Choline acetyltransferase ChAT

If a substance is to be a NT it should be possible to demonstrate appropriate enzymes for its synthesis from a precursor at its site of action, although peptides are transported to their sites of location and action after synthesis in the axon or distal neuronal cell body. The specificity of any enzyme system must also be established, especially if they are to be modified to manipulate the levels of a particular NT, or used as markers for it. Thus choline acetyltransferase (ChAT) may be taken as indicative of ACh and glutamic acid decarboxylase (GAD) of GABA but some of the synthesising enzymes for the monoamines lack such specificity. 

The reaction of choline with mitochondrial bound acetylcoenzyme A is catalysed by the cytoplasmic enzyme choline acetyltransferase (ChAT) (see Fig. 6.1). ChAT itelf is synthesised in the rough endoplasmic reticulum of the cell body and transported to the axon terminal. Although the precise location of the synthesis of ACh is uncertain most of that formed is stored in vesicles. It appears that while ChAT is not saturated with either acetyl-CoA or choline its synthesising activity is limited by the actual availability of choline, i.e. its uptake into the nerve terminal. No inhibitors of ChAT itself have been developed but the rate of synthesis of ACh can, however, be inhibited by drugs like hemicholinium or triethylcholine, which compete for choline uptake into the nerve. 

Acetylcholine is formed from acetyl CoA (produced as a byproduct of the citric acid and glycolytic pathways) and choline (component of membrane lipids) by the enzyme choline acetyltransferase (ChAT). Following release it is degraded in the extracellular space by the enzyme acetylcholinesterase (AChE) to acetate and choline. The formation of acetylcholine is limited by the intracellular concentration of choline, which is determined by the (re)uptake of choline into the nerve ending (Taylor Brown, 1994). 

Acetylcholine synthesis and neurotransmission requires normal functioning of two active transport mechanisms. Choline acetyltransferase (ChAT) is the enzyme responsible for ACh synthesis from the precursor molecules acetyl coenzyme A and choline. ChAT is the neurochemical phenotype used to define cholinergic neurons although ChAT is present in cell bodies, it is concentrated in cholinergic terminals. The ability of ChAT to produce ACh is critically dependent on an adequate level of choline. Cholinergic neurons possess a high-affinity choline uptake mechanism referred to as the choline transporter (ChT in Fig. 5.1). The choline transporter can be blocked by the molecule hemicholinium-3. Blockade of the choline transporter by hemicholinium-3 decreases ACh release,... 

ACh was first proposed as a mediator of cellular function by Hunt in 1907, and in 1914 Dale [2] pointed out that its action closely mimicked the response of parasympathetic nerve stimulation (see Ch. 10). Loewi, in 1921, provided clear evidence for ACh release by nerve stimulation. Separate receptors that explained the variety of actions of ACh became apparent in Dale s early experiments [2]. The nicotinic ACh receptor was the first transmitter receptor to be purified and to have its primary structure determined [3, 4]. The primary structures of most subtypes of both nicotinic and muscarinic receptors, the cholinesterases (ChE), choline acetyltransferase (ChAT), the choline and ACh transporters have been ascertained. Three-dimensional structures for several of these proteins or surrogates within the same protein family are also known. 

Schematic illustration of a generalized cholinergic junction (not to scale). Choline is transported into the presynaptic nerve terminal by a sodium-dependent choline transporter (CHT). This transporter can be inhibited by hemicholinium drugs. In the cytoplasm, acetylcholine is synthesized from choline and acetyl -A (AcCoA) by the enzyme choline acetyltransferase (ChAT). Acetylcholine is then transported into the storage vesicle by a second carrier, the vesicle-associated transporter (VAT), which can be inhibited by vesamicol. Peptides (P), adenosine triphosphate (ATP), and proteoglycan are also stored in the vesicle. Release of transmitter occurs when voltage-sensitive calcium channels in the terminal membrane are opened, allowing an influx of calcium. The resulting increase in intracellular calcium causes fusion of vesicles with the surface membrane and exocytotic expulsion of acetylcholine and cotransmitters into the junctional cleft (see text). This step can he blocked by botulinum toxin. Acetylcholine s action is terminated by metabolism by the enzyme acetylcholinesterase. Receptors on the presynaptic nerve ending modulate transmitter release. SNAPs, synaptosome-associated proteins VAMPs, vesicle-associated membrane proteins.

The activation event Acetylcholine is synthesized from choline and acetyl coenzyme A (Acetyl-CoA) by the enzyme choline acetyltransferase (ChAT) and is immediately stored in small vesicular compartments closely attached to the cytoplasmic side of the presynaptic membranes. 

Fukuyama Y, Okamoto K, Kubo Y, Shida N, Kodama M (1994) New Chlorine-Containing Prenylated C6—C-j Compounds Increasing Choline Acetyltransferase (ChAT) Activity in Culture of Postnatal Rat Septal Neurons from Illicium tashiroi. Chem Pharm Bull 42 2199... 

Another conceivable therapeutic approach for Alzheimer s disease could be based on the administration of neurotrophic factors. Nerve growth factor (NGF) is a 118 amino acid polypeptide with no blood-brain barrier penetrance. Other substances with neurotrophic activity such as epidermal growth factor, brain-derived neurotrophic factor, gangliosides, and the (11-28 peptide of the b-amyloid protein might also have a therapeutic potential. Intracerebroventricular (ICV) administration of NGF has been shown to partially reverse lesion-induced deficits of cortical AChE and choline acetyltransferase (CHAT) activities to promote survival of septal cholinergic neurons after fimbrial transection in adult rats and to reverse behavioral deterioration in rats with such lesions. 

Figure 17-6 Ordered synthesis of acetylcholine (ACh) by choline acetyltransferase (ChAT).

Catalpol. Zhang et al. [233] studied the neuroprotective effects of catalpol, an iridoid glycoside isolated from the fresh rehmannia roots, on the cholinergic system and inflammatory cytokines in the senescent mouse brain induced by D-galactose. Acetylcholinesterase (AChE) activity increased in senescent mouse brain and choline acetyltransferase (ChAT) decreased in the basal forebrain of senescent mouse. Muscarinic acetylcholine receptor Ml (mAChRl) expression declined and the levels of tumor necrosis factor (TNF-a), interleukin-ip (IL-ip), and advanced glycation end products... 

Neuromodulatory transmitter inputs to MOB. Darkfield photomicrographs showing the distribution of cholinergic (a), noradrenergic (b), and serotonergic (c) fibers revealed respectively with immunohistochemistry for choline acetyltransferase (ChAT), dopamine-jS-hydroxylase (DBH), and serotonin (5-HT). Reprinted from Handbook of Chem. Neuroanat. Integrated Sys. CNS, Vol. 12, Part III, Chapter III, The Olfactory System, M. Shipley et al., pp. 469-573, 1996, with permission from Elsevier, Ltd... 

Fig. 2 Acetylcholine production and metabolism. (A) acetylcholine production [by choline acetyltransferase (ChAT)] and (B) breakdown of acetylcholine [by acetylcholinesterases (AChE)]...

Juarez de Ku LM, Sharma-Stokkermans M, Meserve LA. 1994. Thyroxine normalizes polychlorinated biphenyl (PCB) dose-related depression of choline acetyltransferase (ChAT) activity in hippocampus and basal forebrain of 15-day-old rats. Toxicology 94 19-30. 

Provost TL, Juarez De Ku LM, Zender C, et al. 1999. Dose- and age-dependent alterations in choline acetyltransferase (ChAT) activity, learning and memory, and thyroid hormones in 15- and 30-day old rats exposed to 1.25 or 12.5 ppm polychlorinated biphenyl (PCB) beginning at conception. Prog Neuro-Psychopharmacol Biol Psychiat 23 915-928. 

Choline uptake is inhibited by hemicholinium ( in Figure II-2-1). ACh is synthesized from choline and acetyl-CoA via choline acetyltransferase (ChAT) and accumulated in synaptic vesicles. 

Biochemical measurement of distinct levels of acetylcholine (McIntosh, 1941 Kasa et al., 1982) and its biosynthetic enzyme, choline acetyltransferase (ChAT) in cerebellar tissue (Kasa and Silver, 1969 Salvaterra and Foders, 1979 Hayashi, 1987 and others) indicated the presence of a cholinergic innervation in the cerebellum. ChAT activity varies among different lobules with the highest levels in the nodulus and ventral uvula. Following deafferentation of the cerebellar cortex, ChAT activity is considerably de-... 

Fig. 84. Illustrations of choline-acetyltransferase (ChAT)-like immunoreactivity in the rabbit cerebellum. A. Sagittal view of the rabbit cerebellum delineating the lobules according to Larsell (Larsell, 1970). Mean measurements of ChAT activity are indicated by numbers in parentheses. B. Magnified view of the ventral vermis. The vermis contains areas of ChAT-positive mossy fiber terminals (indicated by dots). These areas in lobules 1 and 9d are illustrated in C and D, respectively. E. View of the right paraflocculus of the rabbit. ChAT-like immunoreactivity and ChAT activity was highest in the ventral paraflocculus, particularly lobule 2. The numbers in parentheses are mean measurements of ChAT activity, expressed as mmol of Ach synthe-sized/hr. g tissue at 37°C, for each cerebellar lobule in six rabbits. Barmack et al. (1992a).

Fig. 85. A. Drawing displaying the distribution of mossy rosettes (dots) immunoreactive to monoclonal choline-acetyltransferase (ChAT) antibody. The section (40 fim thick) was cut sagittally through the middle vermis of rat cerebellum. A considerable number of immunoreactive mossy terminals are observed in lobules I through IXab, although they are much fewer than in lobules IXc and X. Calibration bar = 1 mm. B. Drawing of part of lobule IXab shows the overall distribution of immunoreactive fibers. Arrows indicate mossy fibers with glomerular rosettes. Small and large arrowheads point to some varicose fibers distributing in or near the Purkinje cell layer (PCL) and in the molecular layer (ML), respectively. The ML fibers are most frequently observed in this lobule and tend to be restricted to the inner half of the layer. Calibration bar = 200 jum. Ojima et al. (1989).

Fig. 86. A. Distribution and density of choline-acetyltransferase (ChAT)-immunoreactive Golgi cells (dots) in two subsequent 100 //m thick parasagittal sections through the vermis of the cerebellar of the cat. Roman numbers indicate lobules according to Larsell pcs, pedunculus cerebellaris superior. Scale bar = 2 mm. B and C. Drawings of immunoreactive Golgi cells from 100 //m thick cerebellar sections. The uppermost cell is from the hemisphere, the lower one from the dorsal vermis. The arrows point to processes thought to be axons. ML, molecular layer GL, granular layer. The border of gray to white matter is marked by a dashed line. Tiling (1990).

Boutons immunoreactive for choline acetyltransferase (ChAT) make synaptic contacts with striatal spiny neurons as well as other striatal cells (Izzo and Bolam 1988). The cholinergic synapses are symmetric and make contact with the cell somata (20%) dendritic shafts (45%) and with dendritic spines (34%). As with the other symmetrical synapses on dendritic spines, these share the spine with an asymmetrical synapse, usually placed more distally on the spine, similar to afferents from the cerebral cortex and thalamus. 

Fig. 10. A) Diagram of a large aspiny striatal neuron that had been intracellularly filled to reveal its dendrites (black) and axon collateral that arborizes within the striatum (gray). From Wilson et al., 1990. B) Distribution of large aspiny striatal neurons labeled with choline acetyltransferase (ChAT) immunoreactivity in a coronal section of the striatum. The patch compartment was labeled in an adjacent section with calbindin immunoreactivity.

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