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We investigated the ability of GM1 to induce phosphorylation of the tyrosine kinase receptor for neurotrophins, Trk, in rat brain, and activation of possible down-stream signaling cascades. GM1 increased phosphorylated Trk (pTrk) in slices of striatum, hippocampus and frontal cortex in a concentration- and time-dependent manner, and enhanced the activity of Trk kinase resulting in receptor autophosphorylation. The ability of GM1 to induce pTrk was shared by other gangliosides, and was blocked by the selective Trk kinase inhibitors K252a and AG879. GM1 induced phosphorylation of TrkA > TrkC > TrkB in a region-specific distribution. Adding GM1 to brain slices activated extracellular-regulated protein kinases (Erks) in all three brain regions studied. In striatum, GM1 elicited activation of Erk2 > Erk1 in a time-and concentration-dependent manner. The GM1 effect on Erk2 was mimicked by other gangliosides, and was blocked by the Trk kinase inhibitors K252a and AG879. Pertussis toxin, as well as Src protein tyrosine kinase and protein kinase C inhibitors, did not prevent the GM1-induced activation of Erk2, apparently excluding the participation of Gi and Gq/11 protein-coupled receptors. Intracerebroventricular administration of GM1 induced a transient phosphorylation of TrkA and Erk1/2 in the striatum and hippocampus complementing the in situ studies. These observations support a role for GM1 in modulating Trk and Erk phosphorylation and activity in brain.
Gangliosides are components of most cell membranes, and are particularly abundant in the brain where they represent the major lipid constituent of the neuronal surface. They are thought to play a role in development, cell differentiation and oncogenic transformation (Schengrund 1990; Zeller and Marchase 1992). The monosialic acid ganglioside GM1 promotes neuronal growth and differentiation in cell cultures, and enhances phenotypic expression and neuronal repair in animal models of neurotrauma and aging. The pleiotropic neurotrophic activity of GM1 extends to multiple neuronal phenotypes in the central nervous system, including cholinergic, dopaminergic, serotoninergic and noradrenergic neurons (Hadjiconstantinou and Neff 1998).
The mechanisms for the neurotrophic actions of GM1 are not completely understood. The wide range of neuronal populations where GM1 appears to be efficacious, suggests that it may interact with a large number of neurotrophic factors or activate common pathways used by these factors for signaling. Of interest are data indicating that GM1 might interact with neurotrophins and their receptors in vivo and in vitro. Indeed, GM1 potentiates the neurotrophic effect of NGF in vivo (Cuello et al. 1989; Fong et al. 1995; for review see Hadjiconstantinou and Neff 1998, 2000) and enhances the NGF-induced tyrosine kinase receptor for neurotrophin (Trk) phosphorylation and activation (Ferrari et al. 1995; Mutoh et al. 1995), as well as NGF-induced TrkA phosphorylation and dimerization (Rabin and Mocchetti 1995; Farooqui et al. 1997). Tyrosine phosphorylation and activation of TrkA by GM1 alone has been demonstrated in C6-glioma cells expressing TrkA (Rabin and Mocchetti 1995). As the aforementioned observations were made using tumor cell lines, often over-expressing the neurotrophin receptor, under artificial conditions, their biological relevance for the neurotrophic actions of GM1 in brain remains to be demonstrated.
Binding of neurotrophins to Trk induces activation of the receptor tyrosine kinase, dimerization and autophosphorylation, and initiates a complex cascade of signal transduction events. Tyrosine phosphorylated Trk binds to Shc, phospholipase Cγ and phosphatidyl inositol 3-kinase (PI3-kinase) through SH2 domains resulting in their phosphorylation and subsequent activation of intracellular signaling pathways and transcription factors regulating gene expression (for review see Kaplan and Stephens 1994; Klesse and Parada 1999). These pathways transduce signals independently, but also can converge on the same downstream effector. Stimulation of a variety of tyrosine kinase receptors leads to a rapid elevation of the enzymatic activity of a family of structurally related serine–threonine kinases, known as mitogen-activated protein kinases (MAPKs), which convert extracellular stimuli to intracellular signals that control gene expression (Schaeffer and Weber 1999). The Erk (extracellular signal-regulated kinase) subfamily of MAPKs, is activated in response to Trk stimulation via the small G proteins, Ras and Rap1, which are the targets of multiple second messengers and kinases (Grewal et al. 1999). Thus, Erks serve as a convergence point where signals from multiple transduction pathways are integrated and processed to the nucleus. The Erk1 and Erk2 isoforms of MAPKs are activated by neurotrophins and are thought to mediate some of their survival and differentiative actions in specific subsets of peripheral and central neurons (Klesse and Parada 1999).
Our laboratory has a longstanding interest in the neurotrophic actions of exogenous GM1 on brain after an injury and during aging (Hadjiconstantinou and Neff 1988; Hadjiconstantinou et al. 1990, 1992; Goettl et al. 1999). Given the overwhelming evidence that GM1 not only mimics but also potentiates the neurotrophic action of neurotrophins in some central neuronal systems (Hadjiconstantinou and Neff 1998, 2000), we explored whether the ganglioside was capable of inducing tyrosine phosphorylation and activation of Trk and initiating signal transduction similar to that of neurotrophins. Toward this goal, we used brain slices to investigate the ability of GM1 to induce tyrosine phosphorylation of Trk in brain slices in situ, and activate Erk1 and Erk2, a convergence point of Trk transduction signaling. Slices of striatum, frontal cortex and hippocampus of adult rats were treated with GM1, other gangliosides and neurotrophins, and phosphorylation and activation of Trks and Erks estimated. To prove the validity and applicability of our in situ system, the effect of intracerebroventricularly (ICV) administered GM1 on the phopshorylation of TrkA and Erks was studied in vivo. To our knowledge this is the first study of GM1-induced phosphorylation/activation of Trks and Erks in brain tissue.
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Accumulated evidence suggests that GM1 ganglioside, like the neurotrophins, has the potential to protect injured and aged central neurons, up-regulate neuronal phenotypic expression, induce neuronal sprouting, and enhance neuronal function and metabolism (Hadjiconstantinou and Neff 1998, 2000). In contrast to peptide neurotrophic factors, GM1 can be administered systemically and has shown promising efficacy in some clinical trials (Nobile-Orazio et al. 1994; Hadjiconstantinou and Neff 2000). GM1 is an interesting, controversial and enigmatic compound, and has the potential to serve as a model molecule for designing neuroprotective and neurorestorative drugs. Although some steps towards understanding its mechanism(s) of action have been made, how GM1 works in the brain has largely been a matter of speculation. Because of commonalities with the neurotrophic action of NGF, interest has been focused on the interaction of GM1 with TrkA. Existing evidence, however, suggests that the profile of neurotrophic action of GM1 resembles more that of BDNF, NT-3 and NT4/5. Like these neurotrophins, GM1 has a broader phenotypic repertoire of trophic action on central neurons implying a possible interaction with TrkB and TrkC also (Hadjiconstantinou and Neff 2000). These studies, for the first time, demonstrate in situ and in vivo induction of neurotrophin receptor Trk activation/phosphorylation by GM1 in the brain, and provide evidence of GM1-initiated signal transduction pathways that involve Erks.
Using brain slices, Knüsel et al. (1994, 1996) reported that NGF was the most potent of the neurotrophins for inducing tyrosine phosphorylation of Trk, in striatum, hippocampus and frontal cortex; in contrast, BDNF and NT3 presented low efficacy. Our observations are in general agreement with those of Knüsel and colleagues. As our studies focused on GM1, we are not able to compare qualitatively or quantitatively the effect of GM1 to that of the endogenous Trk ligands. However, given that in most systems 100 ng/mL of neurotrophin produces a maximal Trk phosphorylation at 5 min, we draw attention to the following observations regarding GM1: (i) The maximal response was seen at 20 min and was comparable with that of NGF, and (ii) The effect on Trk was relatively brief by comparison to that reported for NGF (Knüsel et al. 1994, 1996). Trk phosphorylation decreased progressively after 30 min, and at 2 h was no longer detectable. As the levels of LDH remained stable over 2 h of incubation, we believe that the action of GM1 on Trk in brain slices is relatively rapid and short-lasting. The increase of pTrk after GM1 was prevented by the Trk kinase inhibitors K252a and AG879, implying that the increase in phosphotyrosine resulted from Trk kinase activation. Indeed, increased pTrk reflected enhanced receptor tyrosine kinase activity, as demonstrated by autophosphorylation of the receptor in an in vitro kinase assay. The maximal increase of pTrk was observed by 20 min with 100 μm of GM1, a concentration similar to that inducing Trk phosphorylation in cell cultures (Ferrari et al. 1995; Rabin and Moccheti 1995). The concentration response curve was bell-shaped, with response attenuation at high GM1 concentrations. This might indicate two interacting receptor sites, or alternatively, high concentrations of GM1 in the incubation mixture could promote the formation of micelles and other aggregates (Saqr et al. 1993) that interfere with the interaction of GM1 with Trk. The tyrosine phosphorylating effect of GM1 was also extended to TrkB and TrkC. In contrast to the consistent and robust phopshorylation of Trk A, the TrkB and TrkC response to GM1 appeared to be variable and region-dependent, perhaps reflecting differential neurotrophin receptor expression, localization and responsiveness.
Taken together, our findings show that exposure of brain slices to GM1 results in tyrosine phosphorylation of all three tyrosine kinase receptors for neurotrophins. The provided evidence is particularly strong for phosphorylation of TrkA by GM1, as has been indirectly suggested from in vivo experiments (Cuello et al. 1989; Fong et al. 1995; for review see Hadjiconstantinou and Neff 1998, 2000). The importance of our findings lies in the fact that for the first time they demonstrate an early induction of pTrk in brain tissue by GM1 alone. The validity of our conclusions from the in situ studies is strengthened by our original observation that ICV administration of GM1 induces a rapid and short-lasting tyrosine phosphorylation of TrkA in brain in vivo. Our in situ and in vivo findings point to a drug/receptor-like interaction between exogenous GM1 and Trk, suggesting a pharmacological action. In support of this interpretation we were not able to detect increased GM1 content in Trk immunoprecipitates under the conditions used to induce phosphorylation of Trk (data not shown). The notion that exogenous GM1 might have pharmacological properties does not contradict the long standing belief that incorporation of GM1 into membrane is a prerequisite for biological action (Saqr et al. 1993). The two mechanisms, pharmacological and biological, could complement each other and work in unison toward achieving and maintaining an effect. For example, a pharmacological action could initiate a rapid activation of early signaling cascades, whereas incorporation and accumulation of the ganglioside in the plasma membrane over time might be important for ensuring long-term activation of initial signaling cascades and/or recruiting new pathways with the same final destination and outcome.
How GM1 interacts with Trk is unknown. Studies in cell lines have mainly investigated the GM1/NGF synergism in phosphorylating and activating TrkA. So far, direct binding of GM1 to NGF or alteration of [125I] NGF binding by GM1 have been ruled out (Ferrari et al. 1983; Farooqui et al. 1997) and Mutoh et al. (1995) have proposed that in PC12 cells, GM1 enhances the NGF-elicited Trk kinase activity via an association with the Trk protein. Our in situ and in vivo findings, however, suggest that exogenous GM1 alone, directly or indirectly, can induce Trk kinase activation and autophosphorylation in brain tissue. Based on reports that GM1 increases the content of NGF in the brain (Duchemin et al. 1997) and elevates intracellular calcium in neurons (Hilbush and Levine 1992), the possibility that the effect of GM1 on Trk phosphorylation is indirect, through pre- or post-synaptically released neurotrophins (Blöchl and Thoenen 1996; Goodman et al. 1996) was investigated. Under the experimental conditions used in our studies, GM1 failed to release NGF (data not shown), ruling out that the phosphorylation of TrkA is due to enhanced release of its endogenous ligand. Taking advantage of recent advances of knowledge, three possible scenarios for GM1/Trk interaction can be put forward for consideration:
i. GM1 might mediate its effects on neurotrophin tyrosine kinases through interaction inside the membrane itself, especially in the glycolipid-enriched domains, caveolae, where both GM1 (Parton 1994) and tyrosine kinase receptors, including Trk (Wu et al. 1997; Peiróet al. 2000) are present.
ii. GM1 could directly interact with the extracellular portion of Trk, whose leucine-rich motifs are thought to bind neurotrophins (Windisch et al. 1995a, 1995b) and immunoglobulin-like domains to confer neurotrophin binding affinity and specificity (Pérez et al. 1995; Urfer et al. 1995). In lymphocytes, GM1 binds to and regulates CD4, a molecule containing immunoglobulin-like domains (Saggioro et al. 1993), and other gangliosides bind to proteins bearing extracellular immunoglobulin-like domains, such as myelin-associated glycoprotein and other members of the sialoadhesin family (Schnaar et al. 1998).
Erks are emerging as important regulators of neuronal function. In addition to their well established role in regulating cell growth, proliferation and differentiation (Schaeffer and Weber 1999), this family of MAP kinases is an important player for activity-dependent processes, such as synaptic plasticity, long-term potentiation and cell survival (Grewal et al. 1999). Erk1 and Erk2 are activated by a diverse array of ligands, through tyrosine kinase receptors, GPCRs and calcium-dependent pathways, and are among the protein kinases most commonly used in signal transduction (English et al. 1999). In neurons, the Ras-MEK1/2-Erk1/2 cascade has been demonstrated to be both necessary and sufficient for NGF-induced differentiation (English et al. 1999; Klesse and Parada 1999), whereas its role for neurotrophin induced-survival is more controversial (Kaplan and Miller 2000). GM1 induced a rapid and transient activation of Erk1/2 in slices prepared from the striatum, hippocampus and frontal cortex of rat brain. As with pTrk, a maximal response was observed with 100 μm GM1, and activity diminished with high concentrations of the ganglioside. The observation that GM1-induced phosphorylation of Trk and Erk in brain slices raises the possibility of an intimate relationship between these two events. Phosphorylation of TrkA by GM1 has been linked to activation of Erks in C6-glioma cells expressing the neurotrophin receptor (Rabin and Mocchetti 1995). In our studies, a number of direct and indirect lines of evidence support a link between Trk phosphorylation and Erk activation by GM1 in brain slices: (i) the selective Trk kinase inhibitors K252a and AG879 reversed the GM1-induced activation of Erk2; (ii) pre-incubation with pan-Trk antibodies partially blocked both the GM1- and NGF-induced activation of Erk2; (iii) blockade of Gi-coupled receptors with pertussis toxin or inhibition of the intermediate Src kinase did not prevent the GM1 effect; (iv) PKC, a mediator of Gq/G11-coupled pertussis-toxin insensitive activation of Erk2, was not involved in the GM1 response; (v) notably, ICV administration of GM1 also induced phosphorylation of Erk1/2 in vivo in a pattern similar to that of Trk validating our in situ studies and providing additional evidence for a role of the signaling molecule for the neurotrophic action of the ganglioside and for an association with Trk activation/phosphorylation.
The specificity profile of the GM1 effect on Trk and Erk support a model for study whereby GM1-induced phosphorylation of Trk initiates a signaling cascade(s) that results in activation of Erk1/2 via Raf and MEK1/2. The intermediate steps and the targets of this putative signaling pathway are under investigation. That the maximal activation of Erks by GM1 in situ precedes that of Trk does not contradict this model, as both molecules might be under different phosphorylation/dephosphorylation regulation; GM1-sustained phosphorylation of Trk might be needed for transducing other signals, such as PI3-kinase (Hadjiconstantinou et al. 2000); and the net result of Erk activation by GM1 might be the sum of the ganglioside-effect on various Trks and on different neural cells. Finally, the in situ brain system and the different methodologies used to evaluate Trk phosphorylation and Erk activation might contribute to the temporal discrepancy. In PC12 cells, NGF induces both transient and sustained activation of Erks, with the latter thought to be important for cell differentiation (Marshall 1995). Accordingly, differentiated mature brain neurons would be expected to respond with transient activation of Erks. Indeed, in the brain slices, NGF induced a transient pErk2 increase that peaked by 5–10 min and lasted for about 60 min (data not shown).
The tyrosine phosphorylation of Trk and the activation of Erk2 are not an exclusive property of GM1 ganglioside. Other major gangliosides present in brain, GD1a, GD1b, GT1b as well as GM2 and GM3 displayed similar action when added to brain slices. This is not a surprising finding as, in addition to GM1, other gangliosides display neurotrophic action as well. A GD1a, GD1b, GT1b and GT1 mix enhance neuritogenesis in cell cultures and promote regenerative and behavioral responses when administered to animal models of central or peripheral neuronal injury (Schengrund 1990); GT1b potentiates the NGF-induced neurite extension in PC12 cells and promotes long-term survival after serum deprivation (Ferrari et al. 1983, 1995); and GM3 increases dopamine and GABA uptake in embryonic mesencephalic cultures (Leon et al. 1988). Our data and the literature suggest that the whole ganglioside molecule is required for Trk or Erk phosphorylation/activation. Our observation that ceramide does not affect Trk phosphorylation in brain tissue questions the suggestion of MacPhee and Baker (1999), that the GM1-dependent enhancement of TrkA activity may be due to liberated ceramide. Van Brocklyn et al. (1997) reported both GM1- and ceramide-mediated activation of Erk2 in glioma cells, but the authors concluded that the contribution of the ceramide to the GM1 effect was at most minimal. Presently, we do not know whether there is a link between the Trk and Erk activation by the various gangliosides in our system. Fukumoto et al. (2000), reported that GD3 synthase gene expression in PC12 cells results in continuous activation of TrkA and Erk1/2, probably due to the increased production of GD1b and GT1b. Furthermore, we do not know whether all gangliosides act at the same site and utilize the same mechanism(s) for initiating common signaling pathways. Recent advances in our understanding of the organization of the cell membranes have provided useful clues for future inquiry in this direction. Of particular interest is the emergence of the sphingolipid-enriched cell membrane domains, such as caveolae and glycosphinglolipid-signaling domains, as unique membrane compartments with biological function. They are thought to provide a spatial environment where various protein and lipid molecules involved in signal transduction interact, and they have been shown to partake in signal transduction upon glycosphingolipid-mediated stimulation (Masserini et al. 1999; Hakomori 2000).
In conclusion, our studies for the first time demonstrate that GM1 induces tyrosine phosphorylation of TrkA, TrkB and TrkC high-affinity receptors for neurotrophins, and initiates signal transduction resulting in activation of the MEK1/2/Erk1/2 pathway in brain slices. More importantly, our studies provide evidence for in vivo tyrosine phosphorylation of TrkA and Erk1/2 in brain tissue after GM1 ICV administration. The prevention of the GM1 effect on Trks and Erks by selective Trk kinase inhibitors suggests a possible link between the two events. This is supported further by the exclusion of Gi-or Gq/11-coupled receptors as possible sites for the GM1 effect. In neurons, the MEK1/2/Erk1/2 pathway coordinates complex cellular responses such as differentiation, survival, synaptic plasticity, long-term potentiation, and learning and memory (Grewal et al. 1999; Sweatt 2001). Usage of the MEK1/2/Erk1/2 pathway by GM1 might explain the diverse actions of the ganglioside on immature and mature neurons. Finally, the observed tyrosine phosphorylation of Trks in brain tissue after GM1, in situ and in vivo, confirms the long-standing speculation that GM1 acts, in part, as a neurotrophin mimetic.