Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
2 Overview of the Glutamatergic System Glutamate is the major excitatory neurotransmitter in the nervous sys- tem. Glutamate pathways are linked to many other neurotransmitter path- ways, and glutamate receptors are found throughout the brain and spinal cord in neurons and glia. As an amino acid and neurotransmitter, glutamate has a large array of normal physiological functions. Consequently, gluta- mate dysfunction has profound effects both in disease and injury. At least 30 proteins at, or near, the glutamate synapse control or modu- late neuronal excitability, noted Darryle Schoepp, senior vice president of neuroscience at Merck. These proteins are membrane-bound receptor or transporter proteins (Figure 2-1). They are strategically situated on several cell types converging on the glutamate synapse: pre- and post-synaptic neu- rons, astrocytes (a type of glial cell), and nearby inhibitory neurons that use γ-Aminobutyric acid (GABA). GABA is the chief inhibitory neurotransmit- ter in the brain, and the major difference between glutamate and GABA is that the latter is synthesized from the former by the enzyme L-glutamic acid decarboxylase. Schoepp said the fact that GABA neurons and glutamate neurons are distinguished by this single enzyme could be an efficient way, in evolutionary terms, to control excitability in the nervous system. Glutamate concentrations in the extracellular space are low and tightly controlled by a large number of mechanisms at the synapse. Perturbations to this regulatory system can have deleterious effects such as excess release of glutamate, which can induce hyperexcitability in post-synaptic neurons to the point of excitotoxicity and cell death (cytotoxicity) (Choi, 1994; Doble, 1999). Glutamate-induced excitotoxicity, particularly in the hippo- campus, has been linked to decreased neuronal regeneration and dendritic 5
6 GLUTAMATE-RELATED BIOMARKERS IN DRUG DEVELOPMENT Glia and astrocytes ? mGlu5 lu 3 mG mG mG lu 3 EAAT lu 7 mGlu 1 Postsynaptic /5 u2 Gl m â cAMP Presynaptic NMDA â Ca2+ Modulation Glu AMPA of excitation vGluT â Na+ Kainate â Clâ â cAMP m G lu 4/8 4/ 8 GA mGlu 5 lu 1/ BA mG A EAAT AB GAB GABAB mGlu3 mGlu7 GABA FIGURE 2-1 Illustration of a hypothetical synapse showing localization and func- tion of glutamatergic receptors and transporters. SOURCE: Swanson et al., 2005. branching, leading to impaired spatial learning (Cortese and Luan Phan, 2005). Disruptions of glutamate uptake from the synapse have been linked to reduced sensitivity to reward, a symptom of depression (Bechtholt- Gompf et al., 2010). For these and other reasons, a neurotransmitter of glutamateâs functional significance must be tightly regulated (Swanson et al., 2005). The complexity of regulating glutamate and its pervasive presence throughout the brain may explain why, over the past decades, only three prescription medications have been developed that specifically target gluta- mate or glutamate receptors, memantine, ketamine, and D-cylcoserine. The potential for side effects from these medications is extremely high, which
7 OVERVIEW OF THE GLUTAMATERGIC SYSTEM in part deters further investment. By contrast, a broad range of drugs have been marketed to modulate other neurotransmitters, such as dopamine, serotonin, and acetylcholine, whose synaptic regulation is less complex and whose roles and pathways in central nervous system (CNS) pathways are not as extensive. This presents the most fundamental obstacle facing glutamate bio- marker development and therapeutics: Any agonist or antagonist has the potential to produce beneficial as well as toxic side effects, depending on its concentration, route of administration, dose-related adverse effects, and other key factors. In terms of drug development, the goal is to carefully select a molecular target that modulates dysfunctional glutamate pathways, without disruption of healthy pathways, and minimizes adverse effects. That challenge to glutamate diagnostics and therapeutics was clearly articu- lated at the outset of the workshop by presenters Schoepp and Dan Javitt, program director in cognitive neuroscience and schizophrenia at the Nathan Kline Institute for Psychiatric Research: ⢠H ow can glutamatergic synaptic transmission be selectively modu- lated in the central nervous system? ⢠C an we selectively target pathological processes involving the glu- tamate system? ⢠C an we monitor the long-term effects of single-target interventions because chronic dosing of any medicine in a system as highly plas- tic as the glutamate system may not sustain the beneficial effects? GLUTAMATE RECEPTORS Glutamate receptors are numerous and highly complex; more than 20 glutamate receptors have been identified in the mammalian central nervous system. They fall into two main categories, ionotropic (voltage sensitive) and metabotropic (ligand sensitive). Each ionotropic or metabotropic re- ceptor has three types, depending on binding specificity, ion permeability, conductance properties, and other factors. Each type has multiple subtypes (Table 2-1). Ionotropic receptors are fast acting and, once opened, can produce large changes in current flow even if the voltage difference across the membrane is small. After glutamate, as a ligand, binds to an ionotropic receptor, the receptorâs channel undergoes a conformational change to al- low an immediate influx of extracellular sodium among other ions and an efflux of potassium ions. This triggers membrane depolarization in the post-synaptic cell sufficient to induce signal transmission. One of the main glutamate ionotropic receptors, N-methyl D-aspartate (NMDA), is perme- able to calcium ions in addition to sodium and potassium ions; calcium ions have both beneficial and toxic effects. The NMDA receptor is unusual because it is a coincidence detector; for the channel to open, glutamate must
8 GLUTAMATE-RELATED BIOMARKERS IN DRUG DEVELOPMENT TABLE 2-1 Glutamate Receptor Protein Subunit Composition and Properties Receptor Protein Subunit Receptor Properties Ionotropic Receptors NR1, NR2A*, NR2B*, NMDAR Heterotetrameric; calcium NR2C, NR2D, NR3A, and permeability high; long NR3B channel open time GluR1*, GluR2 edited, AMPAR Heterotetrameric; calcium GluR2, GluR3*, and permeability low if edited GluR4* GluR2, otherwise moderate; short channel open time GluR5*, GluR6, GluR7, Kainate receptor Homotetrameric or KA1, and KA2 heterotetrameric; calcium permeability low; short channel open time Metabotropic Receptors mGluR1* and mGluR5 Group 1 Homodimeric; signals via phospholipase C; located post-synaptically Group 2 mGluR2 and mGluR3 Homodimeric; signals via adenylyl cyclase; located mostly pre-synaptically; agonists and antagonists mostly distinct from Group 3 Group 3 mGluR4, mGluR6, Homodimeric; signals via mGluR7, and mGluR8 adenylyl cyclase; located mostly pre-synaptically; agonists and antagonists mostly distinct from Group 2 *Glutamate receptor protein subunits for which human autoantibodies have been reported. SOURCES: Kew and Kemp, 2005; Pleasure, 2008. bind to the receptor and the post-synaptic cell must be depolarized because the channel is blocked by magnesium at physiological levels and only opens when the cell is depolarized. Ionotropic receptor channels are formed by assemblies of heterotetra- meric or homotetrameric protein subunits. The three types of ionotropic receptors are named after the ligand that expressly binds to one, but not to the other two: NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainic acid. Once these ligands were discovered, many others, whether agonists or antagonists, were subsequently found (Lesage
9 OVERVIEW OF THE GLUTAMATERGIC SYSTEM and Steckler, 2010). Although their properties differ somewhat, as do their anatomical distribution, glutamate receptors are best known for mediating glutamateâs role in learning and memory through plasticity, or modifica- tion, of channel properties; enhanced glutamate neurotransmission; and gene expression (Barco et al., 2006). Not only are NMDA receptors highly expressed on neurons, but they are also expressed on astrocytes (Lee et al., 2010). The human brainâs expansive capacity for plasticity, learning, memory, and recovery from injury is attributed to improvement in synaptic anatomy and physiology of NMDA signaling, most notably in the hip- pocampus and other regions of the mammalian CNS (Barco et al., 2006). The basic mechanisms underlying plasticity include neurogenesis, activity- dependent refinement of synaptic strength, and pruning of synapses. Metabotropic glutamate receptors are slower acting; they exert their effects indirectly, typically through gene expression and protein synthesis. Those effects are often to enhance the excitability of glutamate cells, to reg- ulate the degree of neurotransmission, and to contribute to synaptic plastic- ity (Lesage and Steckler, 2010). Once glutamate binds with a metabotropic receptor, the binding activates a post-synaptic membrane-bound G-protein, which, in turn, triggers a second messenger system that opens a membrane channel for signal transmission. The activation of the protein also triggers functional changes in the cytoplasm, culminating in gene expression and protein synthesis. There are three broad groups of glutamate metabotropic receptors, distinguished by their pharmacological and signal transduction properties. Altogether, a total of eight metabotropic glutamate receptor subtypes have been cloned thus far. Group I metabotropic receptors are largely expressed on the post- synaptic membrane. They have been implicated in problems with learn- ing and memory, addiction, motor regulation, and Fragile X syndrome (Niswender and Conn, 2010). Group II metabotropic receptors are situated not only on post-synaptic cells, but also on pre-synaptic cells, possibly to suppress glutamate transmission (Swanson et al., 2005). Their dual loca- tion may enable them to exert a greater degree of modulation of glutamate signaling (Lesage and Steckler, 2010). Dysfunction of group II metabotropic receptors have been implicated in anxiety, schizophrenia, and Alzheimerâs disease. Group III metabotropic receptors, like group II, are pre-synaptic and inhibit neurotransmitter release. They are found within the hippo- campus and hypothalamus and may play a role in Parkinsonâs disease and anxiety disorders (Swanson et al., 2005). GLUTAMATE TRANSPORTERS Tight regulation of extracellular glutamate concentrations at both the synapse and in extra-synpatic locations is critical for normal synaptic
10 GLUTAMATE-RELATED BIOMARKERS IN DRUG DEVELOPMENT transmission and to prevent excitotoxicity. Glutamate transporters regulate glutamate concentrations and are situated on both pre- and post-synaptic neurons as well as on surrounding astrocytes, a type of glial cell (Kanai et al., 1994). Five excitatory amino acid transporters (EAATs), previously known as glutamate transporters, have been cloned: EAAT-1 to EAAT-5, with EAAT-2 expressed predominantly on cells in brain regions rich in glu- tamate (Eulenburg and Gomeza, 2010). It is widely accepted that glutamate transporters on glial cells are primarily responsible for maintaining extra- cellular glutamate concentrations. However, the presence of transporters on multiple cell types suggests a high level of cooperation (Eulenburg and Gomeza, 2010; Foran and Trotti, 2009; Tanaka, 2000). Glial cells, most often astrocytes but also microglia and oligodendro- cytes (Olive, 2009), perform a key role in modulating extracellular gluta- mate levels. Under normal conditions, glutamate is recycled continuously between neurons and glia in what is known as the glutamateâglutamine cycle. Excess glutamate in the synapse is taken up by glial cells via EAAT transporters, where it is converted to glutamine. Glutamine is then trans- ported back into neurons, where it is reconverted to glutamate (Rothman et al., 2003). However, glial cells, under certain conditions, may also release glutamate by at least six mechanisms, one of which is reversal of uptake by glutamate transporters (Malarkey and Parpura, 2008). This kind of reverse transport may be involved in brain damage and stroke (Grewer et al., 2008). Finally, altered expression of EAAT-2 is found in amyotrophic lateral sclerosis. The disease is also marked by excess glutamate levels in the cerebral spinal fluid (Rothman et al., 2003). New therapies are being developed to interfere with this pathological process by targeting EAAT-2 on astrocytes (Rothstein et al., 1992). Given the number of receptors and transporters, the range of cell types expressing them, the variety of regulatory controls, and the narrow concentration difference between normal synaptic function and excitotox- icity, many fundamental questions remain about how to choose potential pharmacological targets. One presenter at the workshop, Schoepp, raised a series of questions and concerns that addressed both biomarkers and choices of molecular target. The foremost concern was whether the bio- marker could distinguish between normal physiology versus pathology. What kind of feedback mechanisms and crosstalk at the synaptic cleft must be considered, especially in light of the probable need for chronic dosing of any new medication? Long-term use of any medication might produce un- expected changes, with the potential for side effects or paradoxical effects. The greatest danger is the specter of any glutamate-related drug inducing excitotoxicity and its ramifications.
