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GAP-43 involvement in the sprouting of hippocampal mossy fibers in a model of epileptogenesis
GAP-43 INVOLVEMENT IN THE SPROUTING OF HIPPOCAMPAL MOSSY FIBERS IN A MODEL OF EPILEPTOGENESIS. Bendotti C. 1, Baldessari &l, De Biasi S. 2 and R. Samanin 1 1-Mario Negri Institute, Via Eritrea, 62, 20155 Milan, Italy. 2-Dipt. Fisiol. Bioch. Gen., Via Celoria 26, 20133 Milano, Italy.
MOLECULAR CLONING OF PROTEINTYROSINE KINASES FROM AN IMMORTALIZED, DIENCEPHALIC CELL LINE AND DIFFERENTIATION BY TROPHIC FACTORS. K. Shimoda, T.Oshima, K. Inoue, D. Kaneto T. Kitagawa, H. Takahashi* and J.W. Commissiong. ** Div. of Neurology. National NishiTottori Hospital, Tottori 68902, JAPAN. *Lab. of Histochemistory, Mitsubishi-kasei Institute of Life Sciences. Machida 163, JAPAN. **NTU, LMCN, NINDS, NIH, Bethesda, MD 20892, USA. Many cytokines have recently been identified in the CNS. However, for many, their physiological functions have not been elucidated. We have prepared an immortalized cell line from the E13 diencephalic region of rat, and tested the hypothesis that IL-3 and bFGF may be involved in signal transduction mechanisms. E13 diencephalic cells were immortalized by transfection with the SV40 LT antigen temperature sensitive mutant, carried by a retroviral vector. D e g e n e r a t e oligonucleotide primers were amplified by the PCR methods, and used to clone PTKs.The clones from one cell line designated $1 were sequenced, and used to identified the following PTKs: Jakl, Jak2, bFGFR, hck/bmk and Try10. Using the Northern blotting technique, we identified the following Janus kinases: Jakl, Jak2, Jak3 and Tyk2. When the $1 ceils were cultured and treated with bFGF and IL-3, both of which were coupled to Jak2, they extended processes and diffe~rentiated into cells with a neuron-like morphology. The results strongly suggest that PTKs are involved in signal transduction mechanisms in the $1 cells. They might also have a similar function during normal development in the CNS. The $1 cell line that we have described may therefore become a model for studies of the molecular mechanisms involved in neuronal differentiation and signal transductin in n the CNS.
EXTRACELLULAR MATRIX IN NEURONAL REGENERATION, SELECTIVE ROLE OF GLYCOSAMINOGLYCANS. L. Vergani, E. Germani, B. Tenconi, A. M. Di Giulio and A. Gorio Department of Medical Pharmacology, Via Vanvitelli 32, 20124 Milano
A subcutaneous injection of 12 mg/Kg kainic acid (KA) induces limbic seizures in adult rats and causse sprouting of mossy fibers (MF) in the inner molecular layer (IML) of the dentate gyrus . Under these conditions a rapid and transient induction of the mRNA of GAP-43, a protein involved in the synaptogenesis, occurs in the granule cells that normally do not express it and an increased immunoreactivity for this protein is observed in the IML but not in the normal projection area of granule cells 30 days after the KA injection (Bendotti et al. Eur. J. Neurosci. 6:509,1994). In attempt to find a correlation between these effects, we have analyzed 1) the ultrastructural immunolocalization of the GAP-43 in the hippocampus at the maximal MF sprouting expression revealed by Timm's staining and 2) the effect of MK 801, which partially reduced the neurotoxic effect of KA, on the induction of GAP-43 mRNA in the granule cells and on the GAP-43 immunoreactivity and Timm's staining after KA injection. We found that GAP-43 positive terminals in the IML showed structural characteristics of MF boutons while no GAP-43 -positive MF terminal were found in the normal projection areas of granular cells thus indicating that newly synthesized GAP-43 is transported predominantly at the branches actively undergoing remodelling. MK 801, which significantly inhibited the GAP-43 mRNA levels induction in the granule cells also counteracted the increase in Timm's staining and GAP-43 immunoreactivity in the IML 2 months after the KA treatment. These data clearly indicate an involvement of GAP-43 in the structural remodelling of the MF in this experimental model of epileptogenesis.
GENETICALLY ENGINEERED CNS PROGENITOR CELLS AND PRIMARY NEUROEPITHELIAL CELLS ARE GROWTH RESTRICTED AND DIFFERENTIATE FOLLOWINGTRANSPLANTATION INTO THE FETAL RAT BRA/N.
It is well kuown that extracellular matrix plays a role in peripheral nerve regeneration and in neurite guidance. Among the various glycosaminoglycans (GAGS) tested in vitro, heparin sulfate, chondroitin sulfate and a mixture of GAGs, named Ateroid, were the most effective in promoting neurite for/nation from 5YSY neuroblastoma cells. In serum free colture medium GAGS were effective at concentrations as low as 1 pM. Irreversible sciatic nerve lesions cause retrograde degeneration of sensory neurons with loss of substance P and met-enkephalin in the lumbar spinal cord. Twentyone days after nerve lesion substance P drops from 10.1 to 6.6 ng/mg protein: treatment with GAGS by daily intraperitonel injection at the dose of 0.2 mg/kg conteracts the peptide loss. promoting a huge increase to 18.5 ng/mgprotein. A similar effect was observed for met-enkephalin. The abundance of NGF mRNA increased by 6 fold 7 and 20 days after lesioning, GAGS treatment promoted a 15 fold increase in the lesioned nerve and a 10 fold increase in the contralateral: mRNA abundance of p75 NGF receptor increase 10 fold between 7 and 20 days after nerve lesion/ng, in the nerve of treated animals the increase was 4 fold. Substantial increases of brain derived neurotrophic factor were observed in the lesioned nerve 20 days after transection, in animals treated with GAGS the increase was present in lesioned and contralateral intact nerves. A similar effect was observed for neurotrophin 3 (NT3). GAGS treatment is also effective after chemical sympathectomy with 6-OHDA in youg rats. These data suggest that GAGS treatment prevents retrograde degeneration and promotes neurite formatiofi and regeneration of acutely lesioned peripheral nerves.
--10m
Magrassi L.°,PezzoUa S.°,Butti G.°,Govoni S.* and Catlan¢o E.# °Dept. of Surgery,Section of Neurosurgery,Univ.Pavia,IRCCS Policlinico S. Matteo, *Insk Pharmacol.,Univ. Pavia;#1nst. Pharmacol. Sci.,Univ. Milan, IT We previously reported the development of a transplantation methodology that allows the implant of CNS derived cells into the fetal rat brain and their identification by means of BrdU immunocytochemistry (23rd Neurosc. Meeting, Abstr. 107.6). By following this experimental strategy we transplanted into the CNS of El6 rat fetuses conditionally immortalized CNS progenitor cells and freshly dissected primary cells isolated from different brain areas. In short term survival experiments (5 days after transplantation) it was found that transplanted cells placed into the embryonic environment can survive, migrate and/or compartimentalize into defined structures (thereafter called clusters) while changing their antigenic properties. Indeed both the conditionally immortalized STI4A cells and the primary cells stop dividing in vivo and down regulate nestin, a cytoscheletal antigen specifically expressed in immature cells (Cattaneo et at., Dev. Brain Res,, 1994). We are presently investigating the behaviour of the transplanted cells after 2 and 12 weeks following transplantation. For these experiments donor cells have been double labelled with BrdU and Di[, a liposoluble dye that flmxesces red when excited using a rhodamine filter set and whose fluorescent signal is spread throughout the entire cell body. The analysis of the DiI signal has revealed that in either ST14A and primary cells transplants, the majority of the cells were still organized into clusters even in transplanted animals that survived until 3 months postnatally while a smaller percentage of the donor cells w e ~ found dispersed into the host brain. Clusters of transplanted cells were found integrated in the host tissue. Morphological evaluation of the DiI labelled cells as well as immunocytochemical and Electron Microscopy data will reveal the cellular identity assumed by the transplanted cells. In addition this experimental strategy will allow to investigate if immature striatal progenitors transplanted into the cortex organize autonomously as striatal tissue or reorganize according to the environment. By testing these potentialities informations will be obtained on the abilities of the donor cells to integrate morphologically and biochemically into the host brain and on the possibilities to modulate pharmacologically their fate. These experiments will also provide informations on the possible use of such donor cells as a vehicle for genes of therapeutical interest.
MOLECULAR CLONING OF PROTEINTYROSINE KINASES FROM AN IMMORTALIZED, DIENCEPHALIC CELL LINE AND DIFFERENTIATION BY TROPHIC FACTORS. K. Shimoda, T.Oshima, K. Inoue, D. Kaneto T. Kitagawa, H. Takahashi* and J.W. Commissiong. ** Div. of Neurology. National NishiTottori Hospital, Tottori 68902, JAPAN. *Lab. of Histochemistory, Mitsubishi-kasei Institute of Life Sciences. Machida 163, JAPAN. **NTU, LMCN, NINDS, NIH, Bethesda, MD 20892, USA. Many cytokines have recently been identified in the CNS. However, for many, their physiological functions have not been elucidated. We have prepared an immortalized cell line from the E13 diencephalic region of rat, and tested the hypothesis that IL-3 and bFGF may be involved in signal transduction mechanisms. E13 diencephalic cells were immortalized by transfection with the SV40 LT antigen temperature sensitive mutant, carried by a retroviral vector. D e g e n e r a t e oligonucleotide primers were amplified by the PCR methods, and used to clone PTKs.The clones from one cell line designated $1 were sequenced, and used to identified the following PTKs: Jakl, Jak2, bFGFR, hck/bmk and Try10. Using the Northern blotting technique, we identified the following Janus kinases: Jakl, Jak2, Jak3 and Tyk2. When the $1 ceils were cultured and treated with bFGF and IL-3, both of which were coupled to Jak2, they extended processes and diffe~rentiated into cells with a neuron-like morphology. The results strongly suggest that PTKs are involved in signal transduction mechanisms in the $1 cells. They might also have a similar function during normal development in the CNS. The $1 cell line that we have described may therefore become a model for studies of the molecular mechanisms involved in neuronal differentiation and signal transductin in n the CNS.
EXTRACELLULAR MATRIX IN NEURONAL REGENERATION, SELECTIVE ROLE OF GLYCOSAMINOGLYCANS. L. Vergani, E. Germani, B. Tenconi, A. M. Di Giulio and A. Gorio Department of Medical Pharmacology, Via Vanvitelli 32, 20124 Milano
A subcutaneous injection of 12 mg/Kg kainic acid (KA) induces limbic seizures in adult rats and causse sprouting of mossy fibers (MF) in the inner molecular layer (IML) of the dentate gyrus . Under these conditions a rapid and transient induction of the mRNA of GAP-43, a protein involved in the synaptogenesis, occurs in the granule cells that normally do not express it and an increased immunoreactivity for this protein is observed in the IML but not in the normal projection area of granule cells 30 days after the KA injection (Bendotti et al. Eur. J. Neurosci. 6:509,1994). In attempt to find a correlation between these effects, we have analyzed 1) the ultrastructural immunolocalization of the GAP-43 in the hippocampus at the maximal MF sprouting expression revealed by Timm's staining and 2) the effect of MK 801, which partially reduced the neurotoxic effect of KA, on the induction of GAP-43 mRNA in the granule cells and on the GAP-43 immunoreactivity and Timm's staining after KA injection. We found that GAP-43 positive terminals in the IML showed structural characteristics of MF boutons while no GAP-43 -positive MF terminal were found in the normal projection areas of granular cells thus indicating that newly synthesized GAP-43 is transported predominantly at the branches actively undergoing remodelling. MK 801, which significantly inhibited the GAP-43 mRNA levels induction in the granule cells also counteracted the increase in Timm's staining and GAP-43 immunoreactivity in the IML 2 months after the KA treatment. These data clearly indicate an involvement of GAP-43 in the structural remodelling of the MF in this experimental model of epileptogenesis.
GENETICALLY ENGINEERED CNS PROGENITOR CELLS AND PRIMARY NEUROEPITHELIAL CELLS ARE GROWTH RESTRICTED AND DIFFERENTIATE FOLLOWINGTRANSPLANTATION INTO THE FETAL RAT BRA/N.
It is well kuown that extracellular matrix plays a role in peripheral nerve regeneration and in neurite guidance. Among the various glycosaminoglycans (GAGS) tested in vitro, heparin sulfate, chondroitin sulfate and a mixture of GAGs, named Ateroid, were the most effective in promoting neurite for/nation from 5YSY neuroblastoma cells. In serum free colture medium GAGS were effective at concentrations as low as 1 pM. Irreversible sciatic nerve lesions cause retrograde degeneration of sensory neurons with loss of substance P and met-enkephalin in the lumbar spinal cord. Twentyone days after nerve lesion substance P drops from 10.1 to 6.6 ng/mg protein: treatment with GAGS by daily intraperitonel injection at the dose of 0.2 mg/kg conteracts the peptide loss. promoting a huge increase to 18.5 ng/mgprotein. A similar effect was observed for met-enkephalin. The abundance of NGF mRNA increased by 6 fold 7 and 20 days after lesioning, GAGS treatment promoted a 15 fold increase in the lesioned nerve and a 10 fold increase in the contralateral: mRNA abundance of p75 NGF receptor increase 10 fold between 7 and 20 days after nerve lesion/ng, in the nerve of treated animals the increase was 4 fold. Substantial increases of brain derived neurotrophic factor were observed in the lesioned nerve 20 days after transection, in animals treated with GAGS the increase was present in lesioned and contralateral intact nerves. A similar effect was observed for neurotrophin 3 (NT3). GAGS treatment is also effective after chemical sympathectomy with 6-OHDA in youg rats. These data suggest that GAGS treatment prevents retrograde degeneration and promotes neurite formatiofi and regeneration of acutely lesioned peripheral nerves.
--10m
Magrassi L.°,PezzoUa S.°,Butti G.°,Govoni S.* and Catlan¢o E.# °Dept. of Surgery,Section of Neurosurgery,Univ.Pavia,IRCCS Policlinico S. Matteo, *Insk Pharmacol.,Univ. Pavia;#1nst. Pharmacol. Sci.,Univ. Milan, IT We previously reported the development of a transplantation methodology that allows the implant of CNS derived cells into the fetal rat brain and their identification by means of BrdU immunocytochemistry (23rd Neurosc. Meeting, Abstr. 107.6). By following this experimental strategy we transplanted into the CNS of El6 rat fetuses conditionally immortalized CNS progenitor cells and freshly dissected primary cells isolated from different brain areas. In short term survival experiments (5 days after transplantation) it was found that transplanted cells placed into the embryonic environment can survive, migrate and/or compartimentalize into defined structures (thereafter called clusters) while changing their antigenic properties. Indeed both the conditionally immortalized STI4A cells and the primary cells stop dividing in vivo and down regulate nestin, a cytoscheletal antigen specifically expressed in immature cells (Cattaneo et at., Dev. Brain Res,, 1994). We are presently investigating the behaviour of the transplanted cells after 2 and 12 weeks following transplantation. For these experiments donor cells have been double labelled with BrdU and Di[, a liposoluble dye that flmxesces red when excited using a rhodamine filter set and whose fluorescent signal is spread throughout the entire cell body. The analysis of the DiI signal has revealed that in either ST14A and primary cells transplants, the majority of the cells were still organized into clusters even in transplanted animals that survived until 3 months postnatally while a smaller percentage of the donor cells w e ~ found dispersed into the host brain. Clusters of transplanted cells were found integrated in the host tissue. Morphological evaluation of the DiI labelled cells as well as immunocytochemical and Electron Microscopy data will reveal the cellular identity assumed by the transplanted cells. In addition this experimental strategy will allow to investigate if immature striatal progenitors transplanted into the cortex organize autonomously as striatal tissue or reorganize according to the environment. By testing these potentialities informations will be obtained on the abilities of the donor cells to integrate morphologically and biochemically into the host brain and on the possibilities to modulate pharmacologically their fate. These experiments will also provide informations on the possible use of such donor cells as a vehicle for genes of therapeutical interest.