Activity-Dependent Regulation of Neurotransmitter Synthesis
ACTIVITY-DEPENDENT REGULATION OF NEUROTRANSMITTER SYNTHESIS
Activity-dependent regulation of neurotransmitter synthesis refers to the ability of some nerve cells to change the amount of neurotransmitter synthesized in response to activity. Study of this regulation is prompted by the belief that it is important not only for maintaining a source of neurotransmitter but also for adaptive changes that take place in certain nerve cells during learning and memory. A basic postulate necessary for neurotransmitter synthesis regulation to be a mechanism for learning and memory is that increased neurotransmitter synthesis acts to increase neurotransmitter secretion and, as a consequence, synaptic strength. It has not been technically possible thus far to demonstrate a causal relationship between activity-dependent regulation of neurotransmitter synthesis and an increase in neurotransmitter secretion. Nonetheless, activity-dependent regulation of neurotransmitter synthesis remains a candidate for the cause of neuroplastic changes that underlie learning, memory, and neuroplasticity. Neurotransmitters that have shown activity-dependent regulation of their biosynthesis are acetylcholine, dopamine, and norepinephrine. This entry reviews the mechanisms of the synthesis of these three neurotransmitters and possible roles of their regulatory mechanisms in learning and memory.
Depending upon both the type of nerve cell and the time scale over which adaptation occurs, the cellular and biochemical mechanisms responsible for activity-dependent regulation of neurotransmitter synthesis vary. The time scale of changes ranges from very rapid changes (seconds), in which covalent modification of enzyme protein structure is involved, to more delayed, longer-term changes (days). The latter involve alterations in genetic expression and turnover of enzymes responsible for neurotransmitter biosynthesis. Catecholamines are regulated at both short-and long-term levels, while acetylcholine is regulated only at a short-term level. The regulatory mechanisms are often similar to cellular and biochemical mechanisms used in nonneural cells to regulate the synthesis of hormones or to regulate the biochemical pathways of intermediary metabolism.
Acetylcholine is the neurotransmitter in the autonomic nervous system, the central nervous system (CNS), and at the neuromuscular junction. Even though acetylcholine has one of the highest rates of turnover of any neurotransmitter, its concentration within nerve tissue fluctuates very little. This is because the precursors for acetylcholine synthesis, acetylcoenzyme A and choline, exist in a steady-state equilibrium with choline acetyltransferase (CAT), the enzyme that catalyzes acetylcholine synthesis (Jope, 1979). Consequently, acetylcholine synthesis is well below its maximal possible rate. Therefore any change in the concentration of acetylcholine or its precursors produces a change in acetylcholine synthesis. This has been verified by the demonstration that the transport of choline into the cholinergic neuron controls acetylcholine synthesis. For example, choline addition to slices of brain tissue markedly increases the acetylcholine synthesis rate. In addition, increased neuronal activity increases choline uptake (Simon and Kuhar, 1975). This increased uptake occurs in a manner that persists far beyond the period of increased activity. Thus, stimulation of choline up-take is not due merely to a shift in the equilibrium of the CAT-catalyzed reaction; instead, choline uptake is regulated by neural activity per se. This regulation of choline uptake by nerve activity is predicted to maintain the strength of cholinergic synapses and is a candidate to increase the capacity of these synapses to secrete acetylcholine. Despite this evidence for activity-dependent regulation of choline uptake, little is known concerning how this regulation occurs. Thus, the link between nerve activity and choline transport remains unknown.
Catecholamines—dopamine, norepinephrine, and epinephrine—are neurotransmitters in the sympathetic limb of the autonomic nervous system and in several groups of neurons in the CNS. In contrast with the lack of a mechanism linking nerve activity and the regulation of choline uptake, catecholamine-synthesizing cells have several mechanisms in place to regulate catecholamine levels in response to nerve activity, both in peripheral and central nervous systems and at short-and long-term levels. As one review stated, "An intricate scheme has evolved whereby tyrosine hydroxylase activity is modulated by nearly every documented form of regulation" (Kumer and Vrana, 1996). Regulation occurs at the step in catecholamines synthesis where tyrosine is hydroxylated to form L-dopa. The enzyme catalyzing this reaction, tyrosine hydroxylase, is the first of four enzymatic steps in the catecholamine synthesis pathway. Because tyrosine hydroxylase is present in lower concentration than the other enzymes of the synthesis pathway, it restricts the amount of neurotransmitter synthesized. In addition, tyrosine hydroxylase is constitutively inhibited by the binding of a mole of catecholamine to each mole of enzyme. Two interacting mechanisms are important in short-term, nervous-activity regulation of catecholamine synthesis. One mechanism is catecholamine end-product inhibition of tyrosine hydroxylase activity by the catecholamine products of the pathway. The second is modification of tyrosine hydroxylase structure by the placement of phosphate groups on the tyrosine hydroxylase molecule. The latter process, termed phosphorylation, is catalyzed by at least four separate protein kinases (Kumer and Vrana, 1996), which phosphorylate tyrosine hydroxylase on a combination of three serine residues in the N terminal domain of the enzyme. The phosphorylation alters the properties of tyrosine hydroxylase, increasing its catalytic activity. Phosphorylation is commonly used to modify proteins involved in regulation.
The protein kinases that phosphorylate tyrosine hydroxylase are cyclic adenosine monophosphate-dependent protein kinase (PKA), which phosphorylates serine 40; calcium-calmodulin-dependent protein kinase II (CaM KII), which phosphorylates serine 19; and extracellular receptor activated protein kinase (ERK) (Haycock, Ahn, Cobb, and Krebs, 1992), which phosphorylates serine 31. This phosphorylation has three major effects on the enzyme's function; all three changes enhance tyrosine hydroxylase activity and increase catecholamine synthesis. Serine 40 phosphorylation increases the affinity of the enzyme for tetrahydrobiopterin cofactor (the cofactor is normally present below optimal concentrations) and reduces the catecholamine binding and inhibition of tyrosine hydroxylase (Daubner, Lauriano, Haycock, and Fitzpatrick, 1992). Serine 19 phosphorylation causes the enzyme to interact with an activating protein termed 14-3-3 protein (Ichimura et al., 1987; Itagaki et al., 1999). Serine 31 phosphorylation increases the catalytic activity by an as yet undefined mechanism (Haycock, Ahn, Cobb, and Krebs, 1992).
Which of these mechanisms act to regulate the synthesis of catecholamines in response to neural activity, and how may they relate to learning and memory? Although these questions have been difficult to answer, some conclusions are possible. Under circumstances of cell depolarization, such as when a nerve impulse invades the nerve terminal or during cholinergic stimulation at the adrenal medulla, the phosphorylation and activation of tyrosine hydroxylase by Ca/CAM kinase II appear to be responsible for initial activation of tyrosine hydroxylase (Waymire et al., 1988; Waymire and Craviso, 1993) with a later activation through both PKA and ERK. In addition, evidence indicates that the phosphorylation on serine 19 may facilitate the phosphorylation on serine 40 (Bevilaqua et al., 2001). Thus there appears to be a time-dependent hierarchical phosphorylation and activation of tyrosine hydroxylase that occurs in steps to activate catecholamine biosynthesis. Any condition that increases the size of these phosphorylation events is predicted to enhance the synthesis of catecholamines. This may translate into increased neurotransmitter release and synaptic strength.
Catecholamine synthesis is also regulated at a chronic, long-term level in response to persistent or extreme neural activation. In this case the amount of the enzymes in the catecholamine synthetic pathway, and especially tyrosine hydroxylase, is elevated in response to increased synaptic activity. For example, drugs or conditions such as stress that increase the autonomic nerve activity increase the level of tyrosine hydroxylase in peripheral autonomic cells (Thoenen, Mueller, and Axelrod, 1969). Because cutting the innervation to these cells blocks the increase, the influence is thought to be transynaptic. Because a rise in tyrosine hydroxylase mRNA precedes the increase in protein, the mechanism is believed to be increased transcription of the mRNA encoding tyrosine hydroxylase. The observation that drugs that increase CNS neuronal activity also induce increased levels of CNS tyrosine hydroxylase shows that central tyrosine hydroxylase levels are also regulated by neuronal activity. This nerve activity-dependent regulation of tyrosine hydroxylase synthesis is an attractive candidate for learning and memory because the increase in tyrosine hydroxylase level is expected to increase the strength of the activated synapses. Whether or not this is the case is not yet known. Even so, a considerable effort is being carried out to understand as much as possible about transynaptic regulation of tyrosine hydroxylase level because it is likely the best example known of synaptically mediated regulation of protein synthesis.
Among the issues that remain unresolved concerning the mechanism of the long-term regulation of tyrosine hydroxylase is the nature of the intracellular mechanisms responsible for increased transcription. In a model tissue, the adrenal medulla, acetylcholine and pituitary adenylyl cyclase activating polypeptide (PACAP) are each able to modulate tyrosine hydroxylase level. In this tissue either PACAP or acetylcholine stimulates cAMP level and activates PKA. One hypothesis is that PKA migrates to the cell nucleus to regulate the rate of tyrosine hydroxylase transcription by phosphorylating CREB (Cyclic AMP Response Element Binding), a regulatory protein associated with the tyrosine hydroxylase gene (Kurosawa, Guidotti, and Costa, 1976). Unresolved issues in this simple model are 1. whether acetylcholine stimulates a rise in cAMP or 2. whether other transmitters, such as PACAP, are involved. Because neuropeptides are secreted along with acetylcholine at some cholinergic synapses, it has been suggested that these agonists are responsible for long-term regulation of tyrosine hydroxylase level (Wessels-Reiker, Haycock, Howlett, and Strong, 1991). Also, it is not clear whether transcription is regulated solely through PKA. Because several regulatory domains exist in the tyrosine hydroxylase gene, it appears that protein kinases other than the PKA are likely to be involved. In addition it is possible that the rapidly synthesized protein c-Fos may serve as a protein factor regulating tyrosine hydroxylase transcription. An additional question is whether increased transcription is the principal mechanism of regulation. In isolated adrenal medullary chromaffin cells, transcription increases for only a few hours following either acetylcholine of PACAP stimulation, whereas the increase in mRNA occurs over several days and remains elevated long after transcription has subsided. This raises the issue of the stabilization of the tyrosine hydroxylase mRNA as a major component of the synaptic regulation. Indeed, tyrosine hydroxylase mRNA is capable of regulation through stability, as has been demonstrated for its stabilization by elevated oxygen tension (Paulding and Czyzyk-Krzeska, 1999).
The understanding of the activity-dependent regulation of catecholamines is much more complete than for other neurotransmitters, such as acetylcholine. For some neurotransmitter systems—the amino acids and purines, for example—almost nothing is known about their synthesis regulation, so it is not clear whether it is activity-dependent. This is primarily because these compounds are so intimately associated with intermediary metabolism that it is difficult to separate their neurotransmitter-related metabolism from that associated with general cell function. One generalization emerging from the studies of the mechanisms of activity-dependent regulation of catecholamine synthesis is the prominent position protein phosphorylation plays in both short-and long-term regulation. In the future, studies will likely be directed to applying the understanding being gained of the mechanisms regulating catecholamine synthesis to other neurotransmitters. And although it is important to continue to investigate the mechanisms involved in activity-dependent regulation of neurotransmitter synthesis, it is also important to recognize that the role of neurotransmitter synthesis regulation in higher functions, such as learning and memory, is still hypothetical.
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