Choline Acetyltransferase Activity In Cultures Research Articles

Stimulation of choline acetyltransferase by C3d, a neural cell adhesion molecule ligand

Septal cholinergic neurons project to the hippocampus and release acetylcholine, a neurotransmitter involved in learning and memory. The enzyme choline acetyltransferase (ChAT) is responsible for synthesizing acetylcholine. Promoting ChAT activity and acetylcholine release can lead to new treatments for neurodegenerative diseases with cholinergic deficits, such as Alzheimer's disease. We present evidence that the synthetic molecule C3d, which is a peptide mimetic of the neural cell adhesion molecule (NCAM), promotes ChAT activity in cultures of rat embryonic septal neurons. Our data demonstrate that ChAT activity triggered by C3d is dependent on the fibroblast growth factor receptor (FGFR) and the mitogen-activated protein kinase (MAPK) pathway. C3d did not affect the number of cholinergic neurons in culture, indicating that NCAM homophilic binding enhances ChAT activity, without affecting cholinergic cell survival. In conclusion, the NCAM mimetic peptide C3d promotes ChAT activity in septal neurons through FGFR and MAPK. These findings are relevant to the design of new strategies aimed at stimulating cholinergic function and improving cognition in disorders such as Alzheimer's disease.

Synergistic effects of brain-derived neurotrophic factor and ciliary neurotrophic factor on cultured basal forebrain cholinergic neurons from postnatal 2-week-old rats

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, and ciliary neurotrophic factor (CNTF), a member of the neurocytokine family, are known to have synergistic effects on motoneurons, but such synergistic effect has not been studied in detail especially in the brain. In the present study, we examined the synergistic effects of BDNF and CNTF on the survival of basal forebrain cholinergic neurons cultured from postnatal 2-week-old (P2w) rats. Although BDNF is well-known to promote the survival of basal forebrain cholinergic neurons in P2w culture, CNTF had little effect on the survival of choline acetyltransferase (ChAT)-positive neurons and did not increase ChAT activity in the culture. However, CNTF enhanced BDNF-mediated promotion of cell survival of cholinergic neurons when added concomitantly. BDNF alone induced only a three-fold increase in ChAT activity in control cultures, but the concomitant addition of CNTF resulted in an eight-fold increase. CNTF did not enhance BDNF-mediated cell survival of total neurons from the basal forebrain, hippocampus or cerebellum, suggesting that the synergistic effects of CNTF on the BDNF-mediated increase of viability might be strong in basal forebrain cholinergic neurons. CNTF also enhanced the neurotrophin-4/5-mediated increase of ChAT activity, but not the nerve growth factor (NGF)-mediated one. Furthermore, the BDNF-mediated increase was also enhanced by leukemia inhibitory factor but not by interleukin-6. Similar synergistic pattern between neurotrophins and cytokines were also observed in the induction of ChAT activity in embryonic basal forebrain culture. These results suggest that TrkB, a functional high-affinity receptor of BDNF and NT-4/5, and LIFR β, a receptor component contained in CNTF and LIF receptor complex, might be involved in the observed synergistic effects.

Tricycloillicinone, a novel prenylated c6-c3 compound increasing choline acetyltransferase (ChAT) activity, isolated from Illicium tashiroi.

Tricycloillicinone (1), a novel tricyclic prenylated C6-C3 compound, has been isolated as a neurotrophic substance from the woods of Illicium tashiroi and its structure has been elucidated by spectroscopic analyses. Compound 1 could increase ChAT activity in culture of P10 rat septal neurons.

New chlorine-containing prenylated C6-C3 compounds increasing choline acetyltransferase (ChAT) activity in culture of postnatal rat septal neurons from Illicium tashiroi.

The structures of two new chlorine-containing C6-C3 compounds isolated from the woods of Illicium tashiroi have been established as 2(R)-12-chloro-2,3-dihydroillicinone E (1) and 12-chloroillicinone E (2) by X-ray crystallographic analysis and spectroscopic data, respectively. Compound 1 has been found to significantly increase ChAT activity in culture of P10 rat septal neurons.

Differential effects of insulin on choline acetyltransferase and glutamic acid decarboxylase activities in neuron-rich striatal cultures.

We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.

Role of fibronectin in the inhibitory effect of TGF-β on choline acetyltransferase activity in co-cultures of spinal cord neurons and myotubes

We have recently shown that the enhanced expression of choline acetyltransferase (ChAT) activity in co-cultures of spinal cord motoneurons and muscle cells was blocked by transforming growth factor-β (TGF-β) ( Dev. Brain Res., 57, 129–137, 1990). This study was performed to investigate the role of fibronectin in this effect. TGF-β increased fibronectin level about 2-fold in extracellular matrix of spinal cord cells and skeletal myotubes in culture. Addition of a synthetic polypeptide that competitively inhibits fibronectin binding to its cell surface receptor recovered the TGF-β-induced suppression of ChAT activity in co-cultures. The polypeptide did not affect ChAT activity in cultures of spinal cord cells alone or in co-cultures without TGF-β. These results indicate that TGF-β inhibits the stimulation of ChAT activity in spinal cord neurons in co-culture through a change in the composition and/or amount of fibronectin in the extracellular matrix at neuromuscular contacts.

P-433 - Effects of epidermal growth factor and nerve growth factor on septal choline acetyltransferase activity in culture.

P-433 - Effects of epidermal growth factor and nerve growth factor on septal choline acetyltransferase activity in culture.

Characterization of two factors enhancing choline acetyltransferase activity in cultures of purified rat motoneurons

Motoneurons from E14 rat embryos have been retrogradely labeled with the carbocyanine derivative dil and purified 12-fold by centrifugation on a density gradient made of Nycodenz, as assessed by the increase in CAT activity per cell and in the percentage of dil-labeled cells. A 20- to 36-fold purification was achieved by the microdissection of the labeled lumbar motor columns followed by density-gradient centrifugation. Motoneurons were then purified to near homogeneity by fluorescence-activated cell sorting. In certain experiments, motoneurons were purified by a novel cell-sorting procedure that only uses light-scatter parameters and does not necessitate retrograde labeling. In cultures of motoneurons enriched by centrifugation, muscle-conditioned media (CM) had only a marginal effect on the overall neuronal survival but increased up to 10-fold the number of labeled motoneurons surviving after 7 d in culture. CAT activity per culture was quantitatively stimulated to the same extent, suggesting that the increase in CAT activity caused by CM mostly reflected better survival of motoneurons. In cultures of motoneurons purified by cell sorting, CM increased both survival and CAT expression per motoneuron. We have partially purified 2 factors from CM that stimulate CAT activity per motoneuron 2- to 3-fold without affecting their survival. One of these factors is probably identical to the factor involved in the cholinergic/noradrenergic choice of sympathetic neurons (Fukada, 1985).

Characterization of dissociated monolayer cultures of human spinal cord

Embryonic human spinal cord cells have been grown in dissociated monolayer cultures for 1 to 7 weeks. Using cell type specific markers, it was possible to show that the cultures contain neurons, astrocytes and fibroblasts. Electrical membrane properties were studied with patch electrodes using the whole cell recording technique. Neurons had short duration action potentials that could be blocked by tetrodotoxin. The membrane currents in these neurons were studied in voltage clamp experiments. Three types of voltage-dependent currents were observed: a sodium current, a potassium current made up of two components, I A and I K, and a calcium current. Both cholinergic and GABAergic neurons are present in the cultures. There is more choline acetyltransferase activity in cultures prepared from the anterior as compared to the posterior part of the spinal cord, suggesting that the cultures contain motoneurons. This tissue culture preparation was developed for the study of amyotrophic lateral sclerosis, we have been unable to detect the presence of any toxic agent from the serum of these patients on the cultured cells. Experiments are in progress to purify the motoneurons using Percoll gradients.

Cholinergic neurons in cultures derived from three-, six- or eight-day-old chick embryo: a biochemical and immunocytochemical study

Cultures were prepared by dissociating 3-day-old whole chick embryos, or 6- or 8-day-old chick embryo cerebral hemispheres. The dispersed cells were plated onto poly- l-lysine coated dishes in Dulbecco's modified Eagle's medium fortified with 10% fetal bovine calf serum. Immunocytochemistry, histochemistry and biochemistry were used to characterize the cholinergic neurons in these cultures. Double staining for acetylcholinesterase (AChE) and neurofilament revealed that a high percentage of neurons also contained AChE; fewer neurons though were positive for choline acetyltransferase (ChAT). The presence of AChE in non-cholinergic neurons was interpreted to be indicative of ‘cholinoceptive’ neurons in culture. The developmental profile for ChAT activity in cultures derived from 3-day-old whole chick embryos was previously described; we have reported that activity reached high levels by 7 days in culture. In this study we found that in cultures derived from 6-day-old chick embryos ChAT activity increased slowly up to 6 days in culture and rose markedly thereafter; in contrast, in cultures derived from 8-day-old chick embryos activity was low until day 9 and rose moderately thereafter. The differences in ChAT expression between cultures derived from chick embryos of different developmental stages may reflect a loss of plasticity of the cholinergic population with age.