Acetylcholine (ACh) has a crucial role in the peripheral and central nervous systems. The enzyme choline acetyltransferase (ChAT) is responsible for synthesizing ACh from acetyl-CoA and choline in the cytoplasm and the vesicular acetylcholine transporter (VAChT) uptakes the neurotransmitter into synaptic vesicles. Following depolarization, ACh undergoes exocytosis reaching the synaptic cleft, where it can bind its receptors, including muscarinic and nicotinic receptors. ACh present at the synaptic cleft is promptly hydrolyzed by the enzyme acetylcholinesterase (AChE), forming acetate and choline, which is recycled into the presynaptic nerve terminal by the high-affinity choline transporter (CHT1). Cholinergic neurons located in the basal forebrain, including the neurons that form the nucleus basalis of Meynert, are severely lost in Alzheimer's disease (AD). AD is the most ordinary cause of dementia affecting 25 million people worldwide. The hallmarks of the disease are the accumulation of neurofibrillary tangles and amyloid plaques. However, there is no real correlation between levels of cortical plaques and AD-related cognitive impairment. Nevertheless, synaptic loss is the principal correlate of disease progression and loss of cholinergic neurons contributes to memory and attention deficits. Thus, drugs that act on the cholinergic system represent a promising option to treat AD patients.

/pdf/alzheimer-s-disease-targeting-the-cholinergic-system-4xmxqwus4t.pdf

Alzheimer's disease: Targeting the Cholinergic System

Organizational and activational effects of sex steroids on brain and behavior: A reanalysis

Alzheimer’s disease and the basal forebrain cholinergic system: relations to β-amyloid peptides, cognition, and treatment strategies

Oxidative stress: Free radical production in neural degeneration

Pathological changes in the nucleus of meynert in Alzheimer's and Parkinson's diseases

The understanding of the synthesis of acetylcholine (ACh) in cholinergic neurons developed steadily during the 1970s and early 1980s and major progress has been achieved in the investigation of most of its aspects. The discovery and characterization of choline carriers in neuronal membranes, the clarification of the origin of acetyl groups of ACh, the elucidation of the significance of axonal transport for the function of cholinergic nerve terminals, the purification of the synthesizing enzyme, choline acetyltransferase (ChAT), and the production of monoclonal antibodies against it (enabling investigators to identify cholinergic neurons throughout the CNS) are examples of important achievements in this area of research. The interest in the synthesis of ACh and the factors that control or affect it was much stimulated by the discovery that an impairment in the production of ACh plays a crucial role in the pathogenesis of Alzheimer’s disease. Problems of the synthesis of ACh including its regulation have been repeatedly reviewed and the reader is advised to turn to articles by MacIntosh and Collier (1976), Collier (1977), Haubrich and Chippendale (1977), Jope (1979), Jenden (1979a,b), and TuCek (1983a, 1984), and to books written or edited by Goldberg and Hanin (1976), Tutek (1978, 1979), Barbeau et al. (1979), Davis and Berger (1979), and Hanin and Goldberg (1982) for comprehensive summaries of experimental data; Table 1 in TuCek’s (1984) article lists additional reviews concerning specific aspects of the synthesis of ACh. The present article is a commentary or overview rather than a review. It has been written in the belief that time is ripe to summarize (and partly restate) what appears to be, in the author’s view and in the light of data available in mid-1984, the most likely model of how the synthesis of ACh is regulated. It is hoped that such an outline may be of some use to colleagues who became interested in the problems of ACh synthesis only recently and in a way marginally, mainly in connection with the attempts to unravel the pathogenesis and find a treatment for Alzheimer’s disease. Many statements in the article are necessarily of a generalizing and simplifying nature; more complete arguments may be found in TuCek (1984).

Regulation of acetylcholine synthesis in the brain.

1. The synthesis of acetylcholine (ACh) has been measured in homogenates of the sciatic nerve, normal and denervated extensor digitorum longus (e.d.l.) muscles, and central (innervated) and peripheral (non-innervated) parts of the diaphragm of the rat. The synthesis proceeded under conditions accepted as optimal for the activity of choline acetyltransferase (ChAT). In view of the finding that cardiac carnitine acetyltransferase (CarAT) is able to acetylate choline (White & Wu, 1973), the possible contribution of CarAT to the synthesis of ACh in the muscles was investigated by using bromoacetylcholine (BrACh) as an inhibitor of ChAT and bromoacetylcarnitine (BrACar) as an inhibitor of CarAT.2. BrACh at a concentration of 2 mum inhibited the synthesis of ACh in nerve homogenates by 98%, in the homogenates of normal e.d.l. muscles by 53%, in denervated e.d.l. muscles by less than 5%; in the central part of the diaphragm BrACh inhibited ACh synthesis by 65%, and in the peripheral part by 13%. Comparative inefficiency of BrACh in inhibiting the synthesis of ACh in muscle homogenates was not due to its inactivation; the inhibitory effect of BrACh on the neural synthesis of ACh was preserved in the presence of muscle homogenates.3. BrACar at a concentration of 20 mum inhibited the synthesis of ACh in homogenates of the nerve by 18%, in those of normal e.d.l. muscles by 67%, in denervated e.d.l. muscles by 90%; in the central part of the diaphragm it inhibited the synthesis by 29%, and in the peripheral part by 76%.4. The inhibitory effects of BrACh and BrACar on the synthesis of ACh in muscle homogenates were roughly additive.5. Within 2 days of transection of the sciatic nerve, the BrACh-sensitive synthesis of ACh in the e.d.l. muscle diminished by 28%, whereas the BrACh-insensitive synthesis of ACh did not change. At 4 days after denervation, the rate of BrACh-sensitive synthesis decreased to 3% of control values.6. The results indicate that at least two enzymes are responsible for the synthesis of ACh in muscle homogenates is probably catalysed by CarAT. associated with intramuscular nerves, and probably corresponds to ChAT. The other enzyme is comparatively insensitive to BrACh, is sensitive to BrACar, and is probably localized in the muscle fibres. The BrACh-insensitive and BrACar-sensitive synthesis of ACh in muscle homogenates is probably catalysed by CarAT.7. Under the conditions used in the present experiments, CarAT was responsible for approximately one half of the synthesis of ACh in the homogenates of innervated e.d.l. muscles and for all ACh synthesized after denervation.8. The results provide an explanation for earlier findings of residual ACh synthesis in homogenates of denervated muscles, without resort to the idea that ChAT is localized in muscle fibres. It is proposed that CarAT catalyses some synthesis of ACh also in intact muscles, and that it is responsible for the synthesis of ACh observed during incubation of whole denervated muscles. It is not clear what is the physiological function of the synthesis of ACh catalysed by CarAT.9. Measurements of the BrACh-sensitive portion of the total ACh-synthesizing capacity of muscle homogenates provide a suitable procedure for obtaining information about the activity of neural ChAT in the muscles.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1982.sp014022

The synthesis of acetylcholine in skeletal muscles of the rat

Problems in the organization and control of acetylcholine synthesis in brain neurons.

Slices of rat caudate nuclei were incubated in saline media containing choline, paraoxon, unlabelled glucose, and [1,5-14C] citrate, [1-14C-acetyl]carnitine, [1-14C]acetate, [2-14C]pyruvate, or [U-14C]glucose. The synthesis of acetyl-labelled acetylcholine (ACh) was compared with the total synthesis of ACh. When related to the utilization of unlabelled glucose (responsible for the formation of unlabelled ACh), the utilization of labelled substrates for the synthesis of the acetyl moiety of ACh was found to decrease in the following order: [2-14C]pyruvate greater than [U-14C]glucose greater than [1-14C-acetyl]carnitine greater than [1,5-14C]citrate greater than [1-14C]acetate. The utilization of [1,5-14C]citrate and [1-14C]acetate for the synthesis of [14C]ACh was low, although it was apparent from the formation of 14CO2 and 14C-labelled lipid that the substrates entered the cells and were metabolized. The utilization of [1,5-14C]citrate for the synthesis of [14C]ACh was higher when the incubation was performed in a medium without calcium (with EGTA); that of glucose did not change, whereas the utilization of other substrates for the synthesis of ACh decreased. The results indicate that earlier (indirect) evidence led to an underestimation of acetylcarnitine as a potential source of acetyl groups for the synthesis of ACh in mammalian brian; they do not support (but do not disprove) the view that citrate is the main carrier of acetyl groups from the intramitochondrial acetyl-CoA to the extramitochondrial space in cerebral cholinergic neurons.

Utilization of Citrate, Acetylcarnitine, Acetate, Pyruvate and Glucose for the Synthesis of Acetylcholine in Rat Brain Slices

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1-14C]acetate, [2-14C]acetate, [4-14C]acetoacetate, [1-14C]butyrate, [1, 5-14C]citrate, [2-14C]glucose, [5-14C]glutamate, 3-hydroxy[3-14C]butyrate, [2-14C]lactate, [U-14C]leucine, [2-14C]pyruvate and [3H]acetylaspartate.

The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2-14C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5-14C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh.

The incorporation of label from [1, 5-14C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2-14C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1-14C]acetate into brain lipids and lowered its incorporation into ACh.

Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1-14C]acetate, [2-14C]acetate, [1-14C]butyrate, [1,5-14C]citrate, [3H]acetylaspartate, [U-14C]leucine, and also after [2-14C]pyruvate and [4-14C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected [2-14C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio.

The incorporation of 14C from [1-14C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1-14C]acetate than after [2-14C]pyruvate.

The results indicate that the [1-14C]acetyl-CoA arising from [1-14C]acetate does not enter the same pool as the [1-14C]acetyl-CoA arising from [2-14C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1-14C]acetate was different from that after [1, 5-14C]citrate. This suggests that [1-14C]acetate and [1, 5-14C]citrate are utilized in different subdivisions of the ‘;small’ compartment.

Stanislav Tuček

Papers

Regulation of acetylcholine synthesis in the brain.

The synthesis of acetylcholine in skeletal muscles of the rat

Problems in the organization and control of acetylcholine synthesis in brain neurons.

Utilization of Citrate, Acetylcarnitine, Acetate, Pyruvate and Glucose for the Synthesis of Acetylcholine in Rat Brain Slices

Provenance of the acetyl group of acetylcholine and compartmentation of acetyl-CoA and Krebs cycle intermediates in the brain in vivo.