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286 BRES 15353
Derivatives of ganglioside GM1 as neuronotrophic agents: comparison of in vivo and in vitro effects Michael S. Cannella 1, Barbara Oderfeld-Nowak 2, Matgorzata Gradkowska 2, Matgorzata Skup 2, Lorella Garofalo 3, A. Claudio Cuello 3 and Robert W. Ledeen 1 1Department of Neurology, Albert Einstein College of Medicine, Bronx, NY 10461 (U. S.A.), 2Nencki Institute of Experimental Biology, Warsaw (Poland) and 3Departmentof Pharmacology and Therapeutics, McGill University, Montreal, Que. (Canada) (Accepted 5 September 1989)
Key words: GM1 ganglioside; GM1 derivative; Neuro-2A cell; PC12 cell; Dorsal root ganglia; Septohippocampal animal model; Nucleus basalis magnocellularis-cortex animal model
Exogenously administered gangliosides have been shown to behave as neuronotrophic/neuritogenic agents in a variety of cell culture systems and animal models, but it is not known whether they operate by the same mechanism in vivo and in vitro. To probe this question we have employed two derivatives of GM1 lacking the negative charge: the methyl ester (GM1-CH3) and the NaBH4 reduction product of the latter (GM1-OH) in which the carboxyl group is replaced by a primary alcohol. Both derivatives proved to be as neuritogenic as GM1 in 3 cell culture systems: neuro-2A ceis, PC12 cells and explanted dorsal root ganglia. However, GM1-OH proved ineffective when applied to two animal models involving reduction of cholinergic markers in: (a) hippocampus following lesion of the lateral fimbria and (b) nucleus basalis magnocellularis following cortical lesion; GM1-CHa showed marginal activity in (a) but more in (b), possibly owing to slow hydrolysis to GM1 which was highly active in both animal models. These results indicate the necessity of a negative charge on the ganglioside molecule for in vivo but not in vitro activity and point to different mechanisms for the trophic effects of exogenous gangliosides.
INTRODUCTION The dramatic changes observed in ganglioside content and structural complexity during elaboration of vertebrate neuronal circuitry 16'3°'45'52'54'58'59 have drawn attention to their prospective role(s) in neuronal differentiation. Potentially related observations include the unusual morphological abnormalities apparently caused by accumulated ganglioside in ganglioside storage diseases 41' 56 and the ability of exogenous gangliosides to exert neuronotrophic and/or neuritogenic influences on living cells. A noteworthy aspect of the latter p h e n o m e n o n is the manifestation of such effects at both the in vitro and in vivo levels. Thus, various neuroblastoma cell lines 3' 12,33,37,43 and primary neuronal culture systems 13A5'23'43'47 were shown to respond to gangliosides as neuritogenic agents under appropriate conditions, while several animal models have been used to demonstrate in vivo trophic effects on neurons of both the CNS and PNS (for review, see refs. 7, 29). In view of certain apparent similarities in these trophic p h e n o m e n a it has been thought likely that the in vivo and in vitro effects share a c o m m o n mechanism, although direct evidence for this is lacking. Little is known, in fact,
concerning the mechanistic role of gangliosides as trophic agents in general. One approach to this question, applied thus far to cell culture systems 3-6'53, has been the use of different gangliosides and synthetic sialoglycolipids to assess the structural requirements for neuritogenic activity. In the present study we have used this approach to compare the in vivo and in vitro effects of two synthetic derivatives of GM1 which lack the negative charge: its methyl ester (GM1-CH3) and the NaBH4-reduction product of the latter ( G M 1 - O H ) in which the carboxyl group of GM1 is replaced by a primary alcohol. These derivatives were applied to 3 in vitro- and 2 in vivo systems: the former included neuro-2A neurobtastoma cells, PC12 p h e o c h r o m o c y t o m a cells, and embryonic chick dorsal root ganglia ( D R G ) explants, while the animal models employed were those involving degeneration of cholinergic fibers in the rat hippocampus following lesion of the lateral fimbria 21, and retrograde degeneration of cholinergic neurons in the nucleus basalis magnocellularis (NBM) following unilateral cortical lesion 11"5°. These in vivo models were previously shown 11'21 to respond to GM1 with preservation of the cholinergic markers, choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE), while the 3 cell culture systems
Correspondence: R.W. Ledeen, Dept. of Neurology, Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
287 TABLE I Per cent of neurite bearing neuro-2A and PC12 cells Neuro-2A (7 fields surveyed) and PC12 (6 fields surveyed) cells were grown as described without (control) or with the indicated glycolipid, each field containing approximately 50-100 cells. PC12 studies were done in the presence of NGE Data are mean + S.D. P values relative to controls were
286 BRES 15353
Derivatives of ganglioside GM1 as neuronotrophic agents: comparison of in vivo and in vitro effects Michael S. Cannella 1, Barbara Oderfeld-Nowak 2, Matgorzata Gradkowska 2, Matgorzata Skup 2, Lorella Garofalo 3, A. Claudio Cuello 3 and Robert W. Ledeen 1 1Department of Neurology, Albert Einstein College of Medicine, Bronx, NY 10461 (U. S.A.), 2Nencki Institute of Experimental Biology, Warsaw (Poland) and 3Departmentof Pharmacology and Therapeutics, McGill University, Montreal, Que. (Canada) (Accepted 5 September 1989)
Key words: GM1 ganglioside; GM1 derivative; Neuro-2A cell; PC12 cell; Dorsal root ganglia; Septohippocampal animal model; Nucleus basalis magnocellularis-cortex animal model
Exogenously administered gangliosides have been shown to behave as neuronotrophic/neuritogenic agents in a variety of cell culture systems and animal models, but it is not known whether they operate by the same mechanism in vivo and in vitro. To probe this question we have employed two derivatives of GM1 lacking the negative charge: the methyl ester (GM1-CH3) and the NaBH4 reduction product of the latter (GM1-OH) in which the carboxyl group is replaced by a primary alcohol. Both derivatives proved to be as neuritogenic as GM1 in 3 cell culture systems: neuro-2A ceis, PC12 cells and explanted dorsal root ganglia. However, GM1-OH proved ineffective when applied to two animal models involving reduction of cholinergic markers in: (a) hippocampus following lesion of the lateral fimbria and (b) nucleus basalis magnocellularis following cortical lesion; GM1-CHa showed marginal activity in (a) but more in (b), possibly owing to slow hydrolysis to GM1 which was highly active in both animal models. These results indicate the necessity of a negative charge on the ganglioside molecule for in vivo but not in vitro activity and point to different mechanisms for the trophic effects of exogenous gangliosides.
INTRODUCTION The dramatic changes observed in ganglioside content and structural complexity during elaboration of vertebrate neuronal circuitry 16'3°'45'52'54'58'59 have drawn attention to their prospective role(s) in neuronal differentiation. Potentially related observations include the unusual morphological abnormalities apparently caused by accumulated ganglioside in ganglioside storage diseases 41' 56 and the ability of exogenous gangliosides to exert neuronotrophic and/or neuritogenic influences on living cells. A noteworthy aspect of the latter p h e n o m e n o n is the manifestation of such effects at both the in vitro and in vivo levels. Thus, various neuroblastoma cell lines 3' 12,33,37,43 and primary neuronal culture systems 13A5'23'43'47 were shown to respond to gangliosides as neuritogenic agents under appropriate conditions, while several animal models have been used to demonstrate in vivo trophic effects on neurons of both the CNS and PNS (for review, see refs. 7, 29). In view of certain apparent similarities in these trophic p h e n o m e n a it has been thought likely that the in vivo and in vitro effects share a c o m m o n mechanism, although direct evidence for this is lacking. Little is known, in fact,
concerning the mechanistic role of gangliosides as trophic agents in general. One approach to this question, applied thus far to cell culture systems 3-6'53, has been the use of different gangliosides and synthetic sialoglycolipids to assess the structural requirements for neuritogenic activity. In the present study we have used this approach to compare the in vivo and in vitro effects of two synthetic derivatives of GM1 which lack the negative charge: its methyl ester (GM1-CH3) and the NaBH4-reduction product of the latter ( G M 1 - O H ) in which the carboxyl group of GM1 is replaced by a primary alcohol. These derivatives were applied to 3 in vitro- and 2 in vivo systems: the former included neuro-2A neurobtastoma cells, PC12 p h e o c h r o m o c y t o m a cells, and embryonic chick dorsal root ganglia ( D R G ) explants, while the animal models employed were those involving degeneration of cholinergic fibers in the rat hippocampus following lesion of the lateral fimbria 21, and retrograde degeneration of cholinergic neurons in the nucleus basalis magnocellularis (NBM) following unilateral cortical lesion 11"5°. These in vivo models were previously shown 11'21 to respond to GM1 with preservation of the cholinergic markers, choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE), while the 3 cell culture systems
Correspondence: R.W. Ledeen, Dept. of Neurology, Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
287 TABLE I Per cent of neurite bearing neuro-2A and PC12 cells Neuro-2A (7 fields surveyed) and PC12 (6 fields surveyed) cells were grown as described without (control) or with the indicated glycolipid, each field containing approximately 50-100 cells. PC12 studies were done in the presence of NGE Data are mean + S.D. P values relative to controls were
