Functional roles of gangliosides in bio-signalling

III ELSEVIER

Behavioural Brain Research 66 (1995) 99-104

BEHAVIOURAL BRAIN RESEARCH

Functional roles of gangliosides in bio-signaling Yoshitaka Nagai* Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan and Glycobiology Research Group of the Frontier Research Program, The Institute c?[" Physical and Chemical Research (RIKEN), Saitama, Japan Accepted 15 August 1994

Abstract

Brain gangliosides, a sialic acid-containing glycosphingolipid family enriched in brain, are discriminated from those of extra neural tissues by their characteristic structures of carbohydrate chain with large molecular diversity. Numerous minor components and monoclonal antibodies to them are useful to identify type, distribution and lineage of the cells, as shown in the recent finding of the ganglioside epitope of cholinergic neuron-specific Chol-1 antigens. Various cell biological effects of exogenous gangliosides (bioactive gangliosides) particularly with regard to cell growth and differentiation strongly suggest involvement of gangliosides and possibly their metabolic intermediates as second messenger in signaling pathways. The neuritogenic as well as synaptogenic effects of gangliosides may be interpreted by their action on protein kinases. The analysis of the neuritogenic activity of GQlb ganglioside on human neuroblastoma cell lines strongly indicates the possibility that the action is carried out by coupling of GQlb sugar-specific glycoreceptor of cell surface membrane and a unique, cell surface localized protein kinase (ecto-protein kinase) to phosphorylate cell surface protein(s) with extracellular ATP. This cell surface (ecto) type of protein phosphorylation system which is in contrast to intracelular (endo) type of protein phosphorylation seems to highly develop in neuron. Possible involvement of gangliosides in synaptic function including ion-transport and long-term potentiation is also suggested.

Key words: Ganglioside; Signal transduction; Long-term potentiation; Neuritogenesis; Protein kinase; Cholinergic neuron marker; Glycosphingolipid; Ecto-proteinkinase

1. Molecular diversity of brain gangliosides and their usefulness as cell markers

Brain gangliosides show a great molecular diversity with numerous novel minor components, and are enriched in neuronal, glial, and in particular, synaptic membranes as well as growth cones [1,2]. Among those novel minor components, Chol-1 gangliosides were recently found to be the epitope of the polyclonal antibody to synaptic membranes of electric organs of Torpedo marmoranta, which was established to be specific for cholinergic neuron by Whittaker et al. in 1982 [3,4]. Immunohistochemistry with this antibody showed specific expression of the epitope on the cell body and nerve terminal of the cholinergic neuron. Successful structural determination ofChol-1 revealed that Chol-1 antigen (Chol-l-~ constitutes two novel Chol-l-~

* Corresponding author. Tokyo Metropolitan Institute of Medical Science, 18-22 Honkomagome 3-chome, Bunkyo-ku, Tokyo 113, Japan. Fax: (81) (3) 3823-2965. 0166-4328/95/$9.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0 1 6 6 - 4 3 2 8 ( 9 4 ) 0 0 1 3 0 - 8

gangliosides, G T l a e and G Q l b ~ [5]. (Fig. 1) and three Chol-1-// gangliosides, G D l a ~ , G T l b ~ and G I ~ [6]. Monoclonal antibody (GGR41) to Chol-1-e demonstrated intense staining of the neuropile of dorsal horn on spinal cord being expressed presumably on the cholinergic nerve terminals [7].) The different staining pattern from that with anti-Chol-1 polyclonal antibody suggested that Chol1-e gangliosides are expressed on the different region from Chol-1-/~ on the cholinergic neuron. Thus, minor, in particular, and major [8] gangliosides should be useful as cell markers not only to identify various neural cells but also to analyse their functional roles as well as cell lineage.

2. Carbohydrate signals in the regulation of cellular activities

It is well known that cell surface carbohydrate profile characteristically changes in development, differentiation, organ regeneration, and cell sociological disorders such as

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Y. Nagai / Behavioural Brain Research 66 (1995) 99-104

2), respectively [ 12,13]. In some cases, sphingosine and its N-methyl derivatives [ 2,14,15 ], sphingosine- 1-phosphate [16] and ceramide [17], all of which are metabolically formed on cell stimuli from sphingolipids (e.g., sphingomyelin, and possibly GM3) of cell membrane, are proposed to play a role as second messenger (Table 1, Case C; Fig. 2, Case 3) [ 1,2]. Sphingosine and lyso GM3 inhibit activity of protein kinase C [2]. Ceramide which was either endogenously formed by a similar metabolic pathway or was exogenously added may trigger cell apoptosis (programmed cell death) (Table 1, Case C; Fig. 2, Case 5) [ 18 ]. Such bioactive sphingolipids and their metabolic derivatives may open new vistas in glycosphingolipid research of brain. Ganglioside GD3 which is one of the characteristic brain gangliosides shows several interesting functional behaviors in brain [19]. GD3 is highly expressed in proliferative embryonic neurons followed by rapid decline in its content after birth and is also expressed in astrocytes in active phase and in melanoma cells. Cultured rat embryonic fibroblastic 3Y1 cells which were transfected and transformed with specified retroviral DNA oncogenes (adenovirus EIA, SV40-T, v-myc) newly express GD3 on cell surface [20]. Monoclonal antibody (R24) to GD3 reversibly suppressed proliferation of those transfected and

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tumors, suggesting its significance to understand cell-cell interactions including cell adhesion [ 1,9]. Moreover, specific carbohydrate recognizing ligands such as bacterial toxins, antibodies, and lectins may influence on cellular activity by means of their binding to cell surface carbohydrates (Fig. 2, Case 4). Cholera toxin B subunit which has been regarded to principally bind to GM1 ganglioside regulates proliferation of cultured cells bimodally, that is, inhibitory in growing logarithmic phase and inductive in stationary, confluent phase of the same cells, and interferes with signal transduction mechanism of the cells [ 10,11 ]. Polyclonal antibodies to GM3 and GM 1, respectively, also regulate growth of certain cultured ceils. Growth factor (EGF, PDGF) dependent signal transduction accompanied by protein phosphorylation is also modulated by gangliosides, GM3 and GM1 (Fig. 2, Case

Table 1 Possible modes of participation of glycoconjugates (glycolipids) in biosignaling Case A Modulation by perturbation of lipid matrix Regulation by alteration of physicochemical membrane electrical parameters, etc. Case B Functional modulation of receptor in micro domain Alteration of receptor function by annular (boundary) lipid molecules (e.g. GM3 ganglioside and its derivatives, etc.) Case C Metabotropic modulation Sphingosine and its derivatives Ceramide De-N-acetylation, etc. Case D Direct perturbation or binding by ligand of surface carbohydrate chain Lectin/Antibody/Toxin interactions with glycoconjugates Case E Glycomessage-glycoreceptor interaction (recognizing, transducing, responding) Intracellular (endo) type signal transduction Cell surface (ecto) type signal transduction Case F Direct transfer to cell nucleus (gene) of glycomessage Carbohydrate-mediated modulation of nucleo-protein supramolecular structure Regulation of gene expression (activation of suppression) by glycosylation

Y. Nagai / Behavioural Brain Research 66 (1995) 99-104

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transformed 3Y1 cells but not of non-transfected, parent 3Y1 cells (Y. Sanai and Y. Nagai, unpublished). Thus, increasing evidence suggests that gangliosides and their ligands may play a role as important, signaling molecules (Table 1, Case D).

3. Gangliosides in neuritogenesis, synaptogenesis and synaptic function There are accumulating data indicating involvement of gangliosides in neuritogenesis and possibly in synaptogenesis [ 1,2,21 ]. Among brain gangliosides used for such studies GM 1 and GQ lb are typical in their characteristic, action spectrum and underlying molecular mechanisms. Both gangliosides exogenously added are capable of promoting neurite outgrowth of cultured neuronal cells, such as neuroblastoma and pheochromocytoma cells, and primary cultures of neurons. GM1, however, promoted it most efficiently in mouse neuroblastoma, Neuro-2a, and also in rat pheochromocytoma, PC-12, in which GM1 potentiated inductive and promotive action of nerve growth factor (NGF), while GM 1 alone does not exhibit the activity at all in PC-12 cell line. Concentrations required for the activity were usually in the range of 10 ~~ 10 -6 M. GM1 was proven to influence on Na + and Ca 2 + flux and to activate adenylate cyclase and cyclic nucleotide phosphodiesterase [2]. Endogenous role of GM 1 in neurite outgrowth was demonstrated by the promotive effect of neuraminidase treatment of the cells, which is presumed to express more GM1 on the surface, and then by blocking the effect with anti-GM 1 antibody and also with cholera toxin subunit B which specifically binds to GM1 [2]. The underlying mechanism of the GM1mediated neuritogenesis, however, remains unclear. In this

connection, it is interesting to note that exogenous GM3 and neolactotype gangliosides acted on human promyelocytic leukemia cells, HL-60, as a differentiation inducer like phorbol ester (TPA), vitamin D and dimethylsulfoxide following initial inhibitory effect of them on the cell growth [22]. The former ganglioside guided the cells to differentiate into monocytic, macrophage-like cells, whereas the latter into mature granulocytes. They were active in the range of 105-106 M which issimilar to that observed in GM1 induced neuritogenesis. It has recently been shown that the activity of the excitatory opioid receptor in cultured dorsal root ganglia (DRG) is potentiated by GM1 and that cholera toxin B or anti-GM 1 antibody selectively blocks opioid--but not forskolin-induced prolongation of the action potential duration [2]. It was also reported that GM1 and possibly other gangliosides modulate receptors of Gsqinked membrane type including the fll receptor either positively or negatively by co-localization with receptor molecules in membrane [2]. On the other hand, the characteristics of GQlb-induced neuritogenesis in cultured human neuroblastoma cell lines, GOTO or NB-1, are different from the case of GM 1 in other neuroblastoma cells such as mouse Neuro-2a or rat PC- 12 cells [23]. Optimal concentration of GQ lb for the activity was in the range of a few 10 9 M ( ~ 5 ng/ml), which is comparable to that of nerve growth factor (NGF). The analysis of structure-function relationship indicated the absolute requirement of G Q l b as well as its carbohydrate structure for the activity. From the competitive inhibition study using oligosaccharide portion of G Q l b (oligo GQ lb), existence of the GQ lb recognizing glycoreceptor molecules on the cell surface was presumed (Fig. 3, Case 2).

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Y. Nagai / Behavioural Brain Research 66 (1995) 99-104

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Fig. 3. Involvement of glycosphingolipids, and their derivatives or analogs in signal transduction system-II. PK, protein kinase; Pke, ecto protein kinase; SAC, sialyl cholesterol; P, phosphate. Case 1: Endo type signal transduction. Case 2: Ecto type signal transduction. Case 3: Direct transfer of glycomessage to cell nucleus resulting in the regulation of gene expression (Table 1, F). Synthetic, artificial ganglioside analogs, ~- or fl-N-acetylneuraminyl cholesterol (Fig. 1) exoglynously added to mouse neuroblastoma cells, Neuro-2a cells, was capable of promoting not only neurite outgrowth but also transcriptional gene activity in cell nuclei, into which the compound was found to be promptly transported in a very short time [36]. The transcriptional activity was enhanced two-fold as much as the control, No such transport and the enhancement of transcription were observed with GM 1 in association with neuritogenesis, suggesting potential involvement of sialo compounds in modulating the gene expression machinery. Meanwhile, rapid transport of neuraminidase-cleavable ~-sialyl cholesterol may provide a useful means to transport sialic acid into cell interior across cell surface membrane.

Therapeutic or in vivo administration of gangliosides to enhance or improve neuritogenesis, synaptogenesis, neuronal repair, traumatic lesions of the peripheral nerve tissues, and behavioral and neurological impairment has been well documented, particularly referring to GM1, after the first report by Ceccarelli et al. in 1976 [1,2,21]. However, it is not yet clear whether or not gangliosides which are administered either intraperitoneally or intravenously or intramuscularly can be transported into targetting loci of the central nervous system across blood-brain barrier.

4. Endo vs. ecto type of signal transduction

Most of receptor-mediated signal transduction and ion channel function are accompanied by subsequent protein phosphorylation which is catalyzed by specific protein kinases. Neuritogenic effect of exogenous G Q l b in human neuroblastoma cells was also accompanied by protein phosphorylation. GQ lb itself, however, did not influence on in vitro activities of several protein kinases such as Ca 2+/calmodulin-dependent kinase, cAMP-dependent kinase and protein kinase C of which activity was rather suppressed [23]. There are many other in vitro experi-

ments which showed that various gangliosides except for GQ lb differently influence on several protein kinases (e.g. protein kinase C, Ca 2 +/calmodulin-dependent kinase II, Ca 2 +/phosphatidylserine-independent kinase M, tyrosine kinase, protein kinase acting on myelin basic proteins as substrate, etc.), being dependent on the type of protein kinases and gangliosides used [2,21]. Intraperitoneal administration of GM1 was also shown to influence activity of protein kinase M and to cause the cytosolic to membrane shift of protein kinase C in fetal rats which were subjected to ischemia [24]. Interestingly, above-mentioned GQlb-dependent protein phosphorylation was observed at the external surface of living cells in the presence of extracellular ATP and was catalyzed by a unique protein kinase which exists at cell surface, therefore, called ecto-protein kinase (Fig. 3, Case 2) [24,25,26]. At least three proteins (54, 60 and 64 kDa) of cell surface membrane were served to be substrates for the ecto-enzyme and phosphorylated at cell surface loci. Correlation between ecto-phosphorylation and neuritogeneses was well established with regard to necessity and optimal dose (a few 10 9 M) of GQlb, and dosedependent inhibition of phosphorylation with oligo GQ 1b. More direct evidence for coupling of ecto-phosphorylation and neuritogenesis was recently given by the use of a unique, cell membrane impermeable and non-cytotoxic protein kinase inhibitor, K-252b, the fate of which is easily traceable by its strong fluorescence. Originally this hydrophilic alkaloid was isolated together with cell membrane permeable K-252a from the culture broth of Nocardiopsis sp. as a potent selective inhibitor for protein kinase C in vitro by Kase et al. in 1986. (Fig. 1) [27]. The inhibitor, K-252b, added to the living cells instantly abolished ectoprotein phosphorylation as well as neuritogeneses [28]. It also inhibited functional synapse formation between primary cultured neurons of rat cerebral cortex [29]. Interestingly phorbolester-induced long-term potentiation (LTP) in rat hippocampal slices was inhibited by this inhibitor [30]. Meanwhile, the characteristic post-tetanic (4 pulses, 100 Hz) LTP was observed in the magnitude of CAl-evoked responses by perfusion of guinea pig hippocampal slices with either G Q l b (4/~g/ml) or GM1 (4/~g/ ml) in a lesser degree (H. Kato, personal communication). The effect of G Q l b was greater than that of GM1. It is likely that K-252b is also capable of interferring such an LTP. On the other hand, the most intense immunostaining with anti-GQlb monoclonal antibody was demonstrated to be associated with CA1 pyramidal cells, the stratum oriens and the alveus of the hippocampus but neither with the other fields nor layers of the hippocampal formation [8]. Extracellular ATP-dependent ecto-phosphorylation was observed in other cultured neuronal cells like PC12h, Neuro-2a and NIE-115, and also in glial cells such as

Y. Nagai / Behavioural Brain Research 66 (1995) 99-104

RCR-1 (astrocyte), C-6 (astrocytoma) and 354-A (peripheral glioma-like s c h w a n n o m a ) (Y. Nagai and S. Tsuji, unpublished). In the latter cases, numbers o f phosphorylated proteins were found to be far fewer than those in neuronal cells, suggesting that ecto-phosphorylation system may mostly develop well in neurons. PC12h cells, o f which the neuritogenesis is solely dependable on N G F but neither on G M 1 nor G Q lb, also showed enhancement o f ecto-phosphorylation with N G F . Simultaneous addition of G M 1 potentiated both N G F - d e p e n d e n t neuritogenesis and ecto-phosphorylation. Interestingly, G Q l b - n o n respondable PC-12 cells themselves already contained sufficient a m o u n t of G Q l b , whereas G Q l b - r e s p o n d a b l e G O T O cells did not contain any detectable amount of endogenous G Q I b [26]. It is very likely that the ecto type of signal transduction is coupled with the endo type one. In order to make ecto-phosphorylation experimentally observable, extracellular addition of [ 7 - 3 2 P ] A T P (1/~M) was necessary, while G Q l b - m e d i a t e d neuritogenesis occurs without addition of A T P to the cells. The possibility should be made clear whether or not extracellular secretion of A T P may occur simultaneously in the presence of exogenous G Q lb. Synaptic vesicles which usually contain an extremely high amount (0.1-0.2 M) of A T P together with neurotransmitters, both of which are secreted into the synaptic cleft on stimulation, may be one of the presumable sources for such extracellular ATP. These A T P molecules secreted on stimulation have so far been presumed to act on purinergic receptors at presynaptic m e m b r a n e after its metabolic conversion to A M P or A D P in the synaptic cleft. On the other hand, the hypothesis was proposed that Ca 2 +-ganglioside interactions may play a role as modulators of synaptic transmission and long-term adaptation [ 31,32]. The activity of Ca 2 + -activated A T P a s e [ 3 3 ], protein phosphatase [34] and Ca 2+ channels [35] are modulated by gangliosides.

5. Five different modes of participation of carbohydrate messages or glycolipids in bio-signaling It becomes evident that glycolipids, in particular, gangliosides and their analogs are in variously ways involved in bio-signaling machinery, as summarized in Table 1. A m o n g six cases shown in Table 1, cases B, C, D, and E seem to be particularly important in their possible relevance to neuronal network formation, synaptic contact, plasticity and brain function. It should be also emphasized that little studies have so far been done on glycoreceptor or protein ligand which specifically interacts with the cell surface carbohydrate chain, which potentially embodies extremely high diversity in a very short sequence.

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Carbohydrate chain is synthesized on the sequential addition of a sugar unit by various specific glycosyl transferases but not on templates that assure more rigorous synthesis as evidenced in proteins and nucleic acids. Enzymatic reactions are influenced by the conditions surrounding the enzyme molecules (temperature, pH, salt ions, nucleotide sugar donors, sugar acceptors, and many others). Nature, however, may utilize such a fuzzy character of synthetic potential in the carbohydrate chains for neuronal plasticity as well as the formation of neuronal network based on cell-cell recognition.

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[26] Nagai, Y. and Tsuji, S., Significance of ganglioside-mediated glycosignal transduction in neuronal differentiation and development. In L. Svennerholm, A.K. Asbury, R.A. Reisfeld, K. Sandhoff, G. Tettamanti and G. Toffano (Eds.), Biological Function qf Gangliosides, Progress in Brain Research, Vol. 101, Elsevier, Amsterdam, 1994, pp. 119-126. [27] Kase, H., Iwahashi, K. and Matsuda, Y. K-252a, a potent inhibitor of protein kinase C from microbial origin, J. Antibiotics, 39 (1986) 1059-1065. [28] Tsuji, S., Yamashita, T., Matsuda, Y. and Nagai, Y., A novel glycosignaling system: GQlb-dependent neuritogenesis of human neuroblastoma cell line, GOTO, is closely associated with GQlbdependent ecto-type protein phosphorylation, Neurochem. Int., 21 (1992) 549-554. [29] Muramoto, K., Kobayashi, K., Nakanishi, S., Matsuda, Y. and Kuroda, Y., Functional synapse formation between cultured neurons of rat cerebral cortex: block by a protein kinase inhibitor which does not permeate cell membrane, Proc. Jpn. Acad. Ser. B, 64 (1988) 319-322. [30] Reyman, K.G., Br0demann, R., Kase, H. and Matties, H., Inhibitors of calmodulin and protein kinase C block different phases of hippocampal long-term potentiation, 461 (1988) 388-392. [31] Rahmann, H., Calcium-ganglioside interactions and modulation of neuronal functions. In N.N. Osborne (Ed.), Current Aspects of Neurosciences, Vol. 4, Macmillan Press, London, 1992, pp. 88-125. [32] Thomas, P.P. and Brewer, G.J., Gangliosides and synaptic transmission, Biochim. Biophys. Acta, 1031 (1990) 277-289. [33] Slenzka, K., Appel, R. and Rahmann, H., Influence of exogenousgangliosides (GM1, GDla, GMix) on a Ca 2+-activated Mg2÷dependent ATPase in cellular and subcellular brain fractions of the Djungarian Dwarf Hamster (Phodopus sungorus), Neurochem. Int., 17 (1990) 609-614. [34] Nakaoka, T., Tsuji, S. and Nagai, Y., Bimodal regulation of protein phosphorylation by a ganglioside in rat brain membrane, J. Neurosci. Res., 31 (1992) 724-730. [35] Spiegel, S., Gangliosides are biomodulators of cell growth. In R.W. Ledeen, Hogan, H.L., Tettamanti, G., Yates, A.J. and R.K. Yu (Eds.), New Trends in Ganglioside Research: Neurochemical and Neuroregenerative Aspects. Fidia Research Series, Vol. 14, Liviana Press-Springer Verlag, Padova-Berlin, 1988, pp. 405-421. [36] Yamashita, T., Tsuji, S. and Nagai, Y., Sialyl cholesterol is translocated into cell nuclei and it promotes neurite outgrowth in a mouse neuroblastoma cell line, Glycobiology, l (1991) 149-154.