Purification of a skeletal muscle polypeptide which stimulates choline acetyltransferase activity in cultured spinal cord neurons

Extracts of rat skeletal muscle contain neurotrophic factors which stimulate the development of choline acetyltransferase in embryonic day 14 rat spinal cord cultures. The trophic activity does not bind heparin-Sepharose or lectin affinity columns. However, mild acid treatment separates the trophic activity into soluble and insoluble fractions. The acid-insoluble activity has been purified 5000-fold to apparent homogeneity using preparative sodium dodecyl sulfate gel electrophoresis to achieve final purification. The purified factor migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, with an apparent molecular mass of 20 kDa and a pI of 4.8. The activity and apparent molecular weight of the purified factor are unaltered by treatment with reducing agents or incubation in acidic conditions. Activity, however, is destroyed by heating or protease treatment. Thus, the factor appears to be a single polypeptide without significant levels of glycosylation or charge microheterogeneity. These results represent the first purification of a neurotrophic factor from skeletal muscle. The physical properties and amino acid composition of this factor differ from those of nerve growth factor and heparin-binding growth factors, as well as from the neurotrophic factor from heart cell conditioned medium which induces cholinergic development in sympathetic neurons.     

Pineal cells enhance choline acetyltransferase activity in sympathetic neurons

Cells derived from the neonatal rat pineal gland were cocultured with cells derived from neonatal rat superior cervical ganglia (SCG) in an attempt to determine whether a sympathetic target organ with only adrenergic properties could enhance the development of adrenergic transmitter properties in sympathetic neurons in tissue culture. Choline acetyltransferase was measured as an index of cholinergic differentiation, and tyrosine hydroxylase was measured as an index of adrenergic differentiation. As indices of total cell number and cellular volume, DNA and protein, respectively, were also measured. We found that the pineal-SCG cocultures contained ten times greater choline acetyltransferase activity than sister neuronal cultures cultured without pineal cells, thus indicating that the pineal cells enhanced cholinergic properties in the sympathetic neurons. This cholinergic enhancement was dependent upon the presence of nerve growth factor and could not be obtained with pineal-conditioned medium. Tyrosine hydroxylase activity, measured on cultures sister to those mentioned above, was low in all cultures and decreased somewhat in SCGs cultured alone. TH activity in the pineal-SCG cocultures, however, increased slightly. Some tyrosine hydroxylating activity developed in pineals cultured alone, however, and may have been responsible for the small increase in tyrosine hydroxylase activity noted in the pineal-SCG cocultures. The implications of these results for a determination of the role that target organ plays in the development of the transmitter properties of sympathetic neurons are discussed.     

Immunohistochemical and in situ hybridization studies of choline acetyltransferase in large motor neurons of the human spinal cord

The localization of choline acetyltransferase (ChAT) protein and mRNA was investigated in large motor neurons of the lumbar spinal cord of 10 autopsied individuals without neurological diseases, by immunohistochemistry and in situ hybridization. In the immunohistochemistry using 20 serial tissue sections with a total thickness of 80 microm, about approximately 58-85% (average 67%) of the large motor neurons (30 microm and more in somal minimal diameter) in the ventral horn were stained with the anti-human ChAT antibody. In the positive neurons, most immunoreactive products were observed focally in the perikarya. Occasionally, the perikarya of some neurons were stained diffusely. In situ hybridization with a single 4 microm-thick tissue section showed that almost all large motor neurons had positive signals (approximately 93-100%, average 98%), which were distributed diffusely in the perikarya. The positivity rate in the in situ hybridization was higher than that in the immunohistochemistry for all 10 cases. These results indicate that ChAT mRNA is transcribed in almost all large motor neurons in the ventral horn of the human spinal cord, but ChAT protein cannot always be detected in the cytoplasm by immunohistochemistry.     

Choline acetyltransferase activity of spinal cord cell cultures is increased by diisopropylphosphorofluoridate.

Diisopropylphosphorofluoridate (DFP) increased the specific activity of choline acetyltransferase (ChAT) in mouse spinal cord cell cultures but paraoxon did not, though both toxicants inhibited cholinesterase activity to a comparable extent. This effect of DFP was observed after prolonged exposure but not after short-term application to the cultures. It is postulated that this delayed effect of DFP $\text{in vitro}$ may possibly be related tothe delayed neuropathy caused by DFP in certain species $\text{in vivo}$.     

Polysialic acid limits choline acetyltransferase activity induced by brain-derived neurotrophic factor

Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine, is known to be activated by brain derived neurotrophic factor (BDNF). We found that the specific removal of the carbohydrate polysialic acid (PSA) significantly increased BDNF-induced ChAT-activity in embryonic septal neurons. Using a p75 neurotrophin receptor (p75(NTR)) function-blocking antibody and K252a, a-pan tropomyosin related kinase (Trk) inhibitor, we demonstrate that BDNF-induced ChAT activity requires the stimulation of p75(NTR) and TrkB. PSA removal drastically increased radioactive iodinated ([(125)I])BDNF's maximal binding capacity (Bmax), derived from concentrations of [(125)I]BDNF ranging from 1 pM to 3.2 nM. In the presence of unlabeled nerve growth factor to prevent the binding of [(125)I]BDNF to p75(NTR) sites, the impact of PSA removal on the binding capacity of [(125)I]BDNF was greatly reduced. In conclusion, PSA limits BDNF-induced ChAT activity and BDNF-receptor interactions. BDNF-induced ChAT activity is TrkB and p75(NTR) dependent, and upon PSA removal the additional binding of BDNF to its receptors, especially p75(NTR), likely contributes to the maximal ChAT activity observed. In vivo, the ontogenetic loss of PSA in the postnatal period may allow more interactions between BDNF and its receptors to increase ChAT activity and assure the proper development of the cholinergic septal neurons.     

ERK 1/2 signaling pathway is involved in nicotine-mediated neuroprotection in spinal cord neurons

Evidence indicates that agonists of neuronal nicotinic receptors (nAChRs), including nicotine, can induce neuroprotective and anti-apoptotic effects in the CNS. To study these mechanisms, the present study focused on nicotine-mediated modulation of the extracellular regulated kinase 1 and 2 (ERK1/2) pathway in cultured spinal cord neurons. Exposure to nicotine (0.1-10 microM) for as short as 1 min markedly upregulated levels of phosphorylated ERK1/2 (pERK1/2) and increased total ERK1/2 activity. Inhibition studies with mecamylamine and alpha-bungarotoxin revealed that these effects were mediated by the alpha7 nicotinic receptor. In addition, pre-exposure to U0126, a specific inhibitor of the ERK1/2 signaling, prevented nicotine-mediated anti-apoptotic effects. To indicate if treatment with nicotine also can activate ERK1/2 in vivo, a moderate spinal cord injury (SCI) was induced in rats using a weight-drop device and nicotine was injected 2 h post-trauma. Consistent with in vitro data, nicotine increased levels of pERK1/2 in this animal model of spinal cord trauma. Results of the present study indicate that the ERK1/2 pathway is involved in anti-apoptotic effects of nicotine in spinal cord neurons and may be involved in therapeutic effects of nicotine in spinal cord trauma.     

High activity of choline acetyltransferase induced in neuroblastoma X glia hybrid cells

Hybrids were made between rat glioma X mouse neuroblastoma cell lines and were examined for the specific activities of choline acetyltransferase and acetylcholinesterase. The specific activities of choline acetyltransferase of the hybrids were as high as those in normal brain, whereas neither parent line showed appreciable activities. The electrical excitability of the neuroblastoma cells was retained in the hybrids.     

High affinity choline uptake by spinal cord neurons in dissociated cell culture

Spinal cord-myotube cultures prepared with dissociated embryonic chick spinal cord cells and myoblasts exhibit a high affinity mechanism for accumulating choline. The uptake mechanism has a K_m of 3.4 ± 0.5 μM (7) and a V_m of 40.0 ± 0.1 (7) pmoles/min/mg of protein (mean ± SEM; number of determinations in parentheses). It is inhibited 90–95% by 10 μM hemicholinium-3 or by replacement of Na⁺ in the incubation solution with Li⁺. Part of the choline (10–20%) accumulated by the high affinity system is converted to acetylcholine (ACh). Uptake studies on spinal cord cells and myotubes grown separately demonstrate that the spinal cord cells can account for virtually all of the choline uptake observed in the mixed cultures. Myotubes are unnecessary under these conditions for the expression of the high affinity uptake mechanism by spinal cord cells. Neurons are not the only cell type in culture to exhibit high affinity choline uptake. Chick fibroblasts in both rapidly growing and stationary phase can accumulate choline with kinetics similar to those observed for the high affinity uptake by spinal cord cells. Little if any of the choline accumulated by fibroblasts, however, is converted to ACh. In most uptake studies with spinal cord cells, contributions from fibroblasts were minimized by carrying out the analysis at a time when few non-neuronal cells were present in the spinal cord cultures. These observations suggest that a population of chick central nervous system (CNS) neurons develop a high affinity choline uptake mechanism in cell culture that has many of the properties described for uptake by cholinergic neurons in vivo and that at least part of the choline accumulated by the system can be used for neurotransmitter synthesis.     

Source and target cell specificities of a conditioned medium factor that increases choline acetyltransferase activity in cultured spinal cord cells

A macromolecular factor(s) in muscle conditioned medium (CM), when applied to spinal cord (SC) cells in culture, causes large increases in the activity of choline acetyltransferase (CAT), the enzyme which synthesizes the neurotransmitter acetylcholine. We have found apparent specificity of both species and cell type for the production, release, or action of this CAT stimulation component (CSC). Rat and mouse muscle CMs contained CSC which was active in mouse SC cells; chick muscle CM did not. In addition to muscle CM, the CM from cell cultures of mouse heart, liver, and kidney contained CSC. However, CM from secondary cultures of liver cells contained little if any CSC. These apparent specificities were not due to differences in the protein content of either the cells providing CM or of the CM itself. There was also apparent specificity of response to CSC among cholinergic cells in culture. Cultures of cells from only two of four regions of the mouse central nervous system, and from one of five neuronal cell lines tested, had increased CAT activity after treatment with muscle CM. The response in NG108-15 neuroblastoma-glioma hybrid cells was further characterized, and was used to develop a more convenient and rapid assay for CSC.     

Localization of choline acetyltransferase in human brain stem neurons

Berth's method was used to study the cytochemical activity of choline acetyltransferase in truncus cerebri neurons of 6-8 lunar month-old human fetuses. Three types on neurons were diagnosed in the nuclei of the truncus cerebri with regard to cholinacetyltransferase localization: (1) cholinergic cholinoceptive neurons; (2) cholinergic non-cholinoceptive neurons; (3) non-cholinergic cholinoceptive neurons. The distribution of the neurons in 27 nuclei of the truncus cerebri is described.     

Autoradiographic labeling of spinal cord neurons with high affinity choline uptake in cell culture

Embryonic chick spinal cord neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline. We find that, in addition to acetylcholine synthesis, the accumulated choline is used for the synthesis of metabolites such as lipids that are retained in part by conventional fixation techniques. As a result autoradiographic methods can be used to identify the cells that have the uptake mechanism in spinal cord cultures. About 60% of the neurons are labeled by [³H]choline uptake in cultures prepared with spinal cord cells from 4-day-old embryos, and about 40% are labeled in cultures prepared with cord cells from 7-day-old embryos. Neurons that innervate skeletal myotubes in spinal cord-myotube cultures are consistently labeled by [³H]choline uptake. Neurons unlabeled by the procedure are viable: they exclude the dye trypan blue and accumulate ¹⁴C-amino acids for protein synthesis. Most of the neurons unlabeled by [³H]choline uptake can instead be labeled by uptake of γ-[³H]aminobutyric acid, and vice versa. These results suggest that high affinity choline uptake can be used to label cholinergic neurons in cell culture, and that at least some populations of noncholinergic neurons are not labeled by the procedure. It cannot yet be concluded, however, that all labeled neurons are cholinergic since more labeled neurons are obtained per cord than would be expected from the number of neurons making up identified cholinergic populations in vivo. A three- to fourfold increase in the amount of high affinity choline uptake is observed between Days 3 and 15 in culture for spinal cord cells obtained from 4-day-old embryos. The number of [³H]choline-labeled neurons in such cultures decreases slightly during the same period, suggesting that the increase in uptake reflects neuronal growth or development rather than an increase in population size. Both the magnitude of the uptake and the number of [³H]choline-labeled neurons are the same for spinal cord cells grown with and without skeletal myotubes.     

Developmental changes of choline acetyltransferase (ChAc) activity in chick embryo spinal and sympathetic ganglia

The ChAc activity of spinal and sympathetic ganglia was measured throughout the embryonic life of the chick. In spinal ganglia, the ChAc activity reached a first peak when the maximal proliferation of neuroblasts occurred. Then, the relative ChAc activity decreased. After the 12th day of incubation, the enzyme activity increased again and reached a second peak on the 16th day. In sympathetic ganglia, the general course of the development of ChAc activity was similar to spinal ganglia. However, higher enzymic activity was found. Furthermore, the earlier peak of ChAc activity occurred 48 hr later than the corresponding peak in spinal ganglia. The behaviour of ChAc activity in these two areas of the developing nervous system is interpreted as a function of their histogenesis.     

Neurotrophins differentially enhance acetylcholine release, acetylcholine content and choline acetyltransferase activity in basal forebrain neurons

Several lines of evidence indicate that nerve growth factor is important for the development and maintenance of the basal forebrain cholinergic phenotype. In the present study, using rat primary embryonic basal forebrain cultures, we demonstrate the differential regulation of functional cholinergic markers by nerve growth factor treatment (24–96 h). Following a 96‐h treatment, nerve growth factor (1–100 ng/mL) increased choline acetyltransferase activity (168–339% of control), acetylcholine content (141–185%), as well as constitutive (148–283%) and K⁺‐stimulated (162–399%) acetylcholine release, but increased release was not accompanied by increased high‐affinity choline uptake. Enhancement of ACh release was attenuated by vesamicol (1 µm ), suggesting a vesicular source, and was abolished under choline‐free conditions, emphasizing the importance of extracellular choline as the primary source for acetylcholine synthesized for release. A greater proportion of acetylcholine released from nerve growth factor‐treated cultures than from nerve growth factor‐naïve cultures was blocked by voltage‐gated Ca²⁺ channel antagonists, suggesting that nerve growth factor modified this parameter of neurotransmitter release. Cotreatment of NGF (20 ng/mL) with K252a (200 nm ) abolished increases in ChAT activity and prevented enhancement of K⁺‐stimulated ACh release beyond the level associated with K252a, suggesting the involvement of TrkA receptor signaling. Also, neurotrophin‐3, neurotrophin‐4 and brain‐derived neurotrophic factor (all at 5–200 ng/mL) increased acetylcholine release, although they were not as potent as nerve growth factor and higher concentrations were required. High brain‐derived neurotrophic factor concentrations (100 and 200 ng/mL) did, however, increase release to a level similar to nerve growth factor. In summary, long‐term exposure (days) of basal forebrain cholinergic neurons to nerve growth factor, and in a less‐potent fashion the other neurotrophins, enhanced the release of acetylcholine, which was dependent upon a vesicular pool and the availability of extracellular choline.     

A radiometric microassay for choline acetyltransferase. Some observations on the spinal cord of the chicken embryo

This paper describes cation-exchange methods for separating acetyl[³H] coenzyme A from [acetyl-³H]choline. Blanks for the routine method were approximately 0.05% of the substrate radioactivity; product recoveries were approximately 97%. The cation-exchange method was moreefficient than the standard methods using either anion-exchange chromatography or periodide precipitation. The cation-exchange method was also morespecific than either of the other two standard methods for estimating choline acetyltransferase (ChAT) activity. ChAT activity was detected in the chicken lumbar spinal cord on embryonic day (E) 2 1/4 with the cation-exchange method. This developmental stage is about 6 hours before the final mitosis of any neuroblast in the ventral horn. Total ChAT activity per lumbar spinal cord increased more than 10,000-fold between E 3 and E 18. Changes in ChAT activity in the lumbar spinal cord following limb-bud extirpation appeared to mirror (with a phase lag) the changes in the number of motoneurons in the lateral motor column.     

Brief exposure to neurotrophins produces a calcium-dependent increase in choline acetyltransferase activity in cultured rat septal neurons

We demonstrate that brief (30-min) exposure of cultured embryonic rat septal neurons to neurotrophins (NTs) increases choline acetyltransferase (ChAT) activity by 20-50% for all tested NTs (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4, each at 100 ng/ml). The increase in ChAT activity was first detected 12 h after NT exposure, persisted at least 48 h, and was not mediated by increased neuronal survival or action-potential activity. Under some conditions, the response to brief NT exposure was as great as that produced by continuous exposure. NT stimulation of ChAT activity was inhibited by inhibitors of p75- and Trk kinase-mediated signaling, by removal of extracellular Ca2+ during the period of NT exposure, and by buffering intracellular Ca2+ with BAPTA. Application of nerve growth factor and brain-derived neurotrophic factor transiently increased [Ca2+] within a subpopulation of neurons. NT stimulation of ChAT activity was not affected significantly by cyclic AMP agonists or antagonists. These findings suggest that brief exposure to NTs can have a long-lasting effect on cholinergic transmission, and that this effect requires Ca2+, but not cyclic AMP.     

Partial cloning and expression of mRNA coding choline acetyltransferase in the spinal cord of the goldfish, Carassius auratus

Choline acetyltransferase (ChAT, EC 2.3.1.6) synthesizes a neurotransmitter, acetylcholine in cholinergic neurons. ChAT is considered to be the most specific marker for cholinergic neurons. To obtain a better marker of the neurons, as the first step, we isolated a partial ChAT cDNA from the goldfish (Carassius auratus) brain by RT-PCR methods. The partial cDNA of the goldfish ChAT was composed of 718 nucleotides. The amino acid sequence of the goldfish ChAT is approximately 70% identical to those of mammalian and chicken ChAT. Northern blot analysis demonstrated that ChAT mRNA was expressed in the brain and the spinal cord of the goldfish, and much abundant in the spinal cord. In the spinal cord of the goldfish, ChAT-positive neurons were detected mainly in the ventral horn by in situ hybridization. In addition, fluorescence in situ hybridization combined with a retrograde labeling by using True Blue demonstrated ChAT mRNA positive neurons were exactly motoneurons. In the cord, putative presynaptic sympathetic neurons were also labeled.