Expression Of Choline Acetyltransferase Activity Research Articles

Docosahexaenoic Acid Supports Cell Growth and Expression of Choline Acetyltransferase and Muscarinic Receptors in NG108-15 Cell Line

A large body of evidence indicates that adequate intake of polyunsaturated fatty acids is essential for brain development in early ontogenesis and positively impacts various pathological states connected with aging, as well as other neurodegenerative diseases (Jump, 2002; Bazan, 2003; Ruxton et al., 2004). In the present experiments, we investigated the possible effects of polyunsaturated docosahexanoic acid (DHA [22:6, n = 3]) on the expression of cholinergic phenotype-represented by choline acetyltransferase (ChAT) activity and a number of surface muscarinic receptors-as well as on cell growth in the cholinergic cell line NG108-15(Hamprecht, 1977; Hamprecht et al., 1985). However, chemical composition of different batches of sera is neither stable nor defined, and this fact complicates investigations on in vitro effects of substances that are natural constituents of serum. To avoid this restraint we employed defined medium in which fatty acid-free bovine albumin as a carrier of DHA replaced serum. Growth of most cell lines, as well as cells in primary cultures, depends strictly on the presence of serum in growth medium. As expected, withdrawal of serum resulted in growth arrest exemplified by a decrease in protein content compared with control cells grown in the presence of serum and also caused a decrease in ChAT activity (Fig. 1, lower left). DHA, at a concentration of 10 mumol/L, largely prevented both growth arrest in defined medium with fatty acid-free bovine albumin as a carrier of DHA and the attenuation of ChAT activity. DHA at concentrations 10 times higher had no further effect. At a concentration of 100 mumol/L, DHA also significantly increased the number of surface muscarinic receptors compared with cells grown in serum-containing as well as serum-free medium (Fig. 1, upper right). These data demonstrate the ability of DHA at low micromolar concentrations to support cell growth and expression of ChAT activity. Although it is not possible to stipulate a mechanism of action on the expression of ChAT and muscarinic receptors, a plausible explanation could be prevention of apoptosis, evidenced by a sharp decrease in executive caspase-3 activity (Fig. 1, lower right). Apoptosis is a process with a high requirement for energy. An improved metabolic state of cells consequent to suppression of apoptosis might thus better fulfill requirements for protein synthesis and targeting.

Recovery of acetylcholine release and choline acetyltransferase activity after freezing-induced degeneration of rat soleus muscle

In order to study the effect of synaptic contact on the amounts of choline acetyltransferase (ChAT) and acetylcholine (ACh) in the nerve terminals and on their ability to release ACh, a freeze—thaw procedure was developed as a means to induce long lasting degeneration of rat soleus muscle. It was found that 4 days after the freeze—thaw procedure the preparation did not contract upon direct electric stimulation and the level of creatine kinase (CK) was below detection. The preparation contained about 15% of the ChAT activity and 15% of the ACh content of the controls. The ACh release evoked by 50 mM KCl was 25% of controls, but it was, when expressed as a fraction of the ACh content, about twice as high as that in control muscles. At day 12, the preparation still did not contract and the level of CK was less than 5% of controls. The ChAT activity and the ACh content were 40%) and 20% of controls, respectively. However, no release of ACh could be evoked by 50 mM KCl. At days 28 and 58 the preparation contracted upon stimulation of the nerve; the CK activity had recovered to about 20% and the ACh content to 40%, while the ChAT activity did not increase above 40%. The KCl–evoked ACh release had recovered to 20—30% of controls. The results indicate that freezing destroyed muscle cells and most intramuscular nerve branches. Subsequent regeneration of muscle fibres was slow, probably because freezing had killed many satellite cells in the muscle. Because the ChAT activity at day 12 had recovered when CK was almost absent and the preparation failed to contract, we conclude that there was expression of ChAT activity in ‘nerve terminals’ which do not make contact with regenerated muscle cells, although little if any ACh was released from these sites. ©1998 Elsevier Science Ltd. All rights reserved.

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

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.

Midkine Is a Mediator of Retinoic Acid-Induced Neuronal Differentiation of Embryonal Carcinoma Cells

Midkine (MK) is a novel growth factor and is the product of a retinoic acid responsive gene. When P19 murine embryonal carcinoma (EC) cells were exposed to MK, they differentiated into neurons, and the neuronal differentiation was accompanied by expression of choline acetyltransferase activity. Synthesis and release of MK in the EC cells treated with retinoic acid were shown by Western blot analysis, and rabbit anti-MK antibody attenuated the action of retinoic acid to induce the neuronal differentiation. These results indicate that MK is one of the mediators of retinoic acid action to induce the neuronal differentiation in EC cells.

Gene expression of mouse choline acetyltransferase. Alternative splicing and identification of a highly active promoter region.

Seven types of mRNA for choline acetyltransferase that differ in the 5'-noncoding region were identified in the mouse spinal cord by cDNA cloning and polymerase chain reaction. Among these transcripts, the M-type mRNA corresponding to the previously cloned mouse cDNA was most abundant in the spinal cord of mouse. A mouse genomic DNA clone containing the 5'-region of choline acetyltransferase mRNA was isolated and sequenced. Comparison of the sequences between the cDNAs and the genomic DNA revealed that the different mRNA species were transcribed from different promoter regions and produced by differential splicing. Two murine cholinergic cell lines, NS20Y and NG108-15, were shown to express the M-type mRNA almost exclusively, and were therefore used to study transcription of M-type mRNA. Fragments of the 5'-region of choline acetyltransferase gene were ligated with chloramphenicol acetyltransferase reporter gene and introduced into cultured cells. The fragment from -2752 to +46, which contained the M-type exon, a TATA-box like element upstream of the M-type exon, and the downstream intron, induced a significant expression of CAT activity in neuronal but not in non-neuronal cell lines. This result indicates that this region of choline acetyltransferase gene contains elements that regulate neuron-specific expression of choline acetyltransferase activity. However, there was no parallel correlation between reporter gene expression in the transfected cells and intrinsic choline acetyltransferase activity in these neuronal cell lines. Possible mechanisms that would explain this observation are discussed.

Expression of choline acetyltransferase activity in a co-culture of spinal cord and skeletal muscle cells is inhibited by myogenic differentiation inhibitors

The effect of myogenic differentiation on the expression of choline acetyltransferase (ChAT) activity in co-cultured spinal cord neurons was studied. ChAT activity in spinal cord cells dissociated from 14-day mouse embryos was markedly increased when co-cultured with skeletal myotubes from 20-day embryos. This enhancement of ChAT activity was not observed in the presence of concanavalin A (ConA) or N-methyl-l-deoxynojirimycin (MDJN) which inhibits myoblast fusion, creatine phosphokinase and acetylcholinesterase activities in muscle cells. ChAT activity in spinal cord neurons cultured alone was unaffected by these agents. The inhibitory effect of ConA and MDJN was reversible, with an almost full recovery of ChAT activity following removal of the agents. Addition of ConA or MDJN after myotube formation exerted little inhibitory effect on ChAT activity. The effects of ConA and MDJN on ChAT activity in co-cultures were comparable to those on creatine phosphokinase and acetylcholinesterase. These observations indicate that the neurotrophic effects of skeletal muscle cells on spinal cord neurons are dependent on the differentiation state of the muscle cells.

Influence of the culture substratum on the expression of choline acetyltransferase activity in purified motoneurons from rat embryos

Motoneurons from E14 rat embryos have been retrogradely labelled with a carbocyanine derivative and purified approximately 20-fold by density gradient centrifugation. For certain experiments, motoneurons have been purified to near homogeneity by cell sorting. These neurons were then cultured at low density on various substrata. Laminin stimulated neurite outgrowth of the identified motoneurons and stimulated choline acetyltransferase (ChAT) development by 2-fold; these cultures could not be maintained for more than 4 days. Motoneurons could be cultured for at least 7 days on monolayers of both living and lysed astrocytes from newborn cortex. The cells survived equally well on both substrata but neurite outgrowth was less elaborated on lysed astrocytes on which motoneurons displayed a mono- or bipolar, rather than a multipolar, morphology. Furthermore, ChAT developed at a 2-fold higher level on lysed, rather than living, newborn astrocytes. Such a difference was not observed using astrocytes from E14 rat embryos. In the latter case, both living and lysed astrocytes supported ChAT development at the same rate as lysed newborn astrocytes. Consequently, living astrocytes from newborn rat cortex are relatively less permissive for ChAT expression. On the other hand, both living and lysed astrocytes from newborn cortex induced ChAT expression by sympathetic neurons to the same extent. We propose that, in addition to retrograde factors from muscle origin, cell surface molecules expressed by embryonic astrocytes stimulate ChAT expression by motoneurons. Such molecules would be present in newborn astrocytes in an intracellular form and could be unmasked by cell lysis. Different cell surface molecules may induce ChAT activity in sympathetic neurons.

Regulation of production by primary cultures of rat forebrain astrocytes of a trophic factor important for the development of cholinergic neurons

The possibility was examined for the production of a trophic factor by astrocytes important for the biochemical development of cholinergic neurons. The activity of choline acetyltransferase (ChAT) was used as a marker to study the maturation of cholinergic neurons, while the metabolic state of astrocytes was monitored in terms of glutamine synthetase activity. When the dissociated cells, derived from the septal-diagonal band region of embryonic rat brain, were cultured either on a preformed layer of highly enriched astrocytes or in the presence of astrocyte-conditioned medium, a marked increase was observed in the expression of ChAT activity. Similar effect was not noticed when cerebellar granule cells replaced the astrocytes. The production of cholinergic factor was decreased when the astroglial cells were induced to proliferate by epidermal growth factor, whereas its production was increased when quiescent astrocytes were treated with a low dose of cytosine arabinoside. The results would indicate that a trophic factor important for the development of cholinergic cells is produced by astrocytes, and that exogenously added agents influencing astroglial metabolism have a marked regulatory effect on the production of this trophic factor.