105 cells/well) in Basal Medium Eagle’s supplemented to contain 10% FCS, 2 mM L-glutamine, 50 Ag/ml gentamicin, and 25 mM KCl. Cytosine arabinoside (10 AM) was added 24 h after plating to inhibit growth of non-neuronal cells. Cultures prepared in this manner typically contain <5% nonneuronal cells (Boje et al., 1993; Gallo et al., 1987) and were used at 8 days in vitro (DIV). For survival assessment under serum-free plating conditions, granule neurons were prepared from postnatal day 3 rat pups (Doherty et al., 1990; Skaper et al., 2000). Dissociated cells were seeded in poly-D-lysine-coated 96-well plates (8 104 cells/well) in Sato’s medium without insulin (Doherty et al., 1990; Skaper et al., 2000). Hippocampal and cortical neuron cultures Hippocampal and cortical neurons were prepared from embryonic days 17.5 – 18 gestation Sprague – Dawley rat fetuses, as described (Skaper et al., 1990). Cells were maintained in Neurobasal medium supplemented with B27 additives with antioxidants, 1 mM sodium pyruvate, 2 mM glutamine, 25 AM glutamate, 50 U/ ml penicillin, and 50 Ag/ml streptomycin. Half of the medium was replaced every 3 to 4 days with fresh maintenance medium (Neurobasal supplemented with 50 U/ml penicillin, 50 Ag/ml streptomycin, 2 mM glutamine, and B27 without antioxidants). Cultures were used after 7 – 9 days and comprised approximately 95% neuronal cells at this time.
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Assessment of neuroprotective activities Cerebellar granule neurons For survival assessment under serum-free conditions, the HAVDI peptide was added to the cerebellar granule neuron cultures at the time of plating; cultures supplemented with 10% FCS (100% survival) or FGF-2 served as controls. Cell survival was quantified after 48 h by a colourimetric reaction with MTT (Manthorpe et al., 1986; Skaper et al., 1990, 2000). Death of differentiated granule neurons (DIV 8) was induced by shifting cultures to serum-free medium without supplemental KCl. HAVDI peptides or FGF-2 were added either at this time, or 1 or 24 h before the shift to serum-free medium. Forty-eight hours after the switch to serum-free/low-KCl conditions, cell survival was quantified by MTT. Absolute MTT values obtained were normalised for small differences in inter-experiment plating densities by scaling to the mean of sham-treated sister cultures (defined as 100%). Hippocampal neurons: excitotoxic injury Experiments were performed in 7- to 9-day-old cultures because previous studies showed that at this time in culture, the neurons are vulnerable to glutamate toxicity and can be protected against excitotoxic insult by FGF-2 (Mattson et al., 1989, 1995). Cultures were exposed to the HAVDI peptide or FGF-2 for 1 – 24 h. Cultures were then exposed to glutamate (50 – 100 AM), and neuronal survival was quantified 24 h later by MTT. The MTT technique has been shown to be equivalent to lactate dehydrogenase release in the measurement of neurotoxin-mediated neuronal death in vitro (Patel et al., 1990). Cell death and recovery was confirmed in all cases by morphological examination of the culture under phase contrast microscopy.
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Takeichi, M., 1995. Morphogenetic role of classic cadherins. Curr. Opin. Cell Biol. 7, 619 – 627. Tang, L., Hung, C.P., Schuman, E.M., 1998. A role for the cadherin family of cell adhesion molecules in hippocampal long-term potentiation. Neuron 20, 1165 – 1175. Togashi, H., Abe, K., Mizoguchi, A., Takaoka, K., Chisaka, O., Takeichi, M., 2002. Cadherin regulates dendritic spine morphogenesis. Neuron 35, 77 – 89. Trolice, M.P., Pappalardo, A., Peluso, J.J., 1997. Basic fibroblast growth factor and N-cadherin maintain rat granulosa cell and ovarian surface epithelial cell viability by stimulating the tyrosine phosphorylation of the fibroblast growth factor receptors. Endocrinology 138, 107 – 113. Utton, M.A., Eickholt, B., Howell, F.V., Wallis, J., Doherty, P., 2001. Soluble N-cadherin stimulates fibroblast growth factor receptor dependent neurite outgrowth and N-cadherin and the fibroblast growth factor receptor co-cluster in cells. J. Neurochem. 76, 1421 – 1430. Vlahos, C.J., Matter, W.F., Hui, K.Y., Brown, R.F., 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one (LY294002). J. Biol. Chem. 269, 5241 – 5248. Walicke, P.A., Feige, J.J., Baird, A., 1989. Characterization of the neuronal receptor for basic fibroblast growth factor and comparison to receptors on mesenchymal cells. J. Biol. Chem. 264, 4120 – 4126. Walsh, F.S., Doherty, P., 1997. Neural cell adhesion molecules of the immunoglobulin superfamily: role in axonal growth and guidance. Annu. Rev. Cell Dev. Biol. 13, 425 – 456. Williams, E.-J., Doherty, P., 1999. Evidence for and against a pivotal role of PI 3-kinase in a neuronal cell survival pathway. Mol. Cell. Neurosci. 13, 272 – 280. Williams, E.-J., Furness, J., Walsh, F.S., Doherty, P., 1994a. Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, NCAM, and N-cadherin. Neuron 13, 583 – 594. Williams, E.J., Walsh, F.S., Doherty, P., 1994b. The production of arachidonic acid can account for calcium channel activation in the second messenger pathway underlying neurite outgrowth stimulated by NCAM, N-cadherin, and L1. J. Neurochem. 62, 1231 – 1234. Williams, E., Williams, G., Gour, B.J., Blaschuk, O.W., Doherty, P., 2000a. A novel family of cyclic peptide antagonists suggests that N-cadherin specificity is determined by amino acids that flank the HAV motif. J. Biol. Chem. 275, 4007 – 4012. Williams, E.J., Williams, G., Gour, B., Blaschuk, O., Doherty, P., 2000b. INP, a novel N-cadherin antagonist targeted to the amino acids that flank the HAV motif. Mol. Cell. Neurosci. 15, 456 – 464. Williams, E.-J., Williams, G., Howell, F.V., Skaper, S.D., Walsh, F.S., Doherty, P., 2001. Identification of an N-cadherin motif that can interact with the fibroblast growth factor receptor and is required for axonal growth. J. Biol. Chem. 276, 43879 – 43886. Williams, G., Williams, E.-J., Doherty, P., 2002. Dimeric versions of two short N-cadherin binding motifs (HAVDI) and INPISG) function as Ncadherin agonists. J. Biol. Chem. 277, 4361 – 4367. Williams, E.-J., Walsh, F.S., Doherty, P., 2003. The FGF receptor uses the endocannabinoid signalling system to couple to an axonal growth response. J. Cell Biol. 160, 481 – 486.
A dimeric version of the short N-cadherin binding motif HAVDI promotes neuronal cell survival by activating an N-cadherin/ fibroblast growth factor receptor signalling cascade Stephen D. Skaper, a,* Laura Facci, a Gareth Williams, b Emma-Jane Williams, b Frank S. Walsh, 1 and Patrick Doherty b a
Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research & Development Limited, New Frontiers Science Park, Harlow CM19 5AW, Essex, UK b Molecular Neurobiology Group, Medical Research Council Centre for Developmental Neurobiology, King’s College London, London SE1 1UL, UK Received 21 July 2003; revised 11 December 2003; accepted 19 December 2003 Available online 20 March 2004 The HAVDI and INPISGQ sequences have been identified as functional binding motifs in extracellular domain 1 (ECD1) of N-cadherin. Cyclic peptides containing a tandem repeat of the individual motifs function as N-cadherin agonists and stimulate neurite outgrowth. We now show that the cyclic peptide N-Ac-CHAVDINGHAVDIC-NH2 (SW4) containing the HAVDI sequence in tandem is efficacious also in promoting the in vitro survival of several populations of central nervous system neurons in paradigms where fibroblast growth factor-2 (FGF-2) is active. SW4 supported the survival of rat postnatal cerebellar granule neurons plated in serum-free medium and limited the death of differentiated granule neurons induced to die by switch to low K+ medium. In addition, SW4 rescued embryonic hippocampal and cortical neurons from injury caused by glutamic acid excitotoxicity. The neuroprotective effects of SW4 displayed a concentration dependence similar to those inducing neuritogenesis, were inhibited by a monomeric version of the same motif and by a specific FGF receptor antagonist (PD173074), and were not mimicked by the linear peptide. Inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase), MAP kinase, and p38 kinase signalling pathways did not interfere with SW4 function. These data suggest that SW4 functions by binding to and clustering N-cadherin in neurons and thereby activating and Ncadherin/FGF receptor signalling cascade, and propose that such agonists may represent a starting point for the development of therapeutic agents promoting neuronal cell survival and regeneration. D 2004 Elsevier Inc. All rights reserved.
Introduction Cell – cell interactions mediated by cell adhesion molecules (CAMs) are fundamental to many developmental processes. In the * Corresponding author. Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research & Development Limited, New Frontiers Science Park, Third Avenue, Harlow CM19 5AW, Essex, UK. Fax: +44-1279-622660. E-mail address: Stephen [email protected] (S.D. Skaper). 1 Present address: Discovery Research, Wyeth Research, Collegeville, PA 19426, USA. Available online on ScienceDirect (www.sciencedirect.com.) 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2003.12.015
nervous system, the ability of neurons to extend axons and innervate their targets in an appropriate manner is governed to a large extent by the binding of CAMs on the neuronal growth cone to CAMs on the surface of other axons or nonneuronal cells (Dodd and Jessell, 1988; Goodman and Shatz, 1993). NCAM, N-cadherin, and L1 are CAMs that promote axonal growth during development (Walsh and Doherty, 1997). N-cadherin is a member of the classical cadherin family of transmembrane glycoproteins that mediate cellular recognition via a homophilic binding mechanism (Takeichi, 1995). In the nervous system, N-cadherin function has been implicated in several key events encompassing cell migration (Barami et al., 1994), axonal growth and guidance (Matsunaga et al., 1988; Riehl et al., 1996), and synapse formation and synaptic plasticity (Bozdagi et al., 2000; Inoue and Sanes, 1997; Tang et al., 1998). In addition to homophilic binding, cadherins have been shown to interact with many adaptor or signalling molecules. Many groups have implicated the fibroblast growth factor receptor (FGFR) in N-cadherin function. For example, neurite outgrowth stimulated by N-cadherin is inhibited by a variety of agents that inhibit FGFR function in neurons (Lom et al., 1998; Saffell et al., 1997; Williams et al., 1994a, 2001). N-cadherin can promote ‘‘contact-dependent’’ survival of ovarian granulosa cells in an FGFR-dependent fashion (Trolice et al., 1997). The FGFR requirement may reflect a more direct interaction among these molecules (Doherty and Walsh, 1996), as the antibody clustering of Ncadherin in cells is associated with the co-clustering of the FGFR (Utton et al., 2001) and FGFR and N-cadherin will co-precipitate from cells (Cavallaro et al., 2001). Soluble forms of some adhesion molecules (Doherty et al., 1995), including N-cadherin (Utton et al., 2001), are effective also in promoting axonal growth. The homophilic binding site resides in extracellular domain 1 (ECD1) (Koch et al., 1999; Shan et al., 1999), and peptide mimetics of two linear sequences from ECD1 (HAVDI and INPISGQ) function as highly specific N-cadherin antagonists in a physiologically relevant assay (Williams et al., 2000a,b). More recently, Williams et al. (2002) have shown that cyclic peptides containing a tandem repeat of the individual motifs
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function as N-cadherin agonists that stimulate neurite outgrowth in a similar manner to native N-cadherin. We now demonstrate that these dimeric peptides are capable of promoting the survival of several populations of central nervous system (CNS) neurons in vitro. Moreover, we show that, as with native N-cadherin, the response to the peptide agonists requires the function of both Ncadherin and the FGFR in the target neuron.
Results A dimeric version of the HAVDI motif displays neuron survivalpromoting and neuroprotective properties Peptide mimetics of the HAVDI motif in ECD1 of N-cadherin function as specific N-cadherin antagonists (Williams et al., 2000a), suggesting this sequence to contain a functional N-cadherin binding motif. A cyclic peptide (SW4) containing the HAVDI sequence in tandem (N-Ac-CHAVDINGHAVDIC-NH2), with the potential to promote cis-dimerisation of N-cadherin in the cell membrane, stimulated neurite outgrowth from rat cerebellar granule neurons cultured on monolayers of 3T3 fibroblasts in an FGFRdependent manner (Williams et al., 2002). As FGF-2 is capable of supporting survival of cerebellar granule neurons plated directly in serum-free (Sato’s) medium without insulin (Williams and Doherty, 1999), the dimeric HAVDI peptide SW4 was tested for its ability to promote survival of granule neurons under these conditions. Fig. 1 shows that SW4 increased the survival of granule neurons cultured in serum-free medium. The effect of SW4 was concentration-dependent, being already significant at 10 Ag/ml and maximal at 30 – 50 Ag/ml peptide (5 – 20 AM). When the dimeric HAVDI motif (peptide SW5) was presented to the granule neurons as a linear rather than as a cyclic peptide (N-Ac-HAVDINGHAVDI-NH2), it had no effect on survival (Table 1). Apoptotic death of differentiated cerebellar granule neurons can be induced by removing serum and lowering the extracellular K+ concentration from 25 to 5 mM (D’Mello et al., 1993; Miller and Johnson, 1996). Pretreatment of granule neurons with FGF-2 for
Fig. 1. The dimeric HAVDI peptide (SW4) promotes survival of cerebellar granule neurons plated in serum-free medium. Granule neurons were plated in Sato’s medium without insulin; SW4 peptide was added at this time. Survival was quantified after 48 h by MTT. Data are means F SD (three experiments) expressed relative to 10% FCS (100%). Under these conditions, survival promoted by FGF-2 (50 ng/ml) was 87.6 F 5.2% (n = 3). *P < 0.05 or #P < 0.01 vs. no FCS.
Table 1 Pharmacology of the dimeric HAVDI peptide in promoting neuronal cell survival Treatment
None SW4 (50 Ag/ml) SW4 (50 Ag/ml) + PD173074 (1 AM) SW4 (30 Ag/ml) + Ad126 (250 Ag/ml) SW5 (50 Ag/ml)
Neuronal cell survival (% control) Granule neurons (serum-free)
Granule neurons (low K+)
Hippocampal neurons (+glutamate)
41.9 F 6.6 70.9 F 5.8* 46.7 F 4.0
42.5 F 2.1 71.4 F 5.1* 47.9 F 6.5
45.0 F 5.4 67.5 F 2.6* 42.7 F 6.7
41.4 F 0.5
47.4 F 0.8
42.4 F 5.3
44.1 F 4.3
51.7 F 6.2
39.1 F 1.7
Cerebellar granule neurons were plated in Sato’s medium (‘serum-free’) for 48 h with the indicated test compound. Survival was quantified after 48 h by MTT. Data are means F SD (three experiments) expressed relative to 10% FCS (100%). Granule neurons at DIV 7 were incubated with the indicated test compound for 24 h and then shifted to serum-free/low-K+ medium. Survival was quantified after 48 h by MTT. Data are means F SD (three experiments) expressed relative to cells switched to serum-free/high (25 mM)-K+ medium (100%). Hippocampal cell cultures (DIV 7 – 9) were pretreated with the indicated test compound for 48 h and then exposed to 50 AM glutamate, and neuronal cell survival was quantified 24 h later by MTT. Data are means F SD (three experiments) expressed relative to no glutamate (100%). * P < 0.01 vs. all other conditions.
24 h rescues the majority of cells from death caused by K+ deprivation (Skaper et al., 2000). Addition of the dimeric HAVDI peptide SW4 to granule neurons at DIV 7, followed by switch to low-K+/serum-free medium 24 h later led to a significant increase in the numbers of surviving cells (Fig. 2), in comparison to cultures not pretreated with SW4. The effect of SW4 was concentration-
Fig. 2. The dimeric HAVDI peptide (SW4) rescues cerebellar granule neurons from death induced by switch to serum-free/low-K+ medium. Granule neurons at DIV 7 were incubated with the indicated concentrations of SW4 peptide for 24 h, and then shifted to serum-free/low-K+ medium. Survival was quantified after 48 h by MTT. Data are means F SD (three experiments) expressed relative to cells switched to serum-free/high (25 mM)-K+ medium (100%). In some culture wells, FGF-2 (50 ng/ml) was added at DIV 7, instead of SW4. Under these conditions, survival promoted by FGF-2 was 87.0 F 5.5% (n = 3). *P < 0.01 vs. serum-free/low-K+ medium.
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dependent, again being maximal at approximately 30 – 50 Ag/ml peptide. As with granule neurons plated under serum-free conditions, the dimeric HAVDI motif (SW5) presented as a linear rather than as a cyclic peptide failed to rescue K+-deprived cells (Table 1). Fibroblast growth factor-2 (FGF-2) is capable of protecting cultured rat hippocampal neurons against glutamate excitotoxicity (Mattson et al., 1989, 1995), and these neurons in vitro express high-afinity receptors for FGF-2 (Walicke et al., 1989). The survival of hippocampal neurons approximately doubled when cells were first incubated with FGF-2 for 24 h before exposure to 50 AM glutamate (Fig. 3, legend). Hippocampal neurons were then incubated with the dimeric HAVDI peptide SW4 for 24 h. When subsequently challenged with glutamate for a further 24 h, a concentration-dependent protection was observed, becoming significant already at 10 Ag/ml and maximal with 30 – 50 Ag/ml peptide (Fig. 3). Increasing the glutamate concentration to 100 AM reduced hippocampal neuronal cell survival to 18.8 F 4.2%, which improved to 36.1 F 4.1% (n = 3, P < 0.05) in cultures pretreated for 24 h with 50 Ag/ml SW4 peptide. The dimeric HAVDI motif (SW5) presented as a linear rather than as a cyclic peptide was inactive in rescuing hippocampal neurons against glutamate toxicity (Table 1). Pharmacology of dimeric HAVDI peptide survival-promoting effects As described above, the dimeric HAVDI motif, when presented as a linear cyclic peptide, failed to exhibit a neuroprotective action (Table 1). Moreover, the monomeric cyclic mimetic of the HAVDI sequence (N-Ac-CHAVDIC-NH 2 ) (Ad126) inhibited the survival-promoting function of the agonist peptide SW4 when evaluated in all three neuronal cell culture models (Table 1). Under the same conditions, the monomeric peptide failed to affect the survival response elicited by FGF-2 (data not shown). Similar experiments using function-blocking antibodies that act as well-established cadherin agonists were not
Fig. 3. The dimeric HAVDI peptide (SW4) attenuates glutamate-induced toxicity in cultured hippocampal neurons. Cultures (DIV 7 – 9) were pretreated with the indicated concentrations of SW4 peptide for 48 h and then exposed to 50 AM glutamate, and neuronal survival was quantified 24 h later by MTT. Data are means F SD (three experiments) expressed relative to no glutamate (100%). In some culture wells, FGF-2 (25 ng/ml) was added instead of SW4. Under these conditions, survival promoted by FGF-2 was 82.2 F 1.5% (n = 3). *P < 0.05 or #P < 0.01 vs. glutamate only.
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Fig. 4. Time dependence of neuroprotection by the dimeric HAVDI peptide. Cerebellar granule neurons at DIV 7 were incubated with the dimeric peptide agonist (SW4, 30 Ag/ml) for 1 or 24 h and then shifted to serum-free/low-K+ medium. Survival was quantified after 48 h by MTT. Data are means F SD (three experiments) expressed relative to cells switched to serum-free/high (25 mM)-K+ medium (100%). Hippocampal and cortical cell cultures (DIV 7 – 9) were pretreated with SW4 (30 Ag/ml) for 1 or 48 h and then exposed to 50 AM glutamate. Neuronal cell survival was quantified 24 h later by MTT. Data are means F SD (three experiments) expressed relative to no glutamate (100%). *P < 0.01 vs. all other conditions.
possible, as such reagents are not available. One may predict that in the assay described here, bivalent antibodies would cluster and internalise N-cadherin in the neurons, complicating analysis of the results. The dimeric peptide agonist appeared to promote neuronal cell survival via an FGFR-dependent mechanism, as a highly specific antagonist of the FGFR (PD173074) (Mohammadi et al., 1998; Skaper et al., 2000) fully inhibited the survival responses (Table 1). The ability of FGF-2 to protect hippocampal neurons from excitotoxicity (Mattson et al., 1989, 1995) and cerebellar granule neurons from death by K+ withdrawal (Skaper et al., 2000) requires a pretreatment of at least 24 h. When tested under similar conditions, neurons required a 24-h exposure to the dimeric HAVDI peptide agonist to observe a protective action (Fig. 4). Addition of the agonist peptide 1 h before, or only at the time of glutamate addition (or K+ withdrawal) did not provide any neuroprotective benefit (Fig. 4). The dimeric peptide agonist was efficacious also in limiting the excitotoxic death of cortical neurons, but only when added 24 h before glutamate exposure (Fig. 4). Tyrosine kinase growth factor receptors activate several intracellular signalling pathways. Of these, the MAP kinase and phosphatidylinositol 3-kinase (PI 3-kinase) pathways have been implicated in survival (Kaplan and Miller, 2000). In a previous study (Williams and Doherty, 1999), we established that the PI 3kinase pathway is not required for FGFR-dependent survival responses in cerebellar granule neurons, and that activation of the FGFR does not result in a robust activation of the PI 3-kinase pathway. The structurally distinct PI 3-kinase inhibitors LY294002 (Vlahos et al., 1994) and wortmannin (Okada et al., 1994), which act via distinct mechanisms, clearly failed to block the dimeric HAVDI peptide agonist promotion of survival of granule neurons under serum-free conditions and excitotoxic injury to hippocampal neurons (Fig. 5). In addition, the selective and potent MAP kinase inhibitor U0126 (Favata et al., 1998), and
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Fig. 5. Neuronal cell survival promoted by the dimeric HAVDI peptide is independent of the PI 3-kinase and MAPK kinase pathways. Hippocampal cell cultures (DIV 7) were pretreated 30 min with one of the following inhibitors: LY294002 (5 AM), wortmannin (0.1 AM), U0126 (10 AM), SB-203580 (10 AM) (optimal published concentrations; cf. Vlahos et al., 1994; D’Mello et al., 1997; Williams and Doherty, 1999; Davies et al., 2000). Medium was then replaced with fresh medium containing inhibitor and SW4 (30 Ag/ml). In the case of wortmannin, inhibitor was re-added after 8 h. After 48 h, cells were exposed to 50 AM glutamate. Neuronal cell survival was quantified 24 h later by MTT. Data are means F SD (two experiments) expressed relative to control survival (no glutamate; 100%). Alternatively, granule neurons were plated in Sato’s medium and, after allowing 1 h for cell attachment, were treated 30 min with inhibitor as above. Medium was then replaced with fresh medium containing inhibitor and SW4 (30 Ag/ml). Wortmannin was re-added after 8 h. Survival was quantified after 48 h by MTT. Data are means F SD (two experiments) expressed relative to control survival with 10% FCS (100%). Inhibitors at the concentrations tested were not cytotoxic on their own. P < 0.01 for all drug treatment conditions vs. insult only (glutamate or serum-free culture for hippocampal and cerebellar granule neurons, respectively).
the p38 inhibitor SB-203580 (Cuenda et al., 1995), were ineffective, as well (Fig. 5).
Discussion A principal finding of this study is that a dimeric version of a short N-cadherin binding motif (HAVDI) is capable of promoting the survival of several populations of CNS neurons, under distinct paradigms of injury, in an FGFR-dependent manner. The responses to the dimeric agonist peptide were inhibited by a monomeric version of the same motif (itself a highly specific N-cadherin antagonist; Williams et al., 2000a), and by a specific FGFR antagonist. These data suggest that the dimeric agonist peptide competes for the same binding site on cells as the monomeric antagonist peptide and that the agonist peptide stimulates neuronal cell survival via and N-cadherin-dependent mechanism. Much of the function of the classical cadherins depends on their ability to promote the stable adhesion of cells to each other. However, it is becoming increasingly evident that simple adhesion models cannot readily explain the diverse functions of some cadherins. Rather, some cadherin functions might be explained by their ability to activate signal transduction cascades in cells rather than by adhesion per se. This idea is supported by the known ability of N-cadherin to stimulate neurite outgrowth, where a soluble dimeric form of N-cadherin can stimulate neuritogenesis as effectively as N-cadherin expressed in a transfected cell line (Utton et al., 2001). Rational design of cyclic peptides that contain tandem mimetics of the functional binding motifs in ECD1 domain of N-cadherin has yielded molecules capable of promoting neurite
outgrowth; in the case of the HAVDI agonist peptide, the response was almost as good as that stimulated by native N-cadherin (Williams et al., 2002). Peptides were designed that contained the motifs in an antiparallel manner, based upon molecular modeling studies, which suggested that this orientation would be required for the simultaneous engagement of two cadherin units (Williams et al., 2002). The present findings clearly show that such interaction is critical also for prevention of neuronal cell death. The neural cell adhesion molecules L1 and CHL1 (Chen et al., 1999) and the synthetic NCAM peptide ligand, C3d (Ditlevsen et al., 2003), have been reported to display survival-promoting effects (albeit modest) on cultured CNS neurons. The neurotropic and neurotrophic effects of CAMs strengthens the emerging roles for adhesion molecules such as N-cadherin in synaptic plasticity (for review, see Goda, 2002; Togashi et al., 2002). The molecular basis for the observed neurosurvival effects of the N-cadherin dimeric peptide agonist are not known. The ability of CAMs to activate an FGFR-signalling cascade in growth cones is required for, and sufficient to explain, their positive effects on growth cone motility (Saffell et al., 1997; Williams et al., 1994a). The initial description of the dimeric peptide demonstrated that the effects of the peptide on neurite outgrowth were absolutely dependent on N-cadherin in the neurons; we can therefore conclude that the peptide cannot directly interact and activate the FGFR in neurons (see Williams et al., 2002). The micromolar concentrations of dimeric peptide agonist needed for neuronal survival (present study) and neurite outgrowth (Williams et al., 2002) further support the notion of N-cadherin activation of an FGFR signalling cascade rather than a direct interaction with FGFR, in contrast to FGF engagement
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of its cognate receptor which operates at nanomolar concentrations (cf. Figs. 1 – 3, legends). A role for DAG lipase activity in the control of axonal growth and guidance in vitro and in vivo has been established (Brittis et al., 1996; Lom et al., 1998) and appears to be a key element in FGF (and CAM)-stimulated calcium influx into neuronal growth cones, which in turn is both necessary and sufficient for an axonal growth response (Doherty and Walsh, 1996; Lom et al., 1998; Williams et al., 1994b). Recent evidence proposes that signalling via the CB1 cannabinoid receptor is not only required for, but can also mediate, the neurite outgrowth response stimulated by N-cadherin and FGF-2, and that it does so by coupling DAG hydrolysis to a signalling cascade that depends upon calcium influx into neurons via both N- and L-type calcium channels (Williams et al., 2003). Although CB1 cannabinoid receptor antagonists inhibited axonal growth responses stimulated by N-cadherin and FGF-2 (Williams et al., 2003), the former failed to show any consistent inhibition of neuronal survival responses by the N-cadherin agonist peptide in the cell culture paradigms utilised here (our unpublished observations). Tyrosine kinase growth factor receptors activate several intracellular signalling pathways. Of these, the MAP kinase and phosphatidylinositol 3-kinase (PI 3-kinase) pathways have been implicated in survival (Kaplan and Miller, 2000). However, selective small molecule inhibitors of PI 3-kinase, MAP kinase, and p38 kinase were unable to neutralise neuroprotection by the HAVDI agonist peptide. In a previous study (Williams and Doherty, 1999), we established that the PI 3kinase pathway is not required for FGFR-dependent survival responses in these neurons, and that activation of the FGFR does not result in a robust activation of the PI 3-kinase pathway. It is therefore not surprising that PI 3-kinase inhibitors do not block the peptide. The pathway that couples the activated FGFR to the neuronal survival response remains to be established and might not be the same for all neurons in each of the experimental paradigms. The neuronal cell survival and axonal regeneration signalling pathways linked to N-cadherin function likely comprise, at least in part, nonoverlapping elements. Neuroprotection afforded by the dimeric agonist peptide required, in several cases, a lengthy exposure to be effective. A similar behaviour has been described for neuronal cell survival promotion by FGF-2 (Mattson et al., 1989, 1995; Skaper et al., 2000), suggesting that a transcriptional response is likely to be important for the Ncadherin/FGFR signalling cascade. A comprehensive study on gene regulation by the peptide and FGF-2 will be required to fully understand the signalling mechanisms and is beyond the scope of the present work. FGF-2 can protect cultured central neurons against injury by a variety of insults, including hypoxia/ischemia and hypoglycemia (Mattson and Barger, 1995) and h-amyloid peptide (Mark et al., 1997). Expression of FGFR is induced after trauma or cerebral ischemia (Logan et al., 1992; Masumura et al., 1996), especially in the penumbral regions of the cortex after focal ischemia (Masumura et al., 1996). Exogenous FGF-2 administration is neuroprotective in experimental ischemia models and enhances functional recovery (Ay et al., 1999) and prevents the death of lesioned basal forebrain cholinergic neurons (Anderson et al., 1988), the latter being of relevance to Alzheimer’s disease (Hefti, 1997). Although exogenously given neurotrophic proteins are efficacious in animal models of nervous system injury, their general clinical usefulness remains in doubt, given their lack of blood – brain barrier penetration and side effects.
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Small molecule nonpeptide mimetics that maintain the neurotrophic and neurotropic activities of the protein while having improved pharmacokinetic and disposition characteristics may minimise these problems (Skaper and Walsh, 1998; Swain et al., 1999). The synthetic peptides described here and elsewhere (Williams et al., 2002) can be viewed as a starting point for the development of such non-peptide mimetics with the potential as therapeutic agents for the promotion of cell survival and axonal regeneration.
Experimental methods Materials Neurobasal medium, B27 supplement, and fetal calf serum (FCS) were obtained from Life Technologies (Paisley, UK); all other cell culture media, supplements, and 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma (St. Louis, MO, USA); U0126 was from Promega (Southampton, UK); LY294002, wortmannin, and SB-203580 were from Calbiochem (Nottingham, UK); recombinant human FGF-2 was from R & D Systems (Abingdon, UK); the dimeric HAVDI peptide N-AcCHAVDINGHAVDIC-NH2 (SW4), the linear form of the dimeric HAVDI motif (N-Ac-HAVDINGHAVDI-NH2) (SW5), and the monomeric cyclic mimetic of the HAVDI sequence (N-Ac-CHAVDIC-NH2) (Ad126) have been described previously (Williams et al., 2000a, 2002). Neuronal cell cultures Cerebellar granule neurons Cultures of cerebellar granule neurons were prepared from 8day-old Sprague – Dawley rats (Charles River, Margate, UK) as described previously (Skaper et al., 1990). Dissociated cells were seeded in poly-D-lysine (35,000 – 70,000 Mr)-coated 48-well plates (Nunclon) (3.5 105 cells/well) in Basal Medium Eagle’s supplemented to contain 10% FCS, 2 mM L-glutamine, 50 Ag/ml gentamicin, and 25 mM KCl. Cytosine arabinoside (10 AM) was added 24 h after plating to inhibit growth of non-neuronal cells. Cultures prepared in this manner typically contain <5% nonneuronal cells (Boje et al., 1993; Gallo et al., 1987) and were used at 8 days in vitro (DIV). For survival assessment under serum-free plating conditions, granule neurons were prepared from postnatal day 3 rat pups (Doherty et al., 1990; Skaper et al., 2000). Dissociated cells were seeded in poly-D-lysine-coated 96-well plates (8 104 cells/well) in Sato’s medium without insulin (Doherty et al., 1990; Skaper et al., 2000). Hippocampal and cortical neuron cultures Hippocampal and cortical neurons were prepared from embryonic days 17.5 – 18 gestation Sprague – Dawley rat fetuses, as described (Skaper et al., 1990). Cells were maintained in Neurobasal medium supplemented with B27 additives with antioxidants, 1 mM sodium pyruvate, 2 mM glutamine, 25 AM glutamate, 50 U/ ml penicillin, and 50 Ag/ml streptomycin. Half of the medium was replaced every 3 to 4 days with fresh maintenance medium (Neurobasal supplemented with 50 U/ml penicillin, 50 Ag/ml streptomycin, 2 mM glutamine, and B27 without antioxidants). Cultures were used after 7 – 9 days and comprised approximately 95% neuronal cells at this time.
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Assessment of neuroprotective activities Cerebellar granule neurons For survival assessment under serum-free conditions, the HAVDI peptide was added to the cerebellar granule neuron cultures at the time of plating; cultures supplemented with 10% FCS (100% survival) or FGF-2 served as controls. Cell survival was quantified after 48 h by a colourimetric reaction with MTT (Manthorpe et al., 1986; Skaper et al., 1990, 2000). Death of differentiated granule neurons (DIV 8) was induced by shifting cultures to serum-free medium without supplemental KCl. HAVDI peptides or FGF-2 were added either at this time, or 1 or 24 h before the shift to serum-free medium. Forty-eight hours after the switch to serum-free/low-KCl conditions, cell survival was quantified by MTT. Absolute MTT values obtained were normalised for small differences in inter-experiment plating densities by scaling to the mean of sham-treated sister cultures (defined as 100%). Hippocampal neurons: excitotoxic injury Experiments were performed in 7- to 9-day-old cultures because previous studies showed that at this time in culture, the neurons are vulnerable to glutamate toxicity and can be protected against excitotoxic insult by FGF-2 (Mattson et al., 1989, 1995). Cultures were exposed to the HAVDI peptide or FGF-2 for 1 – 24 h. Cultures were then exposed to glutamate (50 – 100 AM), and neuronal survival was quantified 24 h later by MTT. The MTT technique has been shown to be equivalent to lactate dehydrogenase release in the measurement of neurotoxin-mediated neuronal death in vitro (Patel et al., 1990). Cell death and recovery was confirmed in all cases by morphological examination of the culture under phase contrast microscopy.
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