Modern views on an ancient chemical: serotonin effects on cell proliferation, maturation, and apoptosis

Brain Research Bulletin, Vol. 56, No. 5, pp. 413– 424, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/01/$–see front matter

PII S0361-9230(01)00614-1

Modern views on an ancient chemical: Serotonin effects on cell proliferation, maturation, and apoptosis Efrain C. Azmitia1,2* Departments of 1Biology and 2Psychiatry, Center for Neural Science, New York University, New York, NY, USA ABSTRACT: Evolutionarily, serotonin existed in plants even before the appearance of animals. Indeed, serotonin may be tied to the evolution of life itself, particularly through the role of tryptophan, its precursor molecule. Tryptophan is an indolebased, essential amino acid which is unique in its light-absorbing properties. In plants, tryptophan-based compounds capture light energy for use in metabolism of glucose and the generation of oxygen and reduced cofactors. Tryptophan, oxygen, and reduced cofactors combine to form serotonin. Serotonin-like molecules direct the growth of light-capturing structures towards the source of light. This morphogenic property also occurs in animal cells, in which serotonin alters the cytoskeleton of cells and thus influences the formation of contacts. In addition, serotonin regulates cell proliferation, migration and maturation in a variety of cell types, including lung, kidney, endothelial cells, mast cells, neurons and astrocytes). In brain, serotonin has interactions with seven families of receptors, numbering at least 14 distinct proteins. Of these, two receptors are important for the purposes of this review. These are the 5-HT1A and 5-HT2A receptors, which in fact have opposing functions in a variety of cellular and behavioral processes. The 5-HT1A receptor develops early in the CNS and is associated with secretion of S-100␤ from astrocytes and reduction of cAMP levels in neurons. These actions provide intracellular stability for the cytoskeleton and result in cell differentiation and cessation of proliferation. Clinically, 5-HT1A receptor drugs decrease brain activity and act as anxiolytics. The 5-HT2A receptor develops more slowly and is associated with glycogenolysis in astrocytes and increased Caⴙⴙ availability in neurons. These actions destabilize the internal cytoskeleton and result in cell proliferation, synaptogenesis, and apoptosis. In humans, 5-HT2A receptor drugs produce hallucinations. The dynamic interactions between the 5-HT1A and 5-HT2A receptors and the cytoskeleton may provide important insights into the etiology of brain disorders and provide novel strategies for their treatment. © 2001 Elsevier Science Inc.

exact midline of the brainstem. Serotonergic fibers interact in complex ways with a variety of cell types—neurons, glial cells, endothelial cells, ependymal cells and others— by binding to at least 14 distinct receptor proteins. Furthermore, serotonin neurons are one of the first brainstem neurons to emerge during early development of the brain and spinal cord—present by the sixth week of gestation in humans. In rats, 5-hydroxytryptamine (5-HT) neurons in the brainstem raphe are among the first neurons to differentiate in the brain and play a key role in regulating neurogenesis [64]. The serotonin neurons are the first neuronal system to innervate the primordial cortical plate. During development, 5-HT fibers arrive at the cortical plate during the peak period of mitosis and maturation [42]. Lauder and Krebs [65] reported that parachlorophenylalanine (PCPA), a 5-HT synthesis inhibitor, retarded neuronal maturation, while mild stress, a releaser of hormones, accelerated neuronal differentiation. These workers defined differentiation as the cessation of cell division measured by incorporation of 3H-thymidine. Since then, many other workers have shown a role for serotonin in neuronal differentiation, (e.g., [54,74] and references contained in Whitaker-Azmitia, this issue). Certainly, all these facts suggest a critical role for serotonin in brain function, but is there really something distinct about serotonin, as a chemical? Serotonin is synthesized from tryptophan, which contains an indole ring and a carboxyl-amide side-chain, similar to all amino acids. The indole ring, however, is unique in that it is composed of both a benzene ring and a secondary pentane ring having a central nitrogen. The indole ring, and therefore tryptophan itself, is capable of absorbing light. In plants, tryptophan produces receptor proteins which harness light and thus produce biologically important molecules [61]. Chlorophyll, for example, captures light because it contains tryptophan, and then generates ATP, reduced cofactors (NADH), and oxygen. This entire process is blocked if tryptophan is substituted with another amino acid [84]. Furthermore, in plants tryptophan itself is converted into the tropic factor auxin, by removing the amide group to make indole-acetic acid. Auxin stimulates changes in cell shape and provides movement for plants. The position of the leaves is regulated by auxin, in order that they face the source of light energy, normally the sun (Fig. 1). Thus tryptophan plays a role in capturing energy and in the positioning of the plant to maximize light absorption. This biosynthetic interaction between tryptophan and light may be maintained throughout evolution. For example, in the mammalian brain, se-

KEY WORDS: 5-HT1A, 5-HT2A, Receptor, Cytoskeleton, Protein kinase C (PKC), S-100␤, Astrocytes, BrdU.

INTRODUCTION Serotonin has been implicated in more behaviors, physiological mechanisms, and disease processes than any other brain neurotransmitter. The enormous range of this single brain chemical system may reflect the vast distribution of its fibers in brain, from a small group of large multipolar neurons. The neurons form a collection of clustered cells termed the raphe nuclei, located on the

* Address for correspondence: Efrain C. Azmitia, Ph.D., 10-09 Main Building, 100 Washington Square East, New York, NY 10003, USA. E-mail: [email protected]

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AZMITIA TABLE 1 DIFFERENCES BETWEEN THE 5-HT1A AND 5-HT2A RECEPTORS

Receptor

5-HT1A 5-HT2A

Affinity

Kinase

Glial

Activity

High 10⫺9 M Low 10⫺6 M

2 PKA

1 S-100␤ 1 Glucose

2 IPSP 1 EPSP NMDA

1 PKC

Shape

Mitosis

Apop

Clinical

1 Diff

2

2

Anxiolytic

2 MT 1Synapse

1

1

Hallucination

Affinity values are those reported in mammalian membrane preparations. The changes in phosphorylation (Kinase) is derived from the inhibitory affect of 5-HT1A receptor on adenylate cyclase and the stimulatory affect of the 5-HT2A receptor on Phospholipase-C. PKA depends on cAMP and PKC depends on Ca⫹⫹. Glial cell actions are in response to the actions of the receptors on cultured glial cells. Activity is the most common change in the electirical activity of a postsynaptic neuron as measured in mammalian brain. In the case of the 5-HT2A receptor, there is evidence for potentiation of the excitatory effects of glutamate. The shape column summarizes the overall affect due to actions on the microtubules (MT), the backbone of the cytoskeletal system. Note the increase in differentiation (Diff) by the 5-HT1A receptor and in synapse formation by 5-HT2A receptors. Cell proliferation (Mitosis) is a measure of changes over several cell cycles. Apoptosis (Apop), programmed cell death, is measure of studies on neuronas and non-neuronal cells. Clinical column provides the affects on humans of drugs having specific actions on the receptor.

rotonin and melatonin, which are synthesized from tryptophan, act to entrain endogenous rhythms to the light cycle [79]. The effects of serotonin on morphology have long been known. For more than 50 years, serotonin has been known to constrict blood vessels (indeed, this is the origin of the name) [95] and induce shape changes in skeletal muscle (at both the light and electron microscope level) [93], platelets [69], endothelial cells [121], and fibroblast [28]. In the periphery, serotonin originates largely from mast cells, which can produce, release, and re-uptake serotonin. The released serotonin may then act as a chemotactic, increase vascular permeability, cause vasodilatation, and smooth muscle spasm [82]. In addition to its role in morphological changes, serotonin also has been shown to play a role in cell proliferation. In cultured rat pulmonary artery smooth muscle cells (SMC), serotonin induces DNA synthesis and potentiates the mitogenic effect of plateletderived growth factor-BB [45]. Serotonin effects on cell proliferation may involve the phosphorylation of GTPase-activating protein (GAP), an intermediate signal in serotonin-induced mitogenesis of SMC [68]. The biological mechanism used by serotonin to change cell morphology and induce proliferation may directly target the cytoskeleton. The main component of the cytoskeleton, which gives cells their shape, is microtubules. These microtubules consist of long polymers of tubulin, which spontaneously depolymerize if they are not actively polymerizing [83]. In 1975, Tan and Lagnado [116] found effects of serotonin and related indole alkaloids on brain microtubular proteins. Several years later, it was found that serotonin is taken up by endothelial cells and binds to stress fibers [7]. Here serotonin induces actin polymerization and affects changes in the cytoskeleton. Thus, there is evidence serotonin has a direct role in regulating and maintaining microtubules and microfilaments. The changes reported in serotonin-induced cytoskeletal stability may be partially mediated by microtubule-associated proteins (MAPs). MAPs serve to stabilize the cytoskeleton by binding to tubulin polymers and inhibiting their depolymerization. In undifferentiated human neuroblastoma cells (LAN-5), high levels of serotonin (50 uM) induce a decrease while low levels of serotonin (50 nM) induce an increase in the cytoplasmic tau protein, a MAP found in high concentrations in the axon of neurons [60]. Thus, there is evidence that serotonin is involved in a variety of cellular processes involved in regulating metabolism, proliferation and morphology. The fine integration of these dynamic events appears to involve multiple receptor action.

ADVENT OF RECEPTORS With time, cells developed crude receptor molecules to open ion channels and regulate intermediate metabolism within the cell by regulating cAMP and Ca⫹⫹ levels. Molecular biologist have sequenced over 50 distinct receptor proteins recognizing serotonin. In this chapter, we will discuss only two mammalian receptors in detail, the 5-HT1A and the 5-HT2A receptor. In the brain and spinal cord, serotonin acts on the 5-HT1A and 5-HT2A receptors to regulate neuronal morphology and apoptosis in the adult brain. Our results indicate that these two receptor systems can be viewed as opposing forces, both important for neuronal and mental functioning, but acting at opposite ends of the spectrum (see Table 1). 5-HT1A Receptors The 5-HT1A receptor develops early. It is a “transiently expressed” intronless receptor, that is, at specific times in development or during stress, very high amounts are expressed quickly. The 5-HT1A levels can then be reduced as the cells or animal ages. An early developmental peak in CNS receptor number occurs in rat [41], human [18], and sheep [104]. In human fetal tissue, the peak is found between 16 and 22 weeks of gestation [18]. In rat, the 5-HT1A receptor is first evident at GD12 in brainstem and increases to a peak at GD15, after which time it declines and is never as high again [56]. In regions maturing later, such as the cerebellum [41] and visual cortex [43], the receptor peak also occurs later. The decrease in receptor number is probably due to increased serotonin brain levels, since the 5-HT1A receptor expression is sensitive to autoinhibition [89,125]. The transduction action of the 5-HT1A receptor is usually associated with a decrease in adenylate cyclase activity (Fig. 2). In cultures of hippocampal neurons, 5-HT1A agonists block the forskolin-induced formation of p-CREB, an important transcription factor increased by c-AMP [89]. In adult neurons, the 5-HT1A receptor also is associated with a hyperpolarization of the membrane potential, attributed to opening a K⫹ current [19]. The 5-HT1A receptor uses these cellular mechanisms to differentiate its target cells. Serotonin inhibits cellular growth of pulmonary artery smooth muscle cells (SMC) through its action on 5-HT1A or 5-HT4 receptors [68]. In the kidney, epithelial cells in the ascending thin limb of the loop of Henle are transformed from a cuboidal to a squamous form with a corresponding disappearance of 5-HT1A immunostaining [63]. This loss of 5-HT1A receptor expression as differentiation occurs is seen in a variety of cell

5-HT1A AND 5-HT2 DYNAMICS

FIG. 1. The diagram illustrates the central role played by tryptophan in the evolution of light transduction into biological processes. Tryptophan is the only essentially amino acid, which is capable of the capture of light energy and is used in both chorphyl and rhodopsin for this purpose. Auxin is indole acetic acid formed from tryptophan and produces movement in plant leaves. It functions to move the leaves of plants to capture light energy. Reduced cofactors, such as NADH, and molecular oxygen are produced by photosynthesis, which is dependent on the capture of light. NADH and oxygen combine to form 5-hydroxytryptophan from tryptophan and then serotonin. The 5-HT1A receptor, estimated to be over a billion years old, evolved from the rhodopsin receptor.

types. In the serotonin cell line, RN46A, the 5-HT1A receptor is 20-fold higher in the undifferentiated cell than in the differentiated cell. The authors suggest that the cell body 5-HT1A receptors may mediate autoregulation of serotonin development [44]. Injection of pregnant rats with a 5-HT agonist results in newborn pups having lowered 5-HT1 receptor binding [125]. In adult brain, although glucocorticoids have been implicated in regulation of the 5-HT1A receptor mRNA expression, serotonin inhibitory feedback continues to play a major role [58]. Cultured fetal rat brain can bind 3H-8-OH-DPAT, a specific 5-HT1A receptor agonist [55]. Activation of this receptor in cultured hippocampal cells stimulates the expression of neuronal markers such as MAP-2 and synaptophysin [91]. In identified fetal cholinergic neurons in culture, 5-HT1A agonists increase dendritic length and branching [103]. Support for receptor autoregulation can also be seen in culture. Addition of 8-OH-DPAT and ipsapirone (also a 5-HT1A receptor agonist) decrease both 5-HT1A receptor mRNA and protein levels in hippocampal cultures [89]. Thus, as 5-HT1A receptors induce differentiation, their own expression appears to be stopped. This is consistent with a decreased role of the 5-HT1A receptor with age. In the intact animal, a negative correlation is reported between hippocampal 5-HT1A receptor binding site densities and age and a similar trend for 5-HT1A (but not 5-HT2A) receptor mRNA abundance [33].

415

FIG. 2. The reported transduction mechanism commonly reported for either the 5-HT1A receptor (left side of cell) and the 5-HT2A receptor (right side of cell) are illustrated. The actions of the 5-HT1A receptor favor electrical inactivity by hyperpolarization due to efflux of K⫹ and metabolic rest by reducing cAMP levels, which would reduce both the activity of protein kinase A (PKA) and the transcription factor pCREB. In contrast, the actions of the 5-HT2A receptor promote neuronal firing by enhancing sensitivity to glutamate AMPA receptors and increasing Ca⫹⫹ levels. The metabolic activity of the cell is greatly enhanced by Ca⫹⫹ increase, which leads to activation of protein kinase C (PKC) and the activation of several important transcription factors including c-fos, Jak, and STAT.

function of these receptors in the adult brain [78]. For example, both prenatal and postnatal stress to the mother significantly increases the number of 5-HT2 receptors in the offspring, even after

5-HT2A Receptors The 5-HT2 receptor-mediated production of inositol phosphates is approximately tenfold higher in the immature brain than in the adult brain [39]. The predominant 5-HT2 receptor in the neonatal period is the 5-HT2C receptor, while the 5-HT2A receptor becomes predominant as the animal ages [59]. The 5-HT2 receptor can be referred to as a programmable receptor—that is, events during development may affect the number, affinity, or

FIG. 3. The figure illustrates the actions of two receptors on astrocytes. On the left side, the 5-HT1A receptor induces the maturation of astrocytes and stimulates the release of S-100␤. On the right side, the 5-HT2A receptor promotes proliferation and enhances glycolysis to break down glycogen into glucose. During development, the 5-HT2A receptor has been localized to regions of intense cell proliferation (neural folds), while the 5-HT1A receptor is seen in regions where cells are terminally differentiating (cortical plate).

416

AZMITIA

they have become adults [2,101]. Several researchers have also suggested a trophic role for the 5-HT2A receptor, including actions on synaptogenesis [88]. Treatment from E11-F17 of chick embryos with the 5-HT2A receptor agonist (DOI) increases (20% above control) and an antagonist (ketanserin) decreases (30 – 40% below control) the synaptic density in lateral motor column of the spinal cord. The peak of the 5-HT2A receptor is earlier than the 5-HT2C receptor and the receptor is functional by postnatal day 7 in the rat hippocampus [59]. This time period is too late to influence differentiation, however the receptor may play a role in branching, terminal sprouting, synaptogenesis, mitogenesis, and glycogen breakdown (see section on neuronal dynamic instability [35]. Thus, the 5-HT2A receptor stimulates the plasticity and adaptability of the brain, by a process that is tightly regulated by receptor down-regulation. 5-HT2A receptors can change the intracellular Ca⫹⫹ levels and activate many of the phosphorylation mechanisms in the cell (Fig. 2). The effects of this receptor on Ca⫹⫹ levels are achieved from a variety of pools. For example, the contractile 5-HT2A receptor signal transduction in guinea pig trachea increases calcium influx through L-type voltage-dependent calcium channels, calcium release from the sarcoplasmic reticulum, and activation of a bisindolylmaleimide-sensitive PKC [120]. All of these sources contribute to the movement of the muscle fibrils. In addition, 5-HT2A receptors can increase cyclic AMP accumulation in the A1 neuronal cell line by protein kinase C-dependent and calcium/calmodulin-dependent mechanisms [22]. This would have the effect of increasing PKA activity. The activation of two major kinase enzymes in the cells has implications for most cellular mechanisms, many associated with the cytoskeleton and shape. 5-HT2A receptors activate both cFos and the apoptotic factor JAK/STAT [49]. The 5-HT-induced stimulation of endothelial actin cables, which produces changes in cell shape and movement, is blocked by the 5-HT2 antagonist ketanserin [121]. Serotonin stimulates stress fibers as much as 80%, and increases surface area by 40% in cultured endothelial cells, an effect also blocked by ketanserin [109]. Another 5-HT2 antagonist, cinanserin, blocks the effects of serotonin on platelet cell shape [69]. Arc (activity regulated, cytoskeleton associated protein) is an effector immediate early gene that is selectively localized in the neuronal dendrites. Expression of Arc mRNA is highly responsive to changes in brain 5-HT functions, and may provide a sensitive marker of postsynaptic 5-HT2 (2A and 2C) receptor functions. [99]. The precise role of these cytoskeletal changes may complement those produced by 5-HT1A receptor. ASTROGLIAL CELLS These cells are central to any discussion of plasticity within the brain, since they not only make glucose available to neurons, but also provide adhesion and trophic factors for neuronal growth and migration. Merzak and co-workers [81] found 5-HT1A receptor mRNA in human normal fetal astrocytes by reverse transcription and polymerase chain reaction (RT-PCR). Hirst and co-workers [57] also used reverse transcriptase-polymerase chain reaction to show the expression of 5-HT1A receptor in astrocytes derived from 2-day-old rats and cultured for 10 –12 days. 5-HT1A receptor immunocytochemical labeling is detected both in vivo [15,62,124] and in culture [66,89]. 5-HT1A receptors are present in newborn cultured glial cells [81]. 5-HT1A receptor mRNA is expressed in normal astrocytes from the left hemisphere and in six glioma cell lines, but not in normal astrocytes from the mature cerebellum. However, in the immature cerebellum positive immunocytochemical 5-HT1A receptor labeling of astrocytes is reported [76]. In the adult brain, Hillion and colleagues [56], using a specific 40-mer

biotin-labeled deoxyoligonucleotide complementary to the 5-HT1A receptor mRNA, detected no labeling in glial cells. Thus, as with the neuronal receptor, there is evidence that the highest levels of the receptor are seen in immature glial cells. 5-HT1A receptor agonists release a neurite extension factor from cultured brainstem and cortical astrocytes identified as S-100␤ [123]. Antagonism of 5-HT1A receptors by NAN-190 reduces the amount of core S-100␤ protein, whereas antagonism of 5-HT2A-C receptors by mianserin has no significant effect on levels of S-100 beta [85]. When the 5-HT1A receptor is stimulated, the astroglial cell responds by releasing S-100␤ and attaining a mature morphology with a shift from a flattened morphology to a process-bearing morphology [122,126] (Fig. 3). In this treatment there is an effective feedback inhibition since as the glial cell matures, it loses binding to the 5-HT-1 receptors [122]. Serotonergic and S-100␤ interactions have also been seen in the adult brain. The hippocampal levels of S-100␤ are decreased after PCPA and PCA (para-chloroamphetamine), and increased by fluoxetine [13,52]. Injections of 5-HT1A receptor agonist increase the immunocytochemical staining of S-100␤ in brain after treatment with cocaine [6], PCA [13] or after adrenalectomy [14]. GFAP, an insoluble fibrillary marker protein, shows a small transient increase after 5-HT damage [21,128]. S-100␤ has effects on neurite extension, but not survival of serotonergic neurons. This is shown in culture [11,72,92,110] and after injections of S-100␤ producing C-6 cells [117]. Other trophic effects of S-100␤ have been reported on neurite extension in chick cortical neuronal cultures [64], on survival of spinal cord motoneurons cultures [23], and on cell division in glial cultures [107]. Trophic properties of S100␤ are also seen in the adult animal. Treatment with antibodies raised against S-100␤ blocks cortical [86] and hippocampal [128] synaptogenesis and long-term potentiation (LTP) [17,70]. In a mutant mouse lacking S-100␤, the polydactyl Nagoya mouse, there is little development of the cortical layers and serotonin terminals are absent [118]. 5-HT2 receptors have been detected in normal glial cells and glioma cell lines [57,77,81,129]. Serotonin stimulates the turnover of phosphoinositide in primary cultures of astroglia from the cerebral cortex, striatum, hippocampus, and brain stem. Ketanserin and ritanserin inhibit this stimulation. The actions of the 5-HT2A receptor in cultured astrocytes involve activation of both PI hydrolysis and c-AMP accumulation. Interactions between the cyclic AMP and the inositol phosphate transduction systems were directly investigated. Serotonin, at a concentration ineffective in stimulating the formation of cyclic AMP, increases the betaadrenergic receptor- stimulated accumulation of cyclic AMP. This potentiation is blocked by the 5-HT2 receptor antagonist ketanserin [51]. In addition, cyclic AMP levels are increased in the presence of both serotonin and an alpha 1-adrenergic receptor agonist, neither of which stimulated cyclic AMP alone. These results suggest specific interactions between the cyclic AMP and inositol phosphate systems in cultured astroglial cells mediated by the 5-HT2A receptor. 5-HT2 receptors in glioma cells appear to regulate proliferation, migration, and invasion. Serotonin was found to positively modulate these three processes in vitro [81]. Finally, and relevant to trophic actions, the 5-HT2 receptor on astroglial cells upregulates glycogenolysis [102] (Fig. 3). These results suggest that 5-HT plays an important role in the control of the biological properties of astroglial cells, and may underlie such diseases as Alzheimer’s [16]. CELL PROLIFERATION The role of both the 5-HT1A and 5-HT2A receptors during development suggest influences on cell proliferation [32]. Al-

5-HT1A AND 5-HT2 DYNAMICS

417

FIG. 4. The drawings show the actions of the 5-HT1A and 5-HT2A receptors on cell proliferations. The 5-HT1A receptor (left side) depicts an acceleration of differentiation produced by enhancement and stabilization cytoskeletal formation. The 5-HT2A receptor (right side) illustrates the increase in cell proliferation accompanied by the occurrence of apoptosis; events consist with an influx of Ca⫹⫹ and fluidity in cytoskeletal formation.

though many factors are involved in determining mitosis, the importance of the cytoskeleton, especially the spindle apparatus, is central. The spindle apparatus is composed of tubulin, exactly like the microtubules involved in cellular structure. In fact, the tubulin used in the construction of the spindle apparatus is actually taken from the microtubules of the cytoskeleton. When cells are about to divide, the microtubules in the processes undergo depolymerization and the tubulin is relocated to the cell body area where the

tubulin undergoes polymerization to form the spindle apparatus. Thus, the mitosis-interphase transition occurs by a redistribution of tubulin among different classes of MTs at essentially constant polymer level [130]. These two processes of process retraction and spindle apparatus construction are intimately associated. However, in cultured mouse brain, neuronal tubulin does increase in amount for the elongation of axonal microtubules. Axonal microtubules are independent of a microtubule-organizing center localized in the perikaryon [80,111]. Thus, dendritic and somal, but not axonal,

FIG. 5. The picture illustrates the effects of 5-HT1A receptors and S-100␤ in regulating the mature phenotype of neurons. A model of “Neuronal Instability” characterizes the flux between a mature and immature phenotype [9]. This model assumes the cytoskeleton is biased towards depolymerization if maintenance factors, such as serotonin and S-100␤, are not continually present. In the absence of these factors a cell can regress its major processes, and can even enter apoptosis. The presence of 5-HT1A receptor agonist or S-100␤ can thus be considered anti-apoptotic.

418

FIG. 6. A model of neurotoxicity proposed in 1990 to explain the destruction of nerve fibers by MDMA, a potent 5-HT releaser. The key step in the generation of the toxicity is the build-up of Ca⫹⫹ inside the neuron. The stimulation of the 5-HT2A receptor, by both MDMA and 5-HT, contributes to this process by activation of phospholipase-C (PI-hydrolysis, PIH), which induces release of internal stores of Ca⫹⫹ and helps phosphorylate Ca⫹⫹ channels to increase influx from external stores. Build-up of internal Ca⫹⫹ by phospholipase-C activation is believed to contribute to apoptosis. Details of this model can be found in reference Azmitia et al. [12].

tubulin pools may be shared. In our experiments, spinal cord cultures showed MAP-2 staining in long processes [90]. MAP-2 is associated with dendritic, but not axonal microtubules. Treatment with colchicine, which disrupts microtubules, resulted in a loss of long processes, but a corresponding increase in MAP-2 somal labeling. The involvement of the 5-HT1A receptor in cell proliferation is assumed to be inhibitory given its stimulatory effects on cell differentiation [67]. However, some studies indicate a direct and indirect role for 5-HT1A receptors in cell proliferation. 5-HT1A agonists given in culture accelerate cell division, generate cell foci, and increase DNA synthesis in transfected NIH-3T3 cells [119]. However, this stimulation of cell proliferation was much stronger when tyrosine kinase receptors were activated by treatment with epidermal growth factor. The transforming and mitogenic effects of 5-HT1A agonists involve a pertussis toxin-sensitive G protein but do not seem to be linked to adenylyl cyclase inhibition. Thus, the action of the 5-HT1A agonist may be mediated by an affect on the membrane potential rather than on cell metabolism and phosphorylation. The early studies of serotonin and cell proliferation in culture appear to argue that serotonin may be important for cell differentiation and the inhibition of cell division in the CNS (Fig. 4). The 5-HT1A receptor is uniquely positioned during the early development of the brain to influence neuronal mitosis. In the monkey brain, the 5-HT1 receptor is found in high levels in ventricular and subventricular proliferative zones of the developing occipital lobe during neurogenesis, which peaks at E60 –E93 [71]. The receptor is seen within the cells destined to become both glial and neuronal cells. When 5-HT levels in the cortex are reduced by prenatal treatment with cocaine, the thickness of the cortex of the newborn pups is greatly reduced [5,6,38]. The microencephaly appears to be due to a decrease in neurogenesis, as indicated by a loss of

AZMITIA bromo-deoxyuridine (BrdU)-labeled cells in the cortex and subventricular zone in the cocaine-treated pups [38]. Treatment of the newborn pups for several days after birth with a 5-HT1A agonist, can quickly reverse the microencephaly and increase the amount of S100␤ immunocytochemical staining seen in hippocampus and cortex [6]. Recently, we have shown those postnatal injections of a 5-HT1A agonist results in an increased labeling by BrdU of hippocampal dentate gyrus neurons 1 hour later [105]. In this study, we also observed that a large percentage of these cells appeared to be more mature after the 5-HT1A agonist, raising the interesting possibility that not only was cell division increased, but also the rate of differentiation. Studies by Haring and coworkers [53] have shown that hippocampal granule neurons have more spines and appear more mature in pups treated with a 5-HT1A agonist. In his studies, the main effect of 5-HT1A receptor agonist was produced by an increased availability of the glial protein S-100␤ since antibodies of this protein prevented the stimulatory affects of the 5-HT1A agonist. The role of 5-HT1A agonist, and S-100␤ on neuronal differentiation, especially with respect to maturation, is presented in the paper by Whitaker-Azmitia [127] and will not be discussed here. The 5-HT1A receptors in the adult brain have clearly been shown to be involved in maintaining the mature state of neurons in the mammalian brain [10,14]. It is therefore surprising that serotonin appears to be involved in regulating adult neurogenesis. In the subventricular zone and in the subgranular zone of the adult hippocampus, the numbers of BrdU cells are decreased after either 5,7-dihydroytryptamine (5,7-DHT) lesions or inhibition of 5-HT synthesis with PCPA [30,31]. This suppression is reversed following normal re-innervation or transplantation of 5-HT-producing fetal neurons [31]. These effects on adult BrdU incorporation appear to be mediated by the 5-HT1A receptor [47]. The paradox is, how can a chemical, which stimulates differentiation, also be involved in cell proliferation? This is true for both the 5-HT1A receptor and S-100␤. One explanation may involve the cytoskeleton. Both 5-HT1A receptor agonists and S-100␤ promote the assembly of the tubulin into microtubules, and this may benefit both the assembly of the cytoskeleton in processes which promote maturation and the assembly of the spindle apparatus in the cell body which promotes cell division. Studies recently performed with S-100␤ in our laboratory suggest that the rate of cell division may be enhanced [9]. Thus, cells in prophase would be directed to divide, while cells in G1 would be directed to differentiate. In summary, it appears that 5-HT1A receptors may be involved in accelerating the rate of neurogenesis and the corresponding later differentiation process, and not necessarily increasing cell division over several cycles (see Fig. 4). The results with the 5-HT2A receptor appear to be more directly consistent with increases in cell proliferation (Fig. 4). This receptor in the adult is involved in de-stabilizing the cytoskeleton and promoting process branching or retraction. In the periphery, introduction of exogenous cells stimulates cell proliferation of the host cells. Experimental vein graft induces a rapid hyperplasia of smooth muscle fiber cells [75]. This hyperplasia is significantly reduced by prior treatment with the 5-HT2A antagonist, ketanserin. The mitogenic effect of 5-HT and its synergistic interaction with TXA2 on smooth muscle cell proliferation is abolished by a 5-HT2 receptor antagonist, LY281067, and this drug may be clinically useful for attenuation of restenosis after angioplasty [97]. Endothelial cells are also sensitive to 5-HT2A receptor drugs and the mitogenic effects of 5-HT on endothelial cells are mediated via the 5-HT2 receptor [98]. Stimulation of endothelial cells by serotonin results in an increase in tritiated thymidine uptake and an increase in cell number. LY281067 blocks this mitogenic effect of serotonin [96]. Smooth muscle cell proliferation is significantly

5-HT1A AND 5-HT2 DYNAMICS increased by activated platelets and this effect is reduced by ketanserin. Activated platelets promote smooth muscle cell proliferation in vitro via release of soluble mediators, including serotonin [37]. Finally, blastogenic transformation of murine spleen cells elicited with concanavalin A is suppressed by serotonin 10(⫺12) to 10(⫺6) M, and marginally stimulated by its antagonists ketanserin and propranolol in low concentrations [113]. A role for the 5-HT2 receptor in cell proliferation is consistent with the presence of these receptors in neural fold [34]. These receptors also actively mediate the action of serotonin on embryonic morphogenesis, probably by preventing the differentiation of cranial neural crest cells and myocardial precursor cells [36]. The transduction mechanism appears to involve Ca⫹⫹ increases since the 5-HT2A active in 7d hippocampus is linked to PI hydrolysis [59]. A detailed pathway for renal mesangial cells is proposed: 5-HT2A receptor 3 PKC 3 NAD(P)H oxidase/reactive oxygen species 3 MEK 3 ERK 3 TGF-beta1mRNA (a proliferative and fibrotic signal) [48]. Thus, in contrast to the actions of the 5-HT1A receptor, the 5-HT2A receptor may stimulate cell proliferation for several cycles, which would be consistent with its inhibitory actions on differentiation. CELL DEATH AND APOPTOSIS 5-HT1A Receptor Apoptosis, or programmed cell death, can be induced in immature neurons by a variety of methods. The cells exposed to apoptotic-inducing conditions may actually up-regulate 5-HT1A receptors. Neuronal cell lines stably transfected with a promoterless segment (G-21) of the human 5-HT1A receptor (5-HT1A-R) gene [112] show a 5 to 15-fold increase in the receptor when deprived of nutrient. The temporal correlation between degeneration and the expression of the 5-HT1A receptor is reminiscent of the up-regulation of heat-stress proteins, and suggests intronless proteins may have a protective role in threatening situations. In agreement with a protective role for the 5-HT1A receptor, treatment with a specific receptor agonist inhibits apoptosis induced by serum deprivation in cultured neurons from chick embryo telencephalons [4]. After 24 h of serum withdrawal, there is an increase in the number of apoptotic cells from 12% to 29%. The 5-HT1A receptor agonist 8-OH-DPAT reduces the number of apoptotic cells in a concentration-dependent manner. The anti-apoptotic effect of 8-OH-DPAT is blocked by adding the selective 5-HT1A receptor antagonist MPPI, but not by the dopamine receptor antagonist chlorpromazine or the beta-receptor blocker propranolol. Similar results are seen with Bay x 3702, another specific 5-HT1A agonist [3]. 5-HT1A receptor agonists are also effective in protecting primary hippocampal and cortical cultures exposed to damaging toxins. BAY x 3702 is neuroprotective against 25 nM staurosporine or 0.5 mM L-glutamate [108,114]. These effects of BAY x 3702 are blocked by WAY-100635. Several transduction pathways may explain the protective effects of the 5-HT1A receptor agonist in preventing apoptosis. For one, the 5-HT1A receptor opening of the K⫹ channel which induces hyperpolarization and attenuates the depolarization needed for opening the NMDA and the voltage-dependent Ca⫹⫹ channels. This pathway has been proposed to explain 5-HT1A receptor agonist protection against the cyanide-induced cytotoxic hypoxia and glutamate-induced excitotoxicity in primary neuronal cell cultures from chick embryo cerebral hemispheres [100]. An alternative pathway is described for apoptotic suppression, which involves activation of mitogen-activated protein kinase (MAPK or Erk-2). Anoxia induces apoptosis in HN2-5 cells, a hippocampal neuronal-derived cell line lacking both N-type Ca⫹⫹ channels and NMDA receptors [1]. Treatment with 8-OH-DPAT protects these

419 cells by a mechanism sensitive to pertussis toxin (G-protein linked) and requiring phosphorylation-mediated activation of MAP-K. The Erk-2 pathway is uncovered by the action of PD98059, the MAP kinase inhibitor. Treatment with the 5-HT1A receptor agonist induces a 60% inhibition of caspase-3 in the HN2-5 cells, which is blocked by PD98059. In support of this pathway, 5-HT1A receptor activation of the ERK2 pathway is also seen in Chinese hamster ovary cells, which have Ca⫹⫹ channels [40]. In these cells the activation is dependent on pertussis toxin and enzymes in the phosphatidylinositol pathway (Ca⫹⫹ sensitive). Interestingly, the 5-HT1A receptor induction of the ERK-2 pathway proceeds independently of PKC. The inhibitors bisindolylmaleimide and Ro-31-8220 block ERK-2 activation by phorbol ester (activator of PKC) but have no effect on 5-HT1A receptor activation. The tyrosine kinase inhibitors genistein and herbimycin A also have no significant effect. These effects in culture are consistent with reports in vivo. It has long been known that 5-HT1A receptors are neuroprotective in models of brain ischemia [24] such as induced focal cerebral ischemia by permanent occlusion of the left middle cerebral artery in rats and mice. Forty-eight hours after vessel occlusion, 5-HT1A receptor agonists (8-OH-DPAT, buspirone, gepirone, ipsapirone, and Bay x 1531) applied 30 min before induction significantly decreased cortical infarct size. Similar results are seen in gerbil hippocampus with a transient occlusion model [26]. The effects of the 5-HT1A receptor agonist are as effective as antagonists against NMDA receptors, AMPA receptors, and L-type calcium channels [100]. 5-HT2 receptor drugs are not effective in these models of apoptosis. Conversely, reduced serotonin levels in the hippocampus potentiate ischemic-induced neuronal damage [87]. Apoptosis can be induced by breakdown of the cytoskeleton. Colchicine is a compound, which binds tubulin and promotes depolymerization of the cytoskeleton. In most cells studied, it leads to cell death by apoptosis [11,20]. Colchicine also causes apoptosis in a variety of malignant cell lines [20,115]. The apoptosis produced in culture of cerebellar granule neurons by 1 uM colchicine is blocked by taxol (100 nM), but not blocked by MK801 (NMDA receptor antagonist), inhibitors of NOS or L-type Ca⫹⫹ channel blockers [27]. Furthermore, intraventricular injections lead to apoptosis of granule neurons in hippocampus and cerebellum within 24 h [27,34]. Inhibitors of caspase 3 block the apoptosis in the cerebellum produced by colchicine [46]. The role of the 5-HT1A receptor in blocking apoptosis is also indirect, and mediated by the release of S-100␤ from astrocytes. S-100␤ is effective at restoring normal morphology, and preventing death, using cell lines and primary cultures exposed to colchicine [29]. In order to determine if exogenous S100␤ can directly influence the cytoskeleton of living cells, cultures of N18 (a neuroblastoma clonal cell line) were treated for 30 min in serumfree medium with 10⫺6 M colchicine. The colchicine was then removed and the cells placed in normal media. Colchicine-treated cells showed a rapid retraction of processes, membrane blebbing, nuclear fragmentation, and cell death over the next several hours. A majority of the cells stained positive with the Hoecht Stain, a marker for apoptosis, after 2 hours. The observed cellular changes, apparently due to cytoskeletal collapse after colchicine, are consistent with the loss of processes in cultures and spines in adult hippocampus treated with antibodies against S100␤ [90]. The addition of 2–20 ngm/ml of S100␤ after the initial 30 min exposure to colchicine prevents apoptosis, cytoplasmic bebbing and induces the regrowth of the retracted processes. Our results suggest that extracellular application of the glial protein S100␤ is able to restore damage produced by cytoskeletal damage and prevent the resultant apoptosis of the cells (Fig. 5). The increased levels of S100␤ seen after brain injury and in certain neurological and

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FIG. 7. The model above is a summary of the neuronal and glial actions of the 5-HT1A and 5-HT2A receptors on cytoskeleton stability. The actions of the 5-HT2A receptor (left side) act to release glucose from glial cells and to increase Ca⫹⫹ levels in neurons. These two events would facilitate neuronal activity and promote structural instability. In contrast, the 5-HT1A receptor (right side) increases the release of S-100␤ from astrocytes and reduces the levels of cAMP in neurons. These two events would encourage neuronal rest and stability.

psychiatric disorders may be considered as beneficial for brain recovery. 5-HT2A Toxicity There is not much evidence for a role of the 5-HT2 receptor in neuronal toxicity. One of the first suggestions was the protective role of ketanserin in preventing 3,4-methylenedioxy-methamphetamine (MDMA; ecstasy)-induced toxicity of cultured serotonergic neurons [12] (Fig. 6). Cultured rat fetal serotonergic neurons were damaged within 24 h by the application of 5 ⫻ 10⫺6 M S(⫹)MDMA by a Ca⫹⫹-dependent mechanism (blocked by L-type channel antagonist). Since MDMA has a low affinity for the 5-HT2 receptor and functions as a 5-HT releaser, we investigated the protective properties of a 5-HT2 antagonist ketanersin. The 5-HT2 receptor is linked to increased intracellular Ca⫹⫹ through a second messenger phosphatidylinositol (PI)-hydrolysis mechanism [8,73]. We found significant protection of MDMA-induced toxicity and proposed a mechanism of action involving the 5-HT2 receptor and Ca⫹⫹ buildup in the neuron to explain the mechanism of toxicity (Fig. 6). Studies in the live animal soon confirmed the protective role for a 5-HT2 antagonist in ecstasy-induced loss of 5-HT in the brains of adult rats [106]. These authors showed that the MDMAinduced 5-HT deficits were prevented by the simultaneous administration of 5-HT2 receptor antagonists such as MDL 11,939 or ritanserin. This effect was not region specific as protection was observed in the cortex, hippocampus, and striatum 1 week after the administration of a single dose of MDMA. Apoptosis, programmed cell death, is strongly linked to a Ca⫹⫹ buildup in the cell, and the 5-HT2 receptor linked PI-hydrolysis could play an important role. Phosphoinositide signaling regulates events in endocytosis and exocytosis, vesicular trafficking of pro-

teins, transduction of extracellular signals, remodeling of the actin cytoskeleton, regulation of calcium flux, and apoptosis [94]. The involvement of IP-3 in cell survival is well known, but the link to apoptosis is only just emerging [50]. For example, selective stimulation of type 3 inositol (1,4,5) trisphosphate receptors [IP(3)R3] in lymphocytes induces apoptosis, which is prevented by antisense constructs to IP(3)R3 and increases in mRNA and protein levels for IP(3)R3 are associated with cell death in early postnatal cerebellar granule cells, dorsal root ganglia, embryonic hair follicles, and intestinal villi [25]. Apoptosis in neurons induced by the glutamate agonist kainate or deprivation of nerve growth factor is correlated with increased levels of IP(3)R3. It remains to be seen, however, if 5-HT2 agonists, which are potent stimulators of PIhydrolysis resulting in marked increases in IP-3 increases, are involved in programmed cell death. IMPLICATIONS FOR DRUG DISCOVERY This chapter has not only reviewed the evolutionary role and a function of serotonin, but also advances the notion that serotonin plays a major role in the plasticity of the brain. We have used two receptors, the 5-HT1A and the 5-HT2A, to show how serotonin can produce opposite and complimentary actions on neuronal functioning (Fig. 2), maturation (Fig. 3), proliferation (Fig. 4), and apoptosis (Figs. 5 and 6). The action of the 5-HT1A receptor on the release of S100␤ offers a unique mechanism by which a neurotransmitter can regulate maturation of itself and its target cells. The effects of the 5-HT2A receptor on PI hydrolysis and Ca⫹⫹ levels has implications for both proliferation and apoptosis. The influence of other chemical and hormonal systems serves to provide many layers of trophic support. Understanding these dynamic, complex homeostatic trophic systems will provide new

5-HT1A AND 5-HT2 DYNAMICS approaches to the treatment of many neural disorders [9,101]. Certain neural disorders involve processes that emerge early during development (e.g., Down’s Syndrome and autism) whereas others emerge late in adult life (e.g., Alzheimer’s and Parkinson’s diseases,). Many mental disorders can occur at any ages, such as depression and anorexia. Since these disorders show a loss of normal brain morphology, the cognitive and emotional breakdown may result from loss of neuronal connections due to abnormalities in the same set of molecules that are essential for maturation during development and plasticity. In this chapter, we have shown that often the actions of one trophic receptor are opposed by another, and what might be critical is the balance of these opposing forces. Brain morphology, and neuronal phenotype is basically unstable due to the dynamics of cytoskeletal microtubules, long polymers of tubulin (Fig. 7). The stability of the microtubules is directly dependent on the phosphorylation state of MAPs, which detach from tubulin polymers when phosphorylated. Serotonin, through its 5-HT1A and 5-HT2A receptors on neuronal and glial cells, can regulate the cytoskeleton, and organize the cytoskeleton of its target cell. Shifts in the balance between the 5-HT1A receptor and the 5-HT2A receptor result in shape changes between stable and pulsating morphology. Since the soma and dendrites are most vulnerable to the state of the MAPs, changes in the size and complexity of these structures directly impinge on neuronal connections, and mental functioning. The severity of the abnormality is dependent on the duration of the receptor imbalances, since prolonged exposure to high Ca⫹⫹ can progress from beneficial branching to dendritic regression, and finally to apoptosis. Restoration of serotonin receptor balance can restore normal mature morphology [13], but the recovery time can extend to months or years depending on the initial degree of morphological regression and cell death. Trophic therapy can be achieved through sophisticated application of multiple 5-HT receptor drugs, but the strategies necessary to produce maximum restorative plasticity require detailed knowledge of biochemical mechanisms involved in cytoskeletal assembly, and of pharmacological actions on transduction activation of phosphorylation states. Targeting of a single process will not likely succeed in achieving a normal mental homeostasis unless complementary and opposing processes are monitored. Drug discovery for effective therapeutic intervention in psychiatric disorders having a morphological basis may require more strategy and less cloning.

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