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Nimodipin restores normal neurite outgrowth after a depolarisation-induced disturbance of calcium homeostasis
9thMeetingoftheESN
9
A3
10
NIWODIPIN RESTORES NORWAL NEURITE OUTaWSH IMPROVES AKONAL OUTGROWTH FROM GROWTH AFTER A DEPOLARISATION-INDUCED DORSAL ROOT GANGLIA TRFATED IN VITRO DISTURBANCE OF CALCIUM HOWEOSTASIS. WITH CISPLATIN P.R. Bar, Ii.Renkema and W.H. Gispen. Research Lab Neurology and Rudolf Hagnus Institute, Univ. Utrecht, NL
v. Mandys, W.H. Gispen and P.R. Bar. Inst Exp Fled, CSAS, Prague and Res. Lab Neurology, RWI, Univ. Utrecht, NL
Nimodipin, a Ca-blocker, accelerates recovery of rats after trauma to the nervous system. Apparently excessive Ca'+plays a crucial role in the ontogeny of damage to neurons. We tested in vitro whether we could prove such role of Ca"+ by depolarising cultured slices of rat spinal cord, thus enhancing Ca-influx. Cultures were scored qualitatively by eye, and outgrowth was quantified by measuring neurofilament (NF, a cytoskeletalprotein present only in mature axons and thus a measure for neuritogenesis)after 5 or 7 d in vitro by ELISA. NF decreased during depolarisation (5OmW K') by 20%. Treatment with nimodipin (0.1 @4) restored outgrowth to normal levels, but had no effect on normal cultures . We conclude that nimodipin acts as neuroprotective agent but not as general neurotrophic factor.
We wanted to develop an in vitro model to study cisplatin (CP)-inducedneuropathy and to see whether melanocortins could ameliorate the toxic action of CP in vitro like they do in Vim. We cultured ED12 chick dorsal root ganglia (DRG) in a semi-solid medium and treated them with CP (O-20 pg/ml) w/o aMSH (l-10 nW). Axonal outgrowth was evaluated daily and mean length (l-) and growth rate (r,) of neUriteS were determined. CP alone resulted in a substantial reduction in r, (from 150 to 85 Mm/day) and in l,,(610 vs 340 pm at day 4). In cultures treated with CP+aMSH, a significant improvement of the the axonal outgrowth was observed: initial r, was 53% higher than in CPtreated cultures and the l,, at day 4 was 455 pm. We conclude that el4SHhas a protective effect in vitro which can be easily quantified in this System. 12 PROTEIN SYNTHESIS IN SQUID BRAIN
11
DISTRIBUTIOW
03
A
EPITOPI ON VINENTIN AND DIPFIRI~TIATION
PHOSPHORYLATED
DURING OF
GROWTH
GLIAL CELLS IN CULTURE. J. Ciesielski-Treska,G. Ulrich and D. Aunis
INSERM U. 338, S-rue Blaise Pascal, Centre de Neurochimie, 67084 Strasbourg, France. The N-terminal region of the vimentin spaced molecule contains closely phosphorylation sites bearing consensus We have motifs for several protein kinases. produced a monoclonal antibody 83 which reacts with a residue phosphorylated by activators of protein kinase C. The epitope for B3 has been located between residues 10 and 34 of the N-terminal domain of vimentin. Immunofluorescence studies with 83 antibody demonstrate that the level of phosphorylated glial low in bipotential vimentin is progenitors (A2B5t/GFAP-), and that the epitope is absent in type II (A2B5-/GFAP+) in astrocytes and (A2B5-/cGAL+) oligodendrocytes. In contrast, type I astrocytes react intensely with B3 and their varies during the cell immunoreactivity cycle. It is high in cells arrested at GO/S
SYNAPTOSOMES.
M.CrispiIIo, C. Perrune Capaao. E. Ca&iglt, E. Me&hini and A. Giuditta. Dapartmant of Gonerd aad Envtr~nmentat Physiology, Univemity of Naptea. Naptea, Italy. A synaptosomal fractkn from squid brain incorporates “smethionine into protein in a t&w and dose dependant fashion. The reaction is stmngly inhibited by cydoeximtde and by hypoosmotic treatment of the particles, but is not affected by RNAase. The ionic composition of the medium markedly modulates the incorporatbn ma&ion. lbe synaptocoma~ preparation
contains active polywmescarrying n-t
peptide chains, as shown by sedbnentatk
analyses. Most
boundary and it decreases in the S phase. Treatment of astrocytes with dBcAl4P also This decreases imnwnoreactivity with B3.
labelled synaptosomal prc4etns are readilyoadimanted,and may be resolved into several bands by monodimensional gel electrophoresis. The resulting pattern differs from the analogous patterns obtained from the cell bodies or the axoplasm of the giant axon. Two of the newly synthetized proteins have bean identitbd as neumikment pmteins, as shown by immunoprecip&aUonwtth a manoconsl antibody specificforthe80kOand70kDsquidneumfUamen t-.
decrease could be prevented by cycloheximide, suggesting that the phosphorylation and dephosphorylation of the 83 epitope is synthetically labile.
As these proteins are neumn-apacifk,it is condudadthat the protein synthetic activity of the squid synaptosornal preparatbn is due to the nerve endings.
9
A3
10
NIWODIPIN RESTORES NORWAL NEURITE OUTaWSH IMPROVES AKONAL OUTGROWTH FROM GROWTH AFTER A DEPOLARISATION-INDUCED DORSAL ROOT GANGLIA TRFATED IN VITRO DISTURBANCE OF CALCIUM HOWEOSTASIS. WITH CISPLATIN P.R. Bar, Ii.Renkema and W.H. Gispen. Research Lab Neurology and Rudolf Hagnus Institute, Univ. Utrecht, NL
v. Mandys, W.H. Gispen and P.R. Bar. Inst Exp Fled, CSAS, Prague and Res. Lab Neurology, RWI, Univ. Utrecht, NL
Nimodipin, a Ca-blocker, accelerates recovery of rats after trauma to the nervous system. Apparently excessive Ca'+plays a crucial role in the ontogeny of damage to neurons. We tested in vitro whether we could prove such role of Ca"+ by depolarising cultured slices of rat spinal cord, thus enhancing Ca-influx. Cultures were scored qualitatively by eye, and outgrowth was quantified by measuring neurofilament (NF, a cytoskeletalprotein present only in mature axons and thus a measure for neuritogenesis)after 5 or 7 d in vitro by ELISA. NF decreased during depolarisation (5OmW K') by 20%. Treatment with nimodipin (0.1 @4) restored outgrowth to normal levels, but had no effect on normal cultures . We conclude that nimodipin acts as neuroprotective agent but not as general neurotrophic factor.
We wanted to develop an in vitro model to study cisplatin (CP)-inducedneuropathy and to see whether melanocortins could ameliorate the toxic action of CP in vitro like they do in Vim. We cultured ED12 chick dorsal root ganglia (DRG) in a semi-solid medium and treated them with CP (O-20 pg/ml) w/o aMSH (l-10 nW). Axonal outgrowth was evaluated daily and mean length (l-) and growth rate (r,) of neUriteS were determined. CP alone resulted in a substantial reduction in r, (from 150 to 85 Mm/day) and in l,,(610 vs 340 pm at day 4). In cultures treated with CP+aMSH, a significant improvement of the the axonal outgrowth was observed: initial r, was 53% higher than in CPtreated cultures and the l,, at day 4 was 455 pm. We conclude that el4SHhas a protective effect in vitro which can be easily quantified in this System. 12 PROTEIN SYNTHESIS IN SQUID BRAIN
11
DISTRIBUTIOW
03
A
EPITOPI ON VINENTIN AND DIPFIRI~TIATION
PHOSPHORYLATED
DURING OF
GROWTH
GLIAL CELLS IN CULTURE. J. Ciesielski-Treska,G. Ulrich and D. Aunis
INSERM U. 338, S-rue Blaise Pascal, Centre de Neurochimie, 67084 Strasbourg, France. The N-terminal region of the vimentin spaced molecule contains closely phosphorylation sites bearing consensus We have motifs for several protein kinases. produced a monoclonal antibody 83 which reacts with a residue phosphorylated by activators of protein kinase C. The epitope for B3 has been located between residues 10 and 34 of the N-terminal domain of vimentin. Immunofluorescence studies with 83 antibody demonstrate that the level of phosphorylated glial low in bipotential vimentin is progenitors (A2B5t/GFAP-), and that the epitope is absent in type II (A2B5-/GFAP+) in astrocytes and (A2B5-/cGAL+) oligodendrocytes. In contrast, type I astrocytes react intensely with B3 and their varies during the cell immunoreactivity cycle. It is high in cells arrested at GO/S
SYNAPTOSOMES.
M.CrispiIIo, C. Perrune Capaao. E. Ca&iglt, E. Me&hini and A. Giuditta. Dapartmant of Gonerd aad Envtr~nmentat Physiology, Univemity of Naptea. Naptea, Italy. A synaptosomal fractkn from squid brain incorporates “smethionine into protein in a t&w and dose dependant fashion. The reaction is stmngly inhibited by cydoeximtde and by hypoosmotic treatment of the particles, but is not affected by RNAase. The ionic composition of the medium markedly modulates the incorporatbn ma&ion. lbe synaptocoma~ preparation
contains active polywmescarrying n-t
peptide chains, as shown by sedbnentatk
analyses. Most
boundary and it decreases in the S phase. Treatment of astrocytes with dBcAl4P also This decreases imnwnoreactivity with B3.
labelled synaptosomal prc4etns are readilyoadimanted,and may be resolved into several bands by monodimensional gel electrophoresis. The resulting pattern differs from the analogous patterns obtained from the cell bodies or the axoplasm of the giant axon. Two of the newly synthetized proteins have bean identitbd as neumikment pmteins, as shown by immunoprecip&aUonwtth a manoconsl antibody specificforthe80kOand70kDsquidneumfUamen t-.
decrease could be prevented by cycloheximide, suggesting that the phosphorylation and dephosphorylation of the 83 epitope is synthetically labile.
As these proteins are neumn-apacifk,it is condudadthat the protein synthetic activity of the squid synaptosornal preparatbn is due to the nerve endings.