Purification and Characterization of Neurotrophic Factor for Retinal Cholinergic Neurons Derived from Cultured Hippocampal Neurons

• Published in: Japanese journal of pharmacology 1994, Vol.65(2), pp.153-162
• Main Authors: Yasukura, Tohru, Omori, Kyoko, Mikami, Toshiko, Inoue, Masafumi, Uyama, Masanobu, Inagaki, Chiyoko
• Format: Article
• Language: English
• Subjects: Amacrine cell (retina); Choline acetyltransferase; Hippocampus; Neurotrophic factor; Peptide
• ISSN: 0021-5198; 1347-3506
• DOI: 10.1254/jjp.65.153

A neurotrophic factor that supports the development of cholinergic retinal neurons 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 a serum-free control culture, retinal neurons from 17-day-old rat fetus displayed little increase in the enzyme activity and a 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 ∼sixfold 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 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 stimulated ChAT activity in the retinal cell culture and dissociated cell culture of the septal nucleus. These findings suggest that the developing hippocampal neurons produce a neurotrophic peptide that stimulates the development of cholinergic neurons.