1995 Fiscal Year Final Research Report Summary
PURIFICATION,CHARACTERIZATION AND SIGNAL TRANSDUCTION OF RETINAL CHOLINERGIC NEURON-NEUROTROPHIC FACTOR DERIVED FROM CULTURED HIPPOCAMPAL NEURONAL CELLS
| Project/Area Number |
06671782
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Ophthalmology
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| Research Institution | Kansai Medical University |
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Principal Investigator |
ITAGAKI Takashi Kansai Medical University, Faculty of Medicine, Assistant Professor, 医学部, 講師 (30140255)
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| Co-Investigator(Kenkyū-buntansha) |
YASUKURA Tohru Kansai Medical University, Faculty of Medicine, Reseach Associate, 医学部, 助手 (80257956)
OMORI Kyoko Kansai Medical University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (90152256)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Hippocampus / Neurotrophic factor / choline acetyltransferase / Amacrin cells / Peptide |
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| Research Abstract |
A neurotrophic factor which supports the development of cholinergic retinal neuron (amacrine cells) was purified from media conditioned by a primary culture of embryonic hippocampal neurons.
Retinal choline acetyltransferase (ChAT), which is located exclusively in amacrine cells, served as a marker for the development of retinal cholinergic neurons. In serum-free control culture, retinal neuron from 17-day-old rat fetus displayd a little increase in the enzyme activity and low proportion of neurite-bearing cells (15-20%) within 7 days. The conditioned media, when added to the retinal neuron culture, dose-dependently increased ChAT activity and the number of neurite-bearing cells (40-60%), the maximum ChAT activity being - 6-fold higher than that in the control. The fraction with these stimulatory activities was purified by Sephadex G-15 column chromatography and two times reverse-phase (C18) HPLC.The final fraction showed - 3,000-fold higher purification as compared with that in the Sephadex G-15 fraction. Gas-phase protein sequencing analysis of the final fraction yielded a peptide sequence : Tyr-Leu-Leu-Pro-Ala-Gln-Val-Asn-Ile-Asp. A synthetic peptide with this sequence dose-dependently simulated ChAT activity in retinal cell culture and dissociated cell culture of the septal nucleus. The peptied also showed similar ChAT-stimulating activity in the dissociated cell culture of the septal nucleus. This peptide increased the phosphotyrosine residues of 180-200 kDa protein and the c-Fos synthesis in retinal culture, suggesting that the tyrosine kinase activation is involved in the response to this peptide. These findings suggest that the developing hippocampal neurons produce a neurotrophic peptide which stimulates the development of cholinergic neurons.
Thus hippocampal neurons appear to produce a neurotrophic peptide that promotes the development of cholinergic neurons amacrine cells.
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