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.