Differentiation of cholinergic neurons and physiological role of ciliary neurotrophic factor (CNTF)

T. Deguchi, Hidemi Misawa, R. Takahashi

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Seven types of mRNA that differed in the 5′-non-coding region were identified for choline acetyltransferase of mouse spinal cord. These mRNAs were produced by differential splicing of pre-mRNAs transcribed from three different promotor regions. Two murine cholinergic cell lines, NS20Y and NG108-15, expressed M-type mRNA most abundantly. Using these cell lines, promotor activity in choline acetyltransferase gene was analyzed by transient assay of a reporter gene. The result indicated that there was promotor activity in the region upstream of the M-type exon and enhancer activity in the intron downstream of the M-type exon, and that this region regulated neuron-specific expression of choline acetyltransferase activity. In contrast, R-type mRNA was exclusively expressed in cultured superior cervical ganglion cells and was markedly increased by ciliary neurotrophic factor (CNTF). To analyse the physiological role of CNTF, we constructed and screened a cDNA library from human sciatic nerves and isolated two types of cDNAs for human CNTF. Sequence analysis revealed that one type of cDNA corresponded to the normal mRNA, while the other type contained a 4 bp stretch insertion within the coding region, which caused frameshift from 39th amino acid with a stop codon 24 amino acids downstream. Analysis of genomic DNA for CNTF showed that there was a point mutation from G to A in the intron of the mutated allele, which created a new splice acceptor site and generated a new mRNA species with 4 bp insertion. Analysis of tissue samples from autopsied subjects and transfection study of CNTF minigenes into cultured cells indicated that the mutated allele expressed only the mutated mRNA, but not the normal mRNA species. Thus, this allele represents a null mutation that abolishes expression of normal CNTF protein. CNTF genotypes were analyzed in 400 Japanese subjects. The frequency of three genotypes did not differ between healthy volunteers and patients suffering from various neurological diseases. Nine mutant homozygote subjects so far detected did not show any neurological disturbances and developmental abnormalities until adulthood. These observations indicate that CNTF is dispensable for the development and maintenance of the nervous system, and that the CNTF null mutation is not causally related with any neurological disease.

Original languageEnglish
Pages (from-to)229-233
Number of pages5
JournalJournal of Physiology - Paris
Volume88
Issue number4
DOIs
Publication statusPublished - 1994
Externally publishedYes

Fingerprint

Ciliary Neurotrophic Factor
Cholinergic Neurons
Messenger RNA
Choline O-Acetyltransferase
Alleles
Introns
Exons
Complementary DNA
Genotype
Amino Acids
Cell Line
Superior Cervical Ganglion
RNA Splice Sites
Mutation
Terminator Codon
RNA Precursors
Nerve Growth Factors
Homozygote
Sciatic Nerve
Gene Library

Keywords

  • choline acetyltransferase
  • CNT
  • neurological diseases
  • neuronal differentiation
  • null mutation

ASJC Scopus subject areas

  • Physiology (medical)
  • Neuroscience(all)

Cite this

Differentiation of cholinergic neurons and physiological role of ciliary neurotrophic factor (CNTF). / Deguchi, T.; Misawa, Hidemi; Takahashi, R.

In: Journal of Physiology - Paris, Vol. 88, No. 4, 1994, p. 229-233.

Research output: Contribution to journalArticle

@article{af5ce38efc554c3fbc6e7f586b2c4593,
title = "Differentiation of cholinergic neurons and physiological role of ciliary neurotrophic factor (CNTF)",
abstract = "Seven types of mRNA that differed in the 5′-non-coding region were identified for choline acetyltransferase of mouse spinal cord. These mRNAs were produced by differential splicing of pre-mRNAs transcribed from three different promotor regions. Two murine cholinergic cell lines, NS20Y and NG108-15, expressed M-type mRNA most abundantly. Using these cell lines, promotor activity in choline acetyltransferase gene was analyzed by transient assay of a reporter gene. The result indicated that there was promotor activity in the region upstream of the M-type exon and enhancer activity in the intron downstream of the M-type exon, and that this region regulated neuron-specific expression of choline acetyltransferase activity. In contrast, R-type mRNA was exclusively expressed in cultured superior cervical ganglion cells and was markedly increased by ciliary neurotrophic factor (CNTF). To analyse the physiological role of CNTF, we constructed and screened a cDNA library from human sciatic nerves and isolated two types of cDNAs for human CNTF. Sequence analysis revealed that one type of cDNA corresponded to the normal mRNA, while the other type contained a 4 bp stretch insertion within the coding region, which caused frameshift from 39th amino acid with a stop codon 24 amino acids downstream. Analysis of genomic DNA for CNTF showed that there was a point mutation from G to A in the intron of the mutated allele, which created a new splice acceptor site and generated a new mRNA species with 4 bp insertion. Analysis of tissue samples from autopsied subjects and transfection study of CNTF minigenes into cultured cells indicated that the mutated allele expressed only the mutated mRNA, but not the normal mRNA species. Thus, this allele represents a null mutation that abolishes expression of normal CNTF protein. CNTF genotypes were analyzed in 400 Japanese subjects. The frequency of three genotypes did not differ between healthy volunteers and patients suffering from various neurological diseases. Nine mutant homozygote subjects so far detected did not show any neurological disturbances and developmental abnormalities until adulthood. These observations indicate that CNTF is dispensable for the development and maintenance of the nervous system, and that the CNTF null mutation is not causally related with any neurological disease.",
keywords = "choline acetyltransferase, CNT, neurological diseases, neuronal differentiation, null mutation",
author = "T. Deguchi and Hidemi Misawa and R. Takahashi",
year = "1994",
doi = "10.1016/0928-4257(94)90085-X",
language = "English",
volume = "88",
pages = "229--233",
journal = "Journal de Physiologie",
issn = "0928-4257",
publisher = "Elsevier Masson SAS",
number = "4",

}

TY - JOUR

T1 - Differentiation of cholinergic neurons and physiological role of ciliary neurotrophic factor (CNTF)

AU - Deguchi, T.

AU - Misawa, Hidemi

AU - Takahashi, R.

PY - 1994

Y1 - 1994

N2 - Seven types of mRNA that differed in the 5′-non-coding region were identified for choline acetyltransferase of mouse spinal cord. These mRNAs were produced by differential splicing of pre-mRNAs transcribed from three different promotor regions. Two murine cholinergic cell lines, NS20Y and NG108-15, expressed M-type mRNA most abundantly. Using these cell lines, promotor activity in choline acetyltransferase gene was analyzed by transient assay of a reporter gene. The result indicated that there was promotor activity in the region upstream of the M-type exon and enhancer activity in the intron downstream of the M-type exon, and that this region regulated neuron-specific expression of choline acetyltransferase activity. In contrast, R-type mRNA was exclusively expressed in cultured superior cervical ganglion cells and was markedly increased by ciliary neurotrophic factor (CNTF). To analyse the physiological role of CNTF, we constructed and screened a cDNA library from human sciatic nerves and isolated two types of cDNAs for human CNTF. Sequence analysis revealed that one type of cDNA corresponded to the normal mRNA, while the other type contained a 4 bp stretch insertion within the coding region, which caused frameshift from 39th amino acid with a stop codon 24 amino acids downstream. Analysis of genomic DNA for CNTF showed that there was a point mutation from G to A in the intron of the mutated allele, which created a new splice acceptor site and generated a new mRNA species with 4 bp insertion. Analysis of tissue samples from autopsied subjects and transfection study of CNTF minigenes into cultured cells indicated that the mutated allele expressed only the mutated mRNA, but not the normal mRNA species. Thus, this allele represents a null mutation that abolishes expression of normal CNTF protein. CNTF genotypes were analyzed in 400 Japanese subjects. The frequency of three genotypes did not differ between healthy volunteers and patients suffering from various neurological diseases. Nine mutant homozygote subjects so far detected did not show any neurological disturbances and developmental abnormalities until adulthood. These observations indicate that CNTF is dispensable for the development and maintenance of the nervous system, and that the CNTF null mutation is not causally related with any neurological disease.

AB - Seven types of mRNA that differed in the 5′-non-coding region were identified for choline acetyltransferase of mouse spinal cord. These mRNAs were produced by differential splicing of pre-mRNAs transcribed from three different promotor regions. Two murine cholinergic cell lines, NS20Y and NG108-15, expressed M-type mRNA most abundantly. Using these cell lines, promotor activity in choline acetyltransferase gene was analyzed by transient assay of a reporter gene. The result indicated that there was promotor activity in the region upstream of the M-type exon and enhancer activity in the intron downstream of the M-type exon, and that this region regulated neuron-specific expression of choline acetyltransferase activity. In contrast, R-type mRNA was exclusively expressed in cultured superior cervical ganglion cells and was markedly increased by ciliary neurotrophic factor (CNTF). To analyse the physiological role of CNTF, we constructed and screened a cDNA library from human sciatic nerves and isolated two types of cDNAs for human CNTF. Sequence analysis revealed that one type of cDNA corresponded to the normal mRNA, while the other type contained a 4 bp stretch insertion within the coding region, which caused frameshift from 39th amino acid with a stop codon 24 amino acids downstream. Analysis of genomic DNA for CNTF showed that there was a point mutation from G to A in the intron of the mutated allele, which created a new splice acceptor site and generated a new mRNA species with 4 bp insertion. Analysis of tissue samples from autopsied subjects and transfection study of CNTF minigenes into cultured cells indicated that the mutated allele expressed only the mutated mRNA, but not the normal mRNA species. Thus, this allele represents a null mutation that abolishes expression of normal CNTF protein. CNTF genotypes were analyzed in 400 Japanese subjects. The frequency of three genotypes did not differ between healthy volunteers and patients suffering from various neurological diseases. Nine mutant homozygote subjects so far detected did not show any neurological disturbances and developmental abnormalities until adulthood. These observations indicate that CNTF is dispensable for the development and maintenance of the nervous system, and that the CNTF null mutation is not causally related with any neurological disease.

KW - choline acetyltransferase

KW - CNT

KW - neurological diseases

KW - neuronal differentiation

KW - null mutation

UR - http://www.scopus.com/inward/record.url?scp=0028148441&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028148441&partnerID=8YFLogxK

U2 - 10.1016/0928-4257(94)90085-X

DO - 10.1016/0928-4257(94)90085-X

M3 - Article

C2 - 7874083

AN - SCOPUS:0028148441

VL - 88

SP - 229

EP - 233

JO - Journal de Physiologie

JF - Journal de Physiologie

SN - 0928-4257

IS - 4

ER -