Subcellular Origin of Sphingosine 1-Phosphate Is Essential for Its Toxic Effect in Lyase-deficient Neurons

Abstract

Cerebellar granule cells from sphingosine 1-phosphate (S1P) lyase-deficient mice were used to study the toxicity of this potent sphingolipid metabolite in terminally differentiated postmitotic neurons. Based on earlier findings with the lyase-stable, semi-synthetic, cis-4-methylsphingosine phosphate, we hypothesized that accumulation of S1P above a certain threshold induces neuronal apoptosis. The present studies confirmed this conclusion and further revealed that for S1P to induce apoptosis in lyase-deficient neurons it must also be produced by sphingosine-kinase2 (SK2). These conclusions are based on the finding that incubation of lyase-deficient neurons with either sphingosine or S1P results in a similar elevation in cellular S1P; however, only S1P addition to the culture medium induces apoptosis. This was not due to S1P acting on the S1P receptor but to hydrolysis of S1P to sphingosine that was phosphorylated by the cells, as described before for cis-4-methylsphingosine. Although the cells produced S1P from both exogenously added sphingosine as well as sphingosine derived from exogenous S1P, the S1P from were not the was produced by the was by also was cis-4-methylsphingosine based on studies in neurons or due to the of for S1P, exogenous S1P be this S1P due to a however, this of toxicity also be in as by the of cis-4-methylsphingosine Cerebellar granule cells from sphingosine 1-phosphate (S1P) lyase-deficient mice were used to study the toxicity of this potent sphingolipid metabolite in terminally differentiated postmitotic neurons. Based on earlier findings with the lyase-stable, semi-synthetic, cis-4-methylsphingosine phosphate, we hypothesized that accumulation of S1P above a certain threshold induces neuronal apoptosis. The present studies confirmed this conclusion and further revealed that for S1P to induce apoptosis in lyase-deficient neurons it must also be produced by sphingosine-kinase2 (SK2). These conclusions are based on the finding that incubation of lyase-deficient neurons with either sphingosine or S1P results in a similar elevation in cellular S1P; however, only S1P addition to the culture medium induces apoptosis. This was not due to S1P acting on the S1P receptor but to hydrolysis of S1P to sphingosine that was phosphorylated by the cells, as described before for cis-4-methylsphingosine. Although the cells produced S1P from both exogenously added sphingosine as well as sphingosine derived from exogenous S1P, the S1P from were not the was produced by the was by also was cis-4-methylsphingosine based on studies in neurons or due to the of for S1P, exogenous S1P be this S1P due to a however, this of toxicity also be in as by the of cis-4-methylsphingosine 1-phosphate (S1P) used S1P, sphingosine sphingosine used S1P, sphingosine sphingosine a potent that to a of and that this sphingolipid both as a for a of and the cells, as a described S1P and on with S1P as a and sphingosine are of sphingosine and S1P to be on the of however, of S1P are also present in and and as for a we the sphingosine cis-4-methylsphingosine as a for studies of and added to the culture this and phosphorylated to the cis-4-methylsphingosine phosphate, it by S1P this of as S1P but apoptosis in postmitotic terminally differentiated neurons the that S1P sphingosine were to induce apoptosis in we that cis-4-methylsphingosine phosphorylated by cells a of This was based on results that the apoptosis in the of the apoptosis in the of the S1P, only on of similar the of the was and with the and of the that that accumulation of S1P in neurons in neuronal this be to we to S1P to S1P by the of by not in accumulation of S1P in neurons of of of the present study was to the that S1P induces neuronal apoptosis it a certain threshold by a for used neurons from of lyase-deficient mice The present studies not only confirmed that elevation of S1P but also revealed that the of the S1P was neuronal apoptosis was only by S1P derived from exogenous S1P that was and to S1P by sphingosine (SK2). we that this also the for of cis-4-methylsphingosine that the of S1P of cellular cells from of or in a were as described before that the was from cells by on a also on a S1P in mice cells were by and by a in a The cells were in and and of in cells, we the based on the of to the by the of of was added to culture of of was in culture medium was as described cells were with a in the medium and both and were in of and was to the of the were a was was to were in of and on for addition of of and were for in the of was are to was as described before and from of with were with medium and in the medium with for were with of the sphingolipid or S1P was as described in and by in S1P was by and the and were used to the S1P and of cells with were and as described by the were with for to the addition of of was confirmed by and of from was used in with the and as by the The was used in to The of were used as The and were used for and and and and The were for for for and for of were with sphingosine S1P or for and in the medium as described S1P, and were by as described of and were with are as and as was as or to with of the of S1P, and in Cerebellar of S1P, and was in neurons from and lyase-deficient mice of the of addition of S1P and were the of S1P in neurons from lyase-deficient with that of mice This was to addition of S1P or sphingosine to the culture addition of S1P, a in S1P also in and in neurons and The in S1P was by a in was in lyase-deficient neurons in cells, and in the of sphingosine was in lyase-deficient neurons that similar of and phosphorylated sphingosine be in cells Although addition of S1P and the sphingosine in cells to and it the of S1P, and that the of sphingosine that of S1P as in and also in in granule cells of the and the with in to the sphingosine a of sphingosine and S1P in lyase-deficient in the of cellular be in neurons. addition of S1P to the culture medium a in cellular was in as well as in lyase-deficient neurons addition of sphingosine to the culture medium a in the of and lyase-deficient neurons. that this the and and was in lyase-deficient to of the cells in cellular was also in and neurons exogenous sphingosine and S1P similar in cellular of in lyase-deficient of in neurons in were from and lyase-deficient as described in neurons were with or S1P or sphingosine as cells were were by as described are of and in of or to cells with by the of in the of of to from neurons with of in the of revealed and lyase-deficient granule neurons Cerebellar neurons from were differentiated and a of with either sphingosine and or S1P and not in of and cells, were in of lyase-deficient neurons with S1P but not sphingosine was added in by were incubation of lyase-deficient neurons in the of S1P that similar to be by cis-4-methylsphingosine in neurons exogenously added S1P, but not induces in terminally differentiated lyase-deficient both the of in only in lyase-deficient neurons with are of and and and lyase-deficient and neurons. in neurons were for with S1P or sphingosine and but in Cerebellar the of neurons was of incubation with sphingosine or S1P a in was only in S1P lyase-deficient neurons the in by S1P was with of the of as well as the of were only in lyase-deficient neurons with in neurons from and lyase-deficient were with or S1P or sphingosine as and were or and was as described to neurons. from was by in lyase-deficient neurons of were in and cells, with was by of in lyase-deficient neurons with S1P sphingosine not and of S1P on the of lyase-deficient neurons was with the described neuronal apoptosis by cis-4-methylsphingosine cis-4-methylsphingosine in the in and were similar for cis-4-methylsphingosine in cells and lyase-deficient cells with S1P of neurons with S1P not the of cis-4-methylsphingosine was for both and neurons not of in neurons with cis-4-methylsphingosine and in lyase-deficient neurons with S1P in neurons from and lyase-deficient mice were with cis-4-methylsphingosine and S1P the was as described from in the of S1P in Cerebellar to a of S1P in the of S1P in neurons S1P in not lyase-deficient neurons from apoptosis. and of were of the or of in the culture These results were confirmed by to S1P and were in differentiated granule cells we confirmed findings in differentiated neurons the that was to be in as by in the and also in cells The of the of was confirmed by incubation in the of the The results in that the of the of S1P not on apoptosis by S1P in lyase-deficient are not in the of S1P in lyase-deficient neurons. in neurons from and lyase-deficient mice were with or S1P or or both to as lyase-deficient neurons were in the of to S1P and and and were or and was as described to neurons. from in and from cells with in the in that cis-4-methylsphingosine neuronal apoptosis of the apoptosis in lyase-deficient neurons a similar we the of and and are of in of cells the and of to be in the of apoptosis in granule cells and postmitotic neurons of on the that a the in and to in a and of was in lyase-deficient neurons with S1P, of was not by induces of but not of in lyase-deficient neurons. Cerebellar neurons from and lyase-deficient were in culture with S1P for of and was in by as described were and with to of in of and S1P to and above exogenous S1P induces in lyase-deficient neurons that to for cis-4-methylsphingosine in neurons the that the for cis-4-methylsphingosine was as of S1P addition of sphingosine and S1P, this this we neurons from mice either or to and the of neurons the of addition of sphingosine by with neurons of addition of S1P only by with that of cis-4-methylsphingosine also not in neurons that this sphingosine phosphorylated by in neurons the of addition of sphingosine only of S1P addition by with of cis-4-methylsphingosine by in neurons with to a to the of S1P of sphingosine and S1P, Cerebellar neurons from and or mice were with medium and in the medium for or of sphingosine S1P or cis-4-methylsphingosine were cells were and and cis-4-methylsphingosine phosphate, were by and as described are from and were to from cells of the present study was to the that S1P induces neuronal apoptosis it above a certain a in of S1P we used neurons from of lyase-deficient of the lyase-deficient cells with either S1P or sphingosine in in S1P to as and for that derived from S1P or however, only the elevation in neuronal and the in and the were similar to in neurons with cis-4-methylsphingosine that was to the This to be by in lyase-deficient neurons with S1P, were also sphingosine addition to the culture that in postmitotic terminally differentiated neurons not for the of that elevation of of and to cells as well finding of the present study in addition to a certain the of the S1P for and are by the sphingosine that with cis-4-methylsphingosine and in of the that and of are with the are to the of S1P by the it well in in and as well as in and in the to the and with of cells apoptosis in the and with of and of apoptosis findings the of by the for of in the The of with to the was confirmed by of and also in of cis-4-methylsphingosine in this Based on we the in exogenous S1P by the cells by by and by to by in the and This with the for lyase-deficient exogenous S1P by the S1P with that cis-4-methylsphingosine also produced in the similar to the in lyase-deficient neurons with the the be from the the of the The of cis-4-methylsphingosine in the of sphingosine and by S1P sphingosine S1P S1P results that S1P cis-4-methylsphingosine are by in lyase-deficient neurons cells for cis-4-methylsphingosine and a certain with earlier S1P by to induce it to the as the S1P in lyase-deficient that exogenous S1P the of S1P not only in but also in neurons to a This that a of exogenous S1P, be on to the by The sphingosine in this by results the of of the and of S1P in the of this on also the of studies as cis-4-methylsphingosine and cellular metabolite cis-4-methylsphingosine phosphate, that a 1-phosphate a in neurons 1-phosphate (S1P) used S1P, sphingosine sphingosine used S1P, sphingosine sphingosine a potent that to a of and that this sphingolipid both as a for a of and the cells, as a described S1P and on with S1P as a and sphingosine are of sphingosine and S1P to be on the of however, of S1P are also present in and and as for a we the sphingosine cis-4-methylsphingosine as a for studies of and added to the culture this and phosphorylated to the cis-4-methylsphingosine phosphate, it by S1P this of as S1P but apoptosis in postmitotic terminally differentiated neurons the that S1P sphingosine were to induce apoptosis in we that cis-4-methylsphingosine phosphorylated by cells a of This was based on results that the apoptosis in the of the apoptosis in the of the S1P, only on of similar the of the was and with the and of the that that accumulation of S1P in neurons in neuronal apoptosis. this be to we to S1P to S1P by the of by not in accumulation of S1P in neurons of of The of the present study was to the that S1P induces neuronal apoptosis it a certain threshold by a for used neurons from of lyase-deficient mice The present studies not only confirmed that elevation of S1P but also revealed that the of the S1P was neuronal apoptosis was only by S1P derived from exogenous S1P that was and to S1P by sphingosine (SK2). we that this also the for of cis-4-methylsphingosine that the of S1P of cellular cells from of or in a were as described before that the was from cells by on a also on a S1P in mice cells were by and by a in a The cells were in and and of in cells, we the based on the of to the by the of of was added to culture of of was in culture medium was as described cells were with a in the medium and both and were in of and was to the of the were a was was to were in of and on for addition of of and were for in the of was are to was as described before and from of with were with medium and in the medium with for were with of the sphingolipid or S1P was as described in and by in S1P was by and the and were used to the S1P and of cells with were and as described by the were with for to the addition of of was confirmed by and of from was used in with the and as by the The was used in to The of were used as The and were used for and and and and The were for for for and for of were with sphingosine S1P or for and in the medium as described S1P, and were by as described of and were with are as and as was as or to with cells from of or in a were as described before that the was from cells by on a also on a S1P in mice cells were by and by a in a The cells were in and and of in cells, we the based on the of to the by the of of was added to culture of of was in culture medium was as described cells were with a in the medium and both and were in of and was to the of the were a was was to were in of and on for addition of of and were for in the of was are to was as described before and from of with were with medium and in the medium with for were with of the sphingolipid or S1P was as described in and by in S1P was by and the and were used to the S1P and of cells with were and as described by the were with for to the addition of of was confirmed by and of from was used in with the and as by the The was used in to The of were used as The and were used for and and and and The were for for for and for of were with sphingosine S1P or for and in the medium as described S1P, and were by as described of and were with are as and as was as or to with of the of S1P, and in Cerebellar of S1P, and was in neurons from and lyase-deficient mice of the of addition of S1P and were the of S1P in neurons from lyase-deficient with that of mice This was to addition of S1P or sphingosine to the culture addition of S1P, a in S1P also in and in neurons and The in S1P was by a in was in lyase-deficient neurons in cells, and in the of sphingosine was in lyase-deficient neurons that similar of and phosphorylated sphingosine be in cells Although addition of S1P and the sphingosine in cells to and it the of S1P, and that the of sphingosine that of S1P as in and also in in granule cells of the and the with in to the sphingosine a of sphingosine and S1P in lyase-deficient in the of cellular be in neurons. addition of S1P to the culture medium a in cellular was in as well as in lyase-deficient neurons addition of sphingosine to the culture medium a in the of and lyase-deficient neurons. that this the and and was in lyase-deficient to of the cells in cellular was also in and neurons exogenous sphingosine and S1P similar in cellular of in lyase-deficient of in the of revealed and lyase-deficient granule neurons Cerebellar neurons from were differentiated and a of with either sphingosine and or S1P and not in of and cells, were in of lyase-deficient neurons with S1P but not sphingosine was added in by were incubation of lyase-deficient neurons in the of S1P that similar to be by cis-4-methylsphingosine in neurons exogenously added S1P, but not induces in terminally differentiated lyase-deficient both the of in only in lyase-deficient neurons with are of and and and lyase-deficient and neurons. in neurons were for with S1P or sphingosine and but in Cerebellar the of neurons was of incubation with sphingosine or S1P a in was only in S1P lyase-deficient neurons the in by S1P was with of the of as well as the of were only in lyase-deficient neurons with in neurons from and lyase-deficient were with or S1P or sphingosine as and were or and was as described to neurons. from was by in lyase-deficient neurons of were in and cells, with was by of in lyase-deficient neurons with S1P sphingosine not and of S1P on the of lyase-deficient neurons was with the described neuronal apoptosis by cis-4-methylsphingosine cis-4-methylsphingosine in the in and were similar for cis-4-methylsphingosine in cells and lyase-deficient cells with S1P of neurons with S1P not the of cis-4-methylsphingosine was for both and neurons not of in neurons with cis-4-methylsphingosine and in lyase-deficient neurons with S1P in neurons from and lyase-deficient mice were with cis-4-methylsphingosine and S1P the was as described from in the of S1P in Cerebellar to a of S1P in the of S1P in neurons S1P in not lyase-deficient neurons from apoptosis. and of were of the or of in the culture These results were confirmed by to S1P and were in differentiated granule cells we confirmed findings in differentiated neurons the that was to be in as by in the and also in cells The of the of was confirmed by incubation in the of the The results in that the of the of S1P not on apoptosis by S1P in lyase-deficient are not in the of S1P in lyase-deficient neurons. in neurons from and lyase-deficient mice were with or S1P or or both to as lyase-deficient neurons were in the of to S1P and and and were or and was as described to neurons. from in and from cells with in the in that cis-4-methylsphingosine neuronal apoptosis of the apoptosis in lyase-deficient neurons a similar we the of and and are of in of cells the and of to be in the of apoptosis in granule cells and postmitotic neurons of on the that a the in and to in a and of was in lyase-deficient neurons with S1P, of was not by induces of but not of in lyase-deficient neurons. Cerebellar neurons from and lyase-deficient were in culture with S1P for of and was in by as described were and with to of in of and S1P to and above exogenous S1P induces in lyase-deficient neurons that to for cis-4-methylsphingosine in neurons the that the for cis-4-methylsphingosine was as of S1P addition of sphingosine and S1P, this this we neurons from mice either or to and the of neurons the of addition of sphingosine by with neurons of addition of S1P only by with that of cis-4-methylsphingosine also not in neurons that this sphingosine phosphorylated by in neurons the of addition of sphingosine only of S1P addition by with of cis-4-methylsphingosine by in neurons with to a to the of S1P of sphingosine and S1P, Cerebellar neurons from and or mice were with medium and in the medium for or of sphingosine S1P or cis-4-methylsphingosine were cells were and and cis-4-methylsphingosine phosphate, were by and as described are from and were to from cells of the of S1P, and in Cerebellar of S1P, and was in neurons from and lyase-deficient mice of the of addition of S1P and were the of S1P in neurons from lyase-deficient with that of mice This was to addition of S1P or sphingosine to the culture addition of S1P, a in S1P also in and in neurons and The in S1P was by a in was in lyase-deficient neurons in cells, and in the of sphingosine was in lyase-deficient neurons that similar of and phosphorylated sphingosine be in cells Although addition of S1P and the sphingosine in cells to and it the of S1P, and that the of sphingosine that of S1P as in and also in in granule cells of the and the with in to the sphingosine a of sphingosine and S1P in lyase-deficient in the of cellular be in neurons. addition of S1P to the culture medium a in cellular was in as well as in lyase-deficient neurons addition of sphingosine to the culture medium a in the of and lyase-deficient neurons. that this the and and was in lyase-deficient to of the cells in cellular was also in and neurons exogenous sphingosine and S1P similar in cellular of in lyase-deficient neurons. of in the of revealed and lyase-deficient granule neurons Cerebellar neurons from were differentiated and a of with either sphingosine and or S1P and not in of and cells, were in of lyase-deficient neurons with S1P but not sphingosine was added in by were incubation of lyase-deficient neurons in the of S1P that similar to be by cis-4-methylsphingosine in neurons exogenously added S1P, but not induces in terminally differentiated lyase-deficient both the of in neurons. S1P but in Cerebellar the of neurons was of incubation with sphingosine or S1P a in was only in S1P lyase-deficient neurons the in by S1P was with of the of as well as the of were was by in lyase-deficient neurons of were in and cells, with was by of in lyase-deficient neurons with S1P sphingosine not and The of S1P on the of lyase-deficient neurons was with the described neuronal apoptosis by cis-4-methylsphingosine cis-4-methylsphingosine in the in and were similar for cis-4-methylsphingosine in cells and lyase-deficient cells with S1P of neurons with S1P not the of cis-4-methylsphingosine was for both and neurons not S1P in the of S1P in Cerebellar to a of S1P in the of S1P in neurons S1P in not lyase-deficient neurons from apoptosis. and of were of the or of in the culture These results were confirmed by to S1P and were in differentiated granule cells we confirmed findings in differentiated neurons the that was to be in as by in the and also in cells The of the of was confirmed by incubation in the of the The results in that the of the of S1P not on apoptosis by S1P in lyase-deficient neurons. S1P the in that cis-4-methylsphingosine neuronal apoptosis of the apoptosis in lyase-deficient neurons a similar we the of and and are of in of cells the and of to be in the of apoptosis in granule cells and postmitotic neurons of on the that a the in and to in a and of was in lyase-deficient neurons with S1P, of was not by S1P in of and S1P to and above exogenous S1P induces in lyase-deficient neurons that to for cis-4-methylsphingosine in neurons the that the for cis-4-methylsphingosine was as of S1P addition of sphingosine and S1P, this this we neurons from mice either or to and the of neurons the of addition of sphingosine by with neurons of addition of S1P only by with that of cis-4-methylsphingosine also not in neurons that this sphingosine phosphorylated by in neurons the of addition of sphingosine only of S1P addition by with of cis-4-methylsphingosine by in neurons with neurons. of the present study was to the that S1P induces neuronal apoptosis it above a certain a in of S1P we used neurons from of lyase-deficient of the lyase-deficient cells with either S1P or sphingosine in in S1P to as and for that derived from S1P or however, only the elevation in neuronal and the in and the were similar to in neurons with cis-4-methylsphingosine that was to the This to be by in lyase-deficient neurons with S1P, were also sphingosine addition to the culture that in postmitotic terminally differentiated neurons not for the of that elevation of of and to cells as well finding of the present study in addition to a certain the of the S1P for and are by the sphingosine that with cis-4-methylsphingosine and in of the that and of are with the are to the of S1P by the it well in in and as well as in and in the to the and with of cells apoptosis in the and with of and of apoptosis findings the of by the for of in the The of with to the was confirmed by of and also in of cis-4-methylsphingosine in this Based on we the in exogenous S1P by the cells by by and by to by in the and This with the for lyase-deficient exogenous S1P by the S1P with that cis-4-methylsphingosine also produced in the similar to the in lyase-deficient neurons with the the be from the the results that S1P cis-4-methylsphingosine are by in lyase-deficient neurons cells for cis-4-methylsphingosine and a certain with earlier S1P by to induce it to the as the S1P in lyase-deficient that exogenous S1P the of S1P not only in but also in neurons to a This that a of exogenous S1P, be on to the by The sphingosine in this by results the of of the and of S1P in the of this on also the of studies as cis-4-methylsphingosine and cellular metabolite cis-4-methylsphingosine phosphate, that a 1-phosphate a in neurons The of the present study was to the that S1P induces neuronal apoptosis it above a certain a in of S1P we used neurons from of lyase-deficient of the lyase-deficient cells with either S1P or sphingosine in in S1P to as and for that derived from S1P or however, only the elevation in neuronal and the in and the were similar to in neurons with cis-4-methylsphingosine that was to the This to be by in lyase-deficient neurons with S1P, were also sphingosine addition to the culture that in postmitotic terminally differentiated neurons not for the of that elevation of of and to cells as well finding of the present study in addition to a certain the of the S1P for and are by the sphingosine that with cis-4-methylsphingosine and in of the that and of are with the are to the of S1P by the it well in in and as well as in and in the to the and with of cells apoptosis in the and with of and of apoptosis findings the of by the for of in the The of with to the was confirmed by of and also in of cis-4-methylsphingosine in this Based on we the in exogenous S1P by the cells by by and by to by in the and This with the for lyase-deficient exogenous S1P by the S1P with that cis-4-methylsphingosine also produced in the similar to the in lyase-deficient neurons with the the be from the the results that S1P cis-4-methylsphingosine are by in lyase-deficient neurons cells for cis-4-methylsphingosine and a certain with earlier S1P by to induce it to the as the S1P in lyase-deficient neurons. that exogenous S1P the of S1P not only in but also in neurons to a This that a of exogenous S1P, be on to the by The sphingosine in this by results the of of the and of S1P in the of this on also the of studies as cis-4-methylsphingosine and cellular metabolite cis-4-methylsphingosine phosphate, that a 1-phosphate a in neurons for and for in the and of the mice were by with with