-methyl-D-aspartate (NMDA) and the regulation...

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Progress in Neurobiology 77 (2005) 252–282

N-methyl-D-aspartate (NMDA) and the regulation of

mitogen-activated protein kinase (MAPK) signaling

pathways: A revolving neurochemical

axis for therapeutic intervention?

John J. Haddad a,b,*a Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanon

b Departments of Biology and Biomedical Sciences, Faculty of Arts and Sciences,

Lebanese International University, Beirut, Lebanon

Received 10 November 2003; received in revised form 10 December 2004; accepted 27 October 2005

Abstract

Excitatory synaptic transmission in the central nervous system (CNS) is mediated by the release of glutamate from presynaptic terminals onto

postsynaptic channels gated by N-methyl-D-aspartate (NMDA) and non-NMDA (AMPA and KA) receptors. Extracellular signals control diverse

neuronal functions and are responsible for mediating activity-dependent changes in synaptic strength and neuronal survival. Influx of extracellular

calcium ([Ca2+]e) through the NMDA receptor (NMDAR) is required for neuronal activity to change the strength of many synapses. At the

molecular level, the NMDAR interacts with signaling modules, which, like the mitogen-activated protein kinase (MAPK) superfamily, transduce

excitatory signals across neurons. Recent burgeoning evidence points to the fact that MAPKs play a crucial role in regulating the neurochemistry of

NMDARs, their physiologic and biochemical/biophysical properties, and their potential role in pathophysiology. It is the purpose of this review to

discuss: (i) the MAPKs and their role in a plethora of cellular functions; (ii) the role of MAPKs in regulating the biochemistry and physiology of

NMDA receptors; (iii) the kinetics of MAPK–NMDA interactions and their biologic and neurochemical properties; (iv) how cellular signaling

Abbreviations: AA, arachidonic acid; AIDS, acquired immunodeficiency syndrome; ALS, amyotrophic lateral sclerosis; AMPA, a-amino-3-hydroxy-5-methyl-

4-isoxazolepropionic acid; APV, 2-amino-5-phosphonovalerate; 2-AP5, 2-amino-5-phosphonopentanoic acid; ATF, activating transcription factor; BAPTA-AM, 1,2-

bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid; BDNF, brain-derived neurotrophic factor; BSO, L-buthionine-(S,R)-sulfoximine; CaM, Ca2+/calmodulin;

CaMK, Ca2+/calmodulin-dependent protein kinase; CAT, catalase; CEE, conjugated equine estrogens; CNS, central nervous system; CNQX, 6-cyano-7-nitroqui-

noxaline-2,3-dione; CPP, 3-[(RS)-2-carboxypiperazin-4-yl)]-propyl-1-phosphonate; CREB, cAMP responsive element binding protein; CS, conditioned stimulus;

DA, dopamine; ECS, electroconvulsive shock; EGF, epidermal growth factor; ERK, extracellular receptor kinase; ERT, estrogen replacement therapy; ERT-kinase,

EGF receptor threonine kinase; FGF, fibroblast growth factor; GAP, GTPase activating protein; g-GCS, g-glutamylcysteine synthetase; GDNF, glial cell line-derived

neurotrophic factor; GEF, guanine nuclear exchange factor; GluR, glutamate receptor; GSH, glutathione; GSTp, glutathione S-transferase Pi; HEK, human

embryonic kidney; HFS, high-frequency stimulation; H2O2, hydrogen peroxide; IEG, immediate early gene; IGF, insulin-like growth factor; IL, interleukin; iNOS,

inducible nitric oxide synthase; IRS, insulin receptor-substrate; JIP, JNK-interacting protein; JNK, Jun N-terminal kinase; KA, kainic acid; LPS, lipopolysaccharide-

endotoxin; LTD, long-term depression; LTM, long-term memory; LTP, long-term potentiaion; MAP, microtubule associated protein; MAPK, mitogen-activated

protein kinase; MAPKK, MAP kinase kinase; MAPKKK,MAP kinase kinase kinase; MBP-kinase, myelin basic protein kinase; MCPG, a-methyl-4-carboxy-phenyl

glycine; METH, methamphetamine; MF, mossy fiber; mGluR, metabotropic glutamate receptor; MI, metabolic inhibition; MKP, MAPK phosphatase; MLK, mixed

lineage kinase; MSG, monosodium glutamate; NGF, nerve growth factor; NO, nitric oxide; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; NMDA-EPSP,

NMDA excitatory postsynaptic potential; NRC, NMDA receptor multi-protein complex; PAK, p21-activated kinase; PCP, phencyclidine; PDGF, platelet-derived

growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; PLA2, phospholipase A2; PKA, protein kinase A; PKC, protein kinase C; PPD, paired-pulse depression;

PTK, protein tyrosine kinase; PTX, pertussis toxin; RGC, retinal ganglion cell; RNS, reactive nitrogen species; RSK-kinase, ribosomal S6 protein kinase; RTK,

receptor tyrosine kinase; SAPK, stress-activated protein kinase; SD, succinate dehydrogenase; S-DHPG, (S)-dihydrophenylglycine; SH, Src homology; SIN-1, 3-

morpholinosydnonimine; SNAP, S-nitroso-N-acetylpenicillamine; SNOC, S-nitrosocysteine; SOD, superoxide dismutase; SRF, serum response factor; STM, short-

term memory; TBS, theta-burst; TCF, ternary complex factor; TEA, tetraethyl-ammonium chloride; TGF, transforming growth factor; TNF, tumor necrosis factor;

VSCC, voltage-sensitive Ca2+ channel; z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone

* Present address: In affiliation with Prof. Bared Safieh-Garabedian, Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut

Lebanon. Previous address: Severinghaus-Radiometer Research Laboratories, School of Medicine, University of California, San Francisco, California, USA. Tel.:

+961 1 350000; fax: +961 1 374374.

E-mail address: [email protected].

0301-0082/$ – see front matter # 2005 Published by Elsevier Ltd.

doi:10.1016/j.pneurobio.2005.10.008

J.J. Haddad / Progress in Neurobiology 77 (2005) 252–282 253

pathways, related cofactors and intracellular conditions affect NMDA–MAPK interactions and (v) the role of NMDA–MAPK pathways in

pathophysiology and the evolution of disease conditions. Given the versatility of the NMDA–MAPK interactions, the NMDA–MAPK axis will

likely form a neurochemical target for therapeutic interventions.

# 2005 Published by Elsevier Ltd.

Keywords: Biochemistry; Calcium; CNS; Glutamate; Kinase; MAPK; Neurochemistry; NMDA; Pathophysiology; Phosphorylation; Signaling

Contents

1. Introduction and general background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

1.1. Glutamate receptors (GluRs) in the CNS—physiology and pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

1.2. GluRs in the CNS—the NMDA receptor subtype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

1.3. GluRs and NMDARs—pathway interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

1.4. The purpose of undertaking this survey. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

2. MAPK signaling pathways: an overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

2.1. MAPK identification and nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

2.2. MAPK biochemistry and properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

2.3. MAPK signaling pathways and activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

3. NMDARs: analytical structure, biophysical properties and biochemical interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

3.1. Receptor bio-analytical assessment—biophysics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

3.2. NMDAR biophysical and biochemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

3.3. NMDAR regulation during development, stress and exogenous pharmacological interventions. . . . . . . . . . . . . . . . . . . . 259

3.3.1. Development and distribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

3.3.2. The role of stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

3.3.3. The role of nitration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

3.3.4. NMDA regulation and pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

3.3.5. Pharmacological modulation of the NMDAR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

3.3.6. Ethanol and the modulation of the NMDAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

4. NMDA-mediated regulation of MAPK signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

4.1. NMDA-mediated regulation of G-coupled receptor proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

4.1.1. NMDA, CaMK and MAPK regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

4.1.2. NMDA, AMPA and MAPK regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

4.1.3. NMDA, CREB and MAPK regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

4.1.4. NMDA, KA and MAPK regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

4.2. NMDA, MAPKs and apoptosis-related pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

4.2.1. Insulin-like growth factors and NMDA-mediated excitotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

4.2.2. Egr-1 and NMDA-mediated excitotoxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

4.2.3. MAPKs and NMDA-mediated neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

4.2.4. Neutrophins, MAPKs and NMDA-mediated neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

4.2.5. Caspases, MAPKs and NMDA-mediated neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

4.2.6. IEG (pip), MAPKs and NMDA-mediated neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

4.2.7. PKC, MAPKs and NMDA-mediated neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

4.3. NMDA, MAPKs and long-term potentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

4.3.1. General aspects of NMDA–MAPK interactions in the regulation of LTP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

4.3.2. Genetic control of NMDA–MAPK interactions in the regulation of LTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

4.4. NMDA, MAPKs and long-term depression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

4.5. NMDA, MAPKs and short- and long-term memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

4.6. NMDA, MAPKs and electroconvulsive shock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

4.7. NMDA, MAPKs and oxidative stress: role of oxidants and inflammatory mediators . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

4.7.1. Role for hydrogen peroxide (H2O2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

4.7.2. Role for superoxide anion (O2��) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

4.7.3. Role for nitric oxide (NO). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

4.7.4. Role for ammonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

4.7.5. Role for cytokines and inflammatory-related mediators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

4.8. NMDA, MAPKs and the role of regulatory transcription factors and cofactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

4.9. NMDA, MAPKs and developmental processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

4.10. NMDA, MAPKs and pain/hyperalgesia mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

4.11. NMDA, MAPKs and behavioral mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

4.12. NMDA, MAPKs and obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

4.13. NMDA, MAPKs and pathophysiology: potential interactions in the evolution of disease/injury . . . . . . . . . . . . . . . . . . . 274

J.J. Haddad / Progress in Neurobiology 77 (2005) 252–282254

5. Summary, conclusion and prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

1. Introduction and general background

1.1. Glutamate receptors (GluRs) in the CNS—physiology

and pathophysiology

The glutamate receptors play a key role in brain function

(Gubellini et al., 2004; Oswald, 2004). In the central nervous

system (CNS), most rapid excitatory synaptic transmission is

mediated by GluR channels (Wang and Durkin, 1995; Hatt,

1999; Ahn et al., 2000; Cammarota et al., 2000; Jiang et al.,

2000b; Sala et al., 2000; Chandler et al., 2001; D’Onofrio et al.,

2001; Monfort et al., 2002; Zeng et al., 2002; Barnstable et al.,

2004).

Dysfunction of the glutamatergic pathways has been

implicated in progressive degenerative diseases, such as

Alzheimer’s disease, Huntington’s disease, Parkinson’s disease,

amyotrophic lateral sclerosis, lathyrism, acquired immunode-

ficiency syndrome (AIDS), encephalopathy and dementia

complex and pain/hyperalgesia, as well as schizophrenia and

other psychiatric disorders (Takagi et al., 1997; Adamchik and

Baskys, 2000; Sze et al., 2001; Conn, 2003; Sung et al., 2003;

Lee et al., 2004; Trudeau, 2004).

Excitatory amino acids, which normally participate in

signaling pathways in the CNS, when present at elevated

concentrations are neurotoxic, and have been implicated in both

acute injury, such as caused by epileptic seizure or hypoxia, and

during periods of ischemia and hypoglycemia (Sala et al., 2000;

Chandler et al., 2001; D’Onofrio et al., 2001; Monfort et al.,

2002; Massieu et al., 2003; Tauskela et al., 2003).

Glutamate interacts with at least three related classes of

ionotropic receptor channels, each commonly referred to by

their preferred pharmacological agonists: N-methyl-D-aspartate

(NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic

acid (AMPA) and kainic acid (KA) (Sala et al., 2000; Chandler

et al., 2001; D’Onofrio et al., 2001; Monfort et al., 2002; Nong

et al., 2004). These receptors have also been implicated in

learning and memory acquisition (Adamchik and Baskys,

2000).

1.2. GluRs in the CNS—the NMDA receptor subtype

The NMDA subtype of the GluR family is an excitatory

neurotransmitter receptor whose subunits comprise a generally

non-selective cation channel and exhibits voltage-dependent

blockade by magnesium (Mg2+) (Nong et al., 2004). Unlike

AMPA and KA receptors, the NMDA receptor (NMDAR) is

also permeable to Ca2+, and the resultant increased intracellular

Ca2+ ([Ca2+]i) in neuronal cells upon gating is thought to be

responsible for evoking the receptor’s role in neuronal plasticity

and neurotoxicity (Perez-Otano and Ehlers, 2004). Further-

more, glycine is a coagonist and essential for the NMDAR

activation (Nong et al., 2004).

While it is known that the pathogenesis of many

neurodegenerative conditions and psychiatric disorders is

mediated, at least in part, by the NMDARs (Takagi et al.,

1997; Adamchik and Baskys, 2000; Sze et al., 2001; Sung et al.,

2003; Ueda, 2004), little fundamental information is available

at the basic level of receptor function. There is, therefore, a

substantial need for effective prophylaxis and therapy in acute

and chronic neurodegenerative disorders that involve excito-

toxic mechanisms.

As noted earlier on, the NMDAR is a crucial receptor for the

neurotransmitter glutamate, which is the most important

excitatory transmitter in the brain (Nong et al., 2004). The

NMDAR, moreover, is not only a receptor, but also a channel (it

is thus a ligand-gated ionic channel). N-methyl-D-aspartate is

the well-known agonist of the NMDAR, the latter being named

after it. It is a very complex ligand-gated channel that seems to

be involved in the toxic effects of excessive glutamate and in

many other processes, such as synaptic plasticity and target

recognition (Wang and Durkin, 1995; Ahn et al., 2000; Barros

et al., 2000; Cammarota et al., 2000; Jiang et al., 2000b; Sala

et al., 2000; Chandler et al., 2001; D’Onofrio et al., 2001;

Monfort et al., 2002).

1.3. GluRs and NMDARs—pathway interactions

The interplay between GluRs, particularly NMDARs, and

molecular signaling pathways is often obscured by the fact that

NMDA basically mediates neurotoxic (or excitotoxic) effects in

Ca2+-dependent and independent manners. However, the

relationship between NMDA and signaling mechanisms

involved in a variety of cellular processes is still precarious,

needless to mention its potential involvement in regulating the

mitogen-activated protein kinase (MAPK)-related pathways

(Wang et al., 2004).

Recent accumulating evidence points in the direction of the

fact that MAPKs play a crucial role in regulating the

neurochemistry of NMDARs, their physiologic properties

and their potential role in pathophysiology (Yun et al., 1999;

Cammarota et al., 2000; Chattopadhyay and Brown, 2000;

D’Onofrio et al., 2001; Jiang et al., 2000b; Fuller et al., 2001;

Hardingham et al., 2001; Rush et al., 2002; Waltereit and

Weller, 2003; Wang et al., 2004).

1.4. The purpose of undertaking this survey

In this respect, I have designed this review to discuss: (i) the

MAPK revolutionary role in regulating a plethora of cellular

functions, including the NMDAR-related pathways; (ii) the role

of MAPK modules in regulating the biochemistry and

physiology of NMDARs and related properties; (iii) the

kinetics of NMDA–MAPK interactions and their biologic

properties; (iv) how cellular signaling pathways, related


cofactors (such as transcription factors), and intracellular

conditions within the microenvironment affect NMDA–MAPK

interactions and (v) the role of NMDA–MAPK modules in

pathophysiology and the evolution of disease conditions.

2. MAPK signaling pathways: an overview

2.1. MAPK identification and nomenclature

MAPKs were identified by virtue of their activation in

response to growth factor stimulation of cells in culture, hence

the name mitogen-activated protein kinases (Mordret, 1993;

Cano and Mahadevan, 1995; Kennedy et al., 1999; Lee et al.,

2000; Avruch et al., 2001; Chakraborty, 2001; Pierce et al.,

2001; Haddad, 2004b).

On the basis of in vitro substrates, the MAPKs have been

variously called microtubule associated protein-2 kinase

(MAP-2 kinase), myelin basic protein kinase (MBP kinase),

ribosomal S6 protein kinase (RSK-kinase; i.e., a kinase that

phosphorylates a kinase) and epidermal growth factor (EGF)

receptor threonine kinase (ERT kinase) (Cano and Mahadevan,

1995; Kennedy et al., 1999; Lee et al., 2000; Avruch et al.,

2001; Chakraborty, 2001; Pierce et al., 2001).

2.2. MAPK biochemistry and properties

All of these proteins have similar biochemical properties,

immuno-cross-reactivities, amino acid sequence and ability to

in vitro phosphorylate similar substrates. Maximal MAPK

activity requires that both tyrosine and threonine residues are

phosphorylated. This indicates that MAPKs act as switch (dual)

kinases that transmit information via increased intracellular

tyrosine and serine/threonine phosphorylation (Guan, 1994;

Sugden and Clerk, 1997; Ono and Han, 2000).

Fig. 1. A model of gene regulation where the switch on/off mediates a series of ph

respectively. This sequential propagation of signals occurs in response to a stimul

Although MAPK activation was first observed in response to

activation of EGF, platelet-derived growth factor (PDGF), nerve

growth factor (NGF) and insulin and insulin-like receptors, other

cellular stimuli, such as T cell activation (which signals through

the Lck tyrosine kinase), phorbol esters (that function through

activation of protein kinase C (PKC)), thrombin, bombesin and

bradykinin (that function through G-proteins), as well as

NMDAR activation and electrical stimulation rapidly induce

tyrosine phosphorylation of MAPKs (Mordret, 1993; Guan,

1994; Cano and Mahadevan, 1995; Sugden and Clerk, 1997;

Kennedy et al., 1999; Lee et al., 2000; Ono and Han, 2000;

Avruch et al., 2001; Chakraborty, 2001; Pierce et al., 2001).

2.3. MAPK signaling pathways and activation

MAPKs are not the direct substrates for receptor tyrosine

kinases (RTKs) or receptor-associated tyrosine kinases but are in

fact activated by an additional class of kinases termed MAP

kinase kinases (MAPK kinases) and MAPK kinase kinases

(MAPKK kinases) (Haddad, 2004b). Amodel of gene regulation

where the switch on/off mediates a series of phosphorylation/

dephosphorylation steps regulated by kinases and phosphatases

(including MAPKs), respectively, is shown in Fig. 1. This

sequential propagation of signals occurs in response to a stimulus

over a pre-specified period of time, in a similar manner to the

mechanisms regulating MAPK signalling pathways.

One of the MAPK kinases has been identified as the proto-

oncogenic serine/threonine kinase, Raf (Cobb et al., 1994;

Errede et al., 1995; Marshall, 1995; Morrison, 1995; Schle-

singer et al., 1998; Tibbles and Woodgett, 1999; Kolch, 2000;

McCubrey et al., 2000; Sebolt-Leopold, 2000; Weinstein-

Oppenheimer et al., 2000; Hagemann and Blank, 2001;

Liebmann, 2001; Zhu et al., 2001; English and Cobb, 2002;

Lee and McCubrey, 2002).

osphorylation/dephosphorylation steps regulated by kinases and phosphatases,

us over a pre-specified period of time.


The best-characterized vertebrate MAPKs fall into three

subgroups (Haddad, 2004b). The first subgroup includes the

founding members of the MAPK family, extracellular signal-

regulated kinase-1 (ERK1 or MAPKERK1/p44) and ERK2 (or

MAPKERK2/p42), and their closest relatives (Frye, 1992;

Denhardt, 1996; Peyssonnaux and Eychene, 2001; Belcheva

and Coscia, 2002; Howe et al., 2002). This subgroup is often

referred to as ERKs, although some ERK proteins are not in

fact members of this subgroup family.

The second subgroup is the Jun N-terminal kinases (JNKs),

so called because they can activate the Jun transcription factor

by phosphorylating two residues near its N-terminus (Ip and

Davis, 1998; Noselli, 1998; Leppa and Bohmann, 1999; Noselli

and Agnes, 1999; Davis, 2000; Mielke and Herdegen, 2000;

Rincon et al., 2000; Barr and Bogoyevitch, 2001; Dong et al.,

2001; Harper and LoGrasso, 2001; Okazawa and Estus, 2002;

Weston and Davis, 2002).

The third subgroup is the p38 MAPKs, so named because

of the molecular weight (38 kDa) of the first representative

of the subgroup to be discovered (Lee and Young, 1996;

Lopez-Ilasaca, 1998; English et al., 1999; Ichijo, 1999;

Obata et al., 2000; Haddad, 2001a; Rincon, 2001; Bulavin

et al., 2002).

Fig. 2. The MAPK network and its regulation by upstream and downstream ki

Members of both the MAPKJNK and MAPKp38 pathways are

also classified as stress-activated protein kinases (SAPKs),

because they are activated in response to osmotic shock, UV

irradiation, inflammatory cytokines and other stressful condi-

tions. In all three subgroups, a large number of MAKKKs feed

into the activation of a smaller number ofMAPKKs andMAPKs.

The diversity of the MAPKKKs thus allows a wide variety of

upstream receptors to couple to MAPK cascades (Marshall,

1994; Lewis et al., 1998; Pearson et al., 2001; Haddad, 2002a;

Haddad and Land, 2002; Haddad et al., 2003) (Fig. 2).

It is beyond the scope of this paper, however, to discuss the

entirety of MAPK regulation and their bifurcations. Excellent

reviews, in this regard, have been recently released covering

this topic (Haddad et al., 2003; Boldt and Kolch, 2004;

Bogoyevitch et al., 2004; Haddad, 2004a,b; Kyosseva, 2004;

Ory andMorrison, 2004; Roux and Blenis, 2004; Rubinfeld and

Seger, 2004; Wada and Penninger, 2004). Therefore, I will

concentrate on the pathways mediating NMDA–MAPK

interactions, after having introduced the biophysics of the

NMDAR.

For clarity, MAPK signaling pathways and their ramifica-

tions have been schematized in Fig. 2. MAPKs and their

corresponding substrates are given in Table 1.

nases and signaling molecules (see text for discussion and further details).


Table 1

MAPKs and downstream transcription factors and substrates

Kinase Substrates

MAPKERK Elk-1; SAP-1; Mnk-1/2; MAPKAP-K1/p90Rsk;

MSK-1

MAPKJNK/SAPK c-Jun; ATF-2; Elk-1

MAPKp38 ATF-2; Elk-1; SAP-1; CHOP; MEF2C;

MAPKAP-K2/K3; Mnk-1/2; MSK-1; PRAK

MAPKAP-K1/p90Rsk c-Fos; SRF

MSK-1 CREB; Histone H3 and HMG-14

Abbreviations: ATF, activating transcription factor; CHOP, C/EBP homologous

protein; CREB, cAMP response element binding protein; HMG-14, high

mobility group-14; MAPKERK, mitogen-activated protein kinase/extracellular

signal-regulated kinase; MAPKAP-K, MAPK-activated protein kinase;

MEF2C, myocyte enhancer factor 2C; Mnk, MAPK interacting protein kinase;

MSK, mitogen- and stress-activated protein kinase; PRAK, p38-related/acti-

vated protein kinase; Rsk, ribosomal S6 kinase; SAP-1, SRE accessory protein-

1; SRF, serum response factor..

3. NMDARs: analytical structure, biophysicalproperties and biochemical interactions

3.1. Receptor bio-analytical assessment—biophysics

Glutamate receptors, as indicated above, are the CNS’s

major excitatory neurotransmitter receptors. GluRs are

described as being ionotropic, linked directly to a ligand-

gated ion channel, which mediates fast forms of excitatory

synaptic transmission, or metabotropic, a diverse grouping of

G-protein coupled receptors, which are linked to multiple

second messenger systems regulating synaptic transmission

and neuronal excitability (Nong et al., 2004).

The ionotropic GluRs are further classified into three groups

based upon their electrophysiological and pharmacological

properties: AMPA receptors, KA receptors and NMDARs.

Fig. 3. The analytical structure of the NMDA recept

Metabotropic GluRs induce internal Ca2+ mobilization by

hydrolysis of inositol phosphate. There exist presynaptic

mGluRs whose role is to function as auto-receptors inhibiting

glutamate release, thereby attenuating glutametergic transmis-

sion (Chen et al., 2002).

The subunit composition of these receptors confers the

characteristic pharmacological and electrophysiological prop-

erties inherent to each receptor class. There are at least 15

identified subunits that combine into multimeric complexes that

constitute GluRs (Dingledine and Conn, 1999; Nong et al.,

2004). The metabotropic GluRs act as monomers or homo-

dimers, thus forming three classes containing two or more

individual receptor proteins: Group I consisting of mGluR1 and

mGluR5, Group II consisting of mGluR2 and mGluR3 and

Group III consisting of mGluR4, mGluR6, mGluR7 and

mGluR8.

Ionotropic receptors, on the other hand, are heterotrimers.

AMPA receptors are made up of GluR1–4, and KA receptors

consist of GluR5–7 and KA-1/2. Themost heterogeneous group

of ionotropic receptors is the NMDA class. Two non-

contiguous binding domains called S1 and S2 have been

identified in ionotropic GluRs including NMDAR. A recent

mutagenesis study has identified six sites on NR2A involved in

NMDAR glutamate binding (Lummis et al., 2002) (Fig. 3).

3.2. NMDAR biophysical and biochemical properties

The NMDARs play a major role in synapse development,

plasticity and cell death during ischemia (Nong et al., 2004).

The receptor is composed of an NR1 (z1 or S1) subunit,

expressed in eight splice variants combined with four NR2

subunits. Four classes of NR2 subunits have been identified and

designated as NR2A (e1) NR2B (e2), NR2C (e3) and NR2D

(e4).

or and its interactions and potential antagonists.


NR1 and NR2 are proteins consisting three complete

transmembrane and an intra-membranous loop linking the first

two transmembrane regions. A third type of receptor unit has

recently been described as NR3, which exists in two variants:

NR3A and NR3B (Eriksson et al., 2000; Matsuda et al., 2002;

Sasaki et al., 2002).

In the mature nervous system, NMDARs are complexes

composed primarily of NR1, NR2A, NR2B and NR2C

subunits, while some contain only NR1 combined with either

NR2A or NR2B. A rare class of the receptor contains NR2D.

All NMDARs contain an NR1 subunit without which the

NMDAR cannot function (Luo et al., 1997; Chazot, 2004).

The NR2B/D variants predominate in the immature nervous

system. In newborn hippocampal granule cells of the rat,

NMDAR are heterogeneous groups consisting of NR1/NR2B

diheteromers and NR1/NR2B/D triheteromers (Pina-Crespo

and Gibb, 2002). Binary complexes consisting of only NR1

with NR2D have been described in the thalamus (Dunah et al.,

1998; Miyamoto et al., 2002). NR1 is transported to the surface

of transfected cells and expressed alone. However, NR2A

subunits require the presence of NR1 in order to be transported

to the surface (Garcia-Gallo et al., 2001).

Binding of glutamate to NR2 and glycine to NR1 subunits is

necessary for the activation of NMDARs. The presence of NR2B

conveys a high affinity for glycine and glycine agonists.

Interaction between theNR1 andNR2 subunits regulates glycine

affinity. The NR2A subunit conveys high affinity for glutama-

tergic agonists. By contrast, the NR2C subunit has very low

affinity for these agonists (Lynch and Guttmann, 2002) (Fig. 4).

The NR3A/B subunit appears to be a modulating element

when present in the NMDAR. NR3A is widely expressed, while

NR3B is predominantly found in motor-neurons (Nishi et al.,

2001). The two variants show close homology and share the

functional domains found in GluRs: the channel gate, channel

pore and ligand-binding domain.

NR3A can bind protein phosphatase at its carboxy terminus,

while NR3B may not as this region is different in these subunits

(Matsuda et al., 2002). The presence of an NR3 subunit results

in decreased Ca2+ permeability and decreased Mg2+ sensitivity.

In cell systems recombinantly expressing NR3, it co-assembles

Fig. 4. Biophysical properties and ion interactions mediated by ionotropic

NMDA and AMPA glutamate receptors.

with NR1 to form receptors in COS cells with slightly increased

mean open time compared to NR1/NR2 based receptors, or

receptors in Xenopus oocytes which are excited by glycine but

unaffected by either glutamate or NMDA stimulation

(Chatterton et al., 2002; Sasaki et al., 2002).

NMDA channels can also be subdivided into two classes by

their conductance properties: ‘high conductance channels’ are

formed by NR2A or NR2B subunits, while ‘low conductance

channels’ are formed by NR2C and NR2D subunits. The low

conductance channels are characterized by reduced sensitivity to

Mg2+ block (Cull-Candy et al., 1998; Nong et al., 2004) (Fig. 4).

There is also a difference in channel open probability

between channels composed of NR2A and NR2B subunits. The

peak channel open probability is two- to five-fold higher for

N2A channels, resulting in peak current densities that are four

times greater in channels made up of NR2A subunits than for

NR2B channels (Chen et al., 1999, 2000). Peak amplitude of

postsynaptic excitatory potentials can, therefore, be modulated

by altering the relative contribution of NR2A and NR2B

subunits to NMDARs.

Recombinantly expressed NR1/NR2D receptors have

demonstrated very slow decay kinetics following stimulation

with glutamate (Cheffings and Colquhuon, 2000; Pina-Crespo

and Gibb, 2002; Brickley et al., 2003; Chen et al., 2004). The

implication is that adult neurons expressing NR2D subunits

would have extended postsynaptic excitatory potentials.

Presynaptically expressed NR2D-comprising receptors would

be expected to exert a depression of postsynaptic currents.

NMDARs are modulated by endogenous and exogenous

factors. For instance, PKC potentiates NMDAR function. This

potentiation is mediated by activation of non-RTKs phosphor-

ylating subunits NR2A and NR2B alone or in conjunction

(Grosshans and Browning, 2001). NR2C and NR2D are

unaffected by PKC. This difference in subunits underlies the

variability in PKC action in control of excitotoxicity.

Furthermore, activation of Pyk2, Src andFynkinases has been

shown to increase tyrosine phosphorylation ofNR2A andNR2B.

Phosphorylation of tyrosine at the C-terminus of NR2A affects

current fluxes through theNMDARchannels (Kohr and Seeburg,

1996). Metabotropic glutamate receptor-1 regulates the tyrosine

phosphorylation of NR2A/B by controlling the basal activity of

Pyk2 and Src (Heidinger et al., 2002). The NR1 subunit is not

tyrosine phosphorylated (Lau and Huganir, 1995). PKC directly

phosphorylates NR2B at residues S1303 and S1323 in the C-

terminus, enhancing currents through the NMDAR channel.

However, NR2C is unresponsive to PKC (Liao et al., 2001).

Another mechanism regulating NMDAR Ca2+ permeability

is activation of Ephrin B receptors, which recruit Src kinases.

This results in tyrosine phosphorylation of NR2A/B subunits. It

is theorized that this system may play a role in regulating

synaptic plasticity (Ghosh, 2002).

Classically, Mg2+ blocks NMDA channels in a voltage-

dependent manner (Fig. 4). At positive membrane potential,

Mg2+ acts as a potentiator of NMDA-induced responses. The

blocking action of Mg2+ is determined by specific residues

within theM2 domain of NR2 subunits (Vissel et al., 2002). The

residues mediating this block have been identified as asparagine


and serine. Asparagine is a major determinant of ion specificity

critical to NMDAR function (Mishra et al., 2002).

Cations, such as Na+, K+ and Ca2+, pass through the

NMDAR channel (Fig. 5). NMDARs are partially inhibited by

ambient H+ (Liu and Zhang, 2000). Zinc (Zn2+) is both voltage-

dependent and independent inhibitor of NMDAR. The NR1

subunit is important in mediating the inhibitory effect of Zn2+

and H+, as well as responses to polyamines (Traynelis et al.,

1998; Duguid and Smart, 2004; Kashiwagi et al., 2004).

The lead (Pb2+) cation is also an inhibitor of NMDAR, but

the degree of inhibition is determined by NR2 subunit

composition. Inhibition by Pb2+ is most potent in NR2A

receptors > NR2C > NR2D; Pb2+ is least potent against

NR2A/C complexes, which are actually potentiated by Pb2+

at very low (<1 mM) concentrations (Omelchenko et al., 1997;

Guilarte and McGlothan, 2003).

In excitatory CNS synapses, Ca2+ transients result in Ca2+-

dependent inactivation of the NMDAR activity. This inactiva-

tion is mediated by Ca2+/calmodulin interaction with a-actinin

at the C-terminus of NR1 subunits, which is essential for

inactivation. The NR2 subunit modulates Ca2+-dependent

inactivation since this does not occur in NR2C-containing

receptors, but does occur in the presence of NR2A. This action

Fig. 5. Electrically active cells, such as neurons and muscle cells, maintain a

resting membrane potential of approximately �70 mV. Neurotransmitters

depolarize or hyperpolarize the cell membrane by opening ion channels within

the membrane that are either an integral part of the receptor molecule (ligand-

gated ion channels) or that are linked to the receptor through a G-protein

mediated mechanism (ion channel-linked receptors). Occupation of the ligand

recognition site of a ligand-gated ion channel induces changes in the con-

formation of the channel-forming protein such that ion flux across the mem-

brane is increased. In contrast, voltage-gated channels open or close in response

to a change in voltage across the adjacent cell membrane. These channels are

responsible for the generation of action potentials in electrically excitable cells.

If Na+ permeability is increased, Na+ moves into the cell down its concentration

gradient and the membrane becomes depolarized in the region of the open

channel. In contrast, if Cl� permeability is increased, the membrane will

become hyperpolarized in the region of the open channel. Depolarization is

associated with generation of action potentials, degranulation, neurotransmis-

sion and muscle contraction. Hyper-polarization is associated with inhibition of

these processes. The resting membrane potential is restored and maintained by

activation of the Na+-K+-ATPase pump that actively extrudes Na+ from the cell.

has been ascribed to M2-3 loop of NR2, which is presumed to

interact with the NR1 C-terminal (Vissel et al., 2002).

Desensitization of NMDARs, furthermore, is modulated by

calcineurin, through dephosphorylation of the C-terminus of

NR2A (Krupp et al., 2002). The N-terminal domain may also

play a role in desensitizing NMDARs. Domains flanking the

agonist-binding domain control glycine-independent desensi-

tization: a short four-residue domain in the first transmembrane

(M1) region and a domain containing a leucine/isoleucine/

valine-binding motif. This pre-M1 region may serve as a link

between ligand binding and channel gating (Krupp et al., 1998).

Voltage-gated and ion channels involving the regulation of

ion flow are schematized in Fig. 5.

3.3. NMDAR regulation during development, stress and

exogenous pharmacological interventions

The subunit composition of NMDARs undergoes modifica-

tion during development and aging, as well as in response to

physiological stress or exogenously applied pharmacological

reagents.

3.3.1. Development and distribution

The developmental profile of the various NMDA subunits

shows regional, temporal and tissue specific variability, and

postnatal alterations in subunit composition may confer the

basis for physiological characteristic of specific regions of the

brain, as well as synaptic plasticity (Sheng et al., 1994;

Simeone et al., 2004).

In adult brain NR2D is restricted to diencephalon,

mesencephalon and brain stem structures, such as globus

pallidus, thalamus, subthalamic nuclei and superior colliculus.

It is transiently expressed in other brain regions, such as

hippocampus, thalamic ventrobasal comples, brainstem reti-

cular formation and inferior colliculus (Wenzel et al., 1997).

NR2C is confined to cerebellum, thalamus and olfactory

bulb in adult rat brain (Wenzel et al., 1995). In cultured cortical

neurons, the developmental profile of subunits mimics the in

vivo pattern. NR1 and NR2B units are the first to appear with

NR2A expression being delayed relative to the other two

(Li et al., 1998). In cortex and striatum, NR2A expression is

absent at birth but develops postnatally, in contrast to the

hindbrain where low levels of NR2A predominate while NR2B

is only transiently expressed during the early postnatal period

(Portera-Cailliau et al., 1996; Simeone et al., 2004).

In the rat brain, NR1 and NR2B/D predominate at all stages

of CNS development prenatally, while NR2A/C is first

detectable prior to birth. As the brain develops, NR2A becomes

near ubiquitous, while NR2B becomes progressively restricted

in its expression to forebrain structures (Wenzel et al., 1995,

1997). In the hippocampus, NMDARs at birth are NR1/NR2B

diheteromers and NR1/NR2B/D triheteromers (Pina-Crespo

and Gibb, 2002). The adult hippocampus shows NR2A and

NR2B prominent in the CA1 and CA3 pyramidal cells, while

NR2C and NR2D are expressed in a different class of

interneurons (Monyer et al., 1994)


In the adult cerebellum, NMDA subunits show differential

distribution patterns. NR2C/D are restricted to cerebellar

granule cells, with some NR2C also found in thalamus and

olfactory bulb. NR2C is barely expressed in rat forebrain. Three

variants of NR2C have been described in developing

cerebellum, one of which does not contribute to the

development of a functional receptor channel (Rafiki et al.,

2002; Simeone et al., 2004).

NR2B is prominent in Purkinje cells but absent from granule

cells. NR2A is lightly distributed in granule cells but heavier in

Purkinje cells and Bergmann glia (Thompson et al., 2000). In

the dorsal root ganglion, two classes of NMDARs have been

identified. The C-fibers express NMDARs consisting of NR1/

NR2D and some NR2C subunits, while A and C fibers both

express NR1/NR2B receptors (Marvizon et al., 2002). The

NR3B subunit is highly expressed in midbrain, pons, medulla

and spinal chord. Throughout development its expression level

remains constant. However, NR3A decreases sharply post-

natally (Matsuda et al., 2002).

With advancing age, changes in subunit expression have

been observed which may underlie age related the NMDAR

function alteration. In mouse cerebral cortex significant

decreases in both NR1 and NR2A subunits were observed

between 10 and 30 months of age. In the hippocampus,

NR2B subunit expression increased while NR1 decreased

over the same time period. Synaptophysin levels also

decreased concomitant with subunit alteration (Magnusson

et al., 2002).

3.3.2. The role of stress

Physiological stress can alter the expression patterns and

phosphorylation states of NMDA subunits. For instance,

ischemia lowers the expression of NR2A/B receptors with

concomitant diminished NMDAR contribution to the excitatory

postsynaptic potential. The degree of alteration shows regional

variation within the hippocampus (Zhang et al., 1997; De Biasi

and Dani, 2003).

Transient ischemia results in increased tyrosine phosphor-

ylation of NR2A/B subunits, with the degree of phosphoryla-

tion varying between regions of the brain (Takagi et al., 1997).

Increased phosphorylation of NR1 following hypoxia-ischemia

proportional to the degree of neuronal injury has been reported

(Guerguerian et al., 2002). Graded hypoxia results in

diminished number of glutamate sites, increased affinity of

the ion channel, decreased activation by glycine, increased

inhibition by Zn2+, and increased activation by glutamate which

correlate with the tissue energy state (Fritz et al., 2002).

Brief in vitro hypoxic hypoclycemia induced the expression

of NR2C subunits (Perez-Velazquez and Zhang, 1994).

Sublethal ischemic episodes can precondition neurons so that

they are protected against a second more severe insult. When

neurons were subjected to a non-lethal potassium cyanide dose,

no changes in NR1/NR2A/B message levels were recorded.

This pre-conditioning did not raise NR2C message levels,

unlike the more severe hypoxic-hypoglycemia (Aizenman

et al., 2000), possibly suggesting that NR2C is recruited in

response to severe insult.

3.3.3. The role of nitration

Nitration appears to be a key mechanism of hypoxia-

mediated modification of the NMDAR (Kosenko et al., 2003).

Peroxynitrite, for example, increases the affinity of both the ion

channel and glutamate binding sites (Zanelli et al., 2000;

Mishra et al., 2002). In order for nitration to proceed, tyrosine

dephosphorylation must first occur, since the nitration site is

located at the adjacent ortho position on tyrosine residues.

Dephosphorylation possibly removes a steric hindrance to

nitration (Mishra et al., 2002).

Increased nitration of NR1, NR2A and NR2B has been

correlated with decreased high-energy phosphates, ATP and

phosphocreatine during hypoxia in cerebrum. Suppressing

nitration by blocking nitric oxide synthase (NOS) prevents the

characteristic NMDAR-mediated Ca2+ influx during hypoxia

(Zanelli et al., 2002). Thus, it appears that as the neurons energy

is depleted by hypoxia, maintaining phosphorylation of the

NMDAR subunits is compromised and nitration increases,

resulting in greater Ca2+ entry. Nitration is, therefore, a

mediator of excitotoxicity.

It could be theorized that in animals that survive extended

periods of hypoxia such as theWestern painted turtle Chrysemis

picta belli, an anoxia-tolerant invertebrate (Haddad, 2004a),

suppression of nitration could be a possible mechanism

employed to prevent catastrophic Ca2+ influx through NMDA

channels during low oxygen exposure.

3.3.4. NMDA regulation and pathophysiology

Pathological conditions can also affect the NMDAR subunit

expression. Peripheral nerve lesions, for instance, result in

reduced expression of NR2A in rat dorsal horn (Karlsson et al.,

2002). Traumatic brain injury results in significant loss of NR1

and NR2A/B subunits from the hippocampus post injury,

followed with recovery. The loss is due to non-transcriptional

changes in the NMDAR, since mRNA levels remain unchanged

during the post injury period. This loss of receptor subunits may

explain temporary memory loss after injury (Kumar et al.,

2002).

Alzheimer’s disease also results in selective and differen-

tially distributed reduction in the NMDAR subunits. Significant

reductions in NR1 and NR2B have been demonstrated in the

hippocampus, with loss of NR2A/B from entorhinal cortex. No

significant changes in NR2A/B were noted in cerebellum,

occipital cortex or caudate (Sze et al., 2001; Bi and Sze, 2002).

The losses of both phosphorylated and non-phosphorylated

subunit proteins correlated with changes in the presynaptic

protein synaptobrevin. These losses were positively correlated

with cognitive deficits and disease stage (Sze et al., 2001; Bi

and Sze, 2002). Elimination of synaptic input has been

demonstrated to alter subunit expression profiles in the

developing visual cortex of the cat. Dark-rearing of kittens,

for example, resulted in depressed NR2A levels (Chen et al.,

2000).

3.3.5. Pharmacological modulation of the NMDAR

The function of one glutamate receptor type can affect

functioning of other receptors based on subunit type. mGluR1


can reduce the NMDAR function in terms of internal Ca2+ rise

and neurotoxicity. The effect of mGluR1 is mediated by PKC

activation, but requires the presence of NR2C subunits (Pizzi

et al., 1999). Activation of mGluR1 in the hippocampus results

in rapid loss of NMDA and AMPA receptors (Snyder et al.,

2001). Receptor antagonists can also affect the NMDAR

expression in rat hippocampus. Systemic treatment with GYK-

152466 and aniracetam changed NR2B mRNA and, subse-

quently, binding of ifrenpodil (Healy and Meador-Woodruff,

2000).

Monosodium glutamate (MSG) has been shown to alter

subunit expression. Administration of subcutaneous MSG to

neonatal rats resulted in an upregulation of NR2C and NR2D

expression in adult hippocampus and striatum, but not cerebral

cortex (Bas-Zarate et al., 2002). In cultured cerebellar granule

cells, brain-derived neurotrophic factor (BDNF) and fibroblast

growth factor (FGF2) protect against the excitotoxicity of

NMDA by down regulating NR2A/C subunits. A concomitant

decrease in NMDA mediated rise in [Ca2+]i occurs, suggesting

that the neuroprotectivemechanism of these trophic factors is to

reduce NMDA subunit expression (Johnson et al., 1993;

Brandoli et al., 1998).

3.3.6. Ethanol and the modulation of the NMDAR

Ethanol is a potent inhibitor of NMDARs, but its precise

mode of action remains obscure (Nagy, 2004). Ethanol does

not cause open channel block by itself and fails to interact

with Mg2+ at its active site (Wirkner et al., 1999). Complicating

the issue, the sensitivity of NMDARs to ethanol also shows

regional variability. Ethanol sensitivity is influenced by

subunit composition, phosphorylation and protein–protein

interactions.

Ethanol inhibition of NR1/NR2B receptors is unaffected by

Fyn tyrosine kinase, in contrast to NR1/NR2A where ethanol

inhibition is reduced by Fyn-mediated phosphorylation of

NR2A subunits (Anders et al., 1999). Chronic ethanol exposure

also upregulates NR2B, but does not affect its phosphorylation

(Hardy et al., 1999; Kalluri and Ticku, 1999; Narita et al.,

2000).

Ethanol affects the developmental profile of subunits

(Kalluri and Ticku, 2003). For instance, ethanol delayed

developmental switchover from NR2B to NR2A in cultured

cerebellar granule cells (Snell et al., 2001). In postnatal rat

hippocampus, ethanol significantly raised NR1 and NR2D,

while decreasing NR2C (Naassila and Daoust, 2002). Due to

the modulatory effects of these subunits, this alteration may be

a factor in neuro-developmental disorders of fetal alcohol

exposure. Moreover, in rat cerebral cortex, ethanol resulted in

increased NR2B expression and altered NR1 splicing, but was

without effect on NR2A/C.

A mutagenesis experiment demonstrated the importance of

the transmembrane-3 domain in NMDA subunits in reducing

ethanol inhibition in the NMDARs expressed in oocytes

(Ronald et al., 2001). Chronic ethanol exposure leads to

adaptive upregulation of NMDARs and AMPA receptors, but

not kainate receptors (Chandler et al., 1999). During chronic

ethanol exposure, the NR2B subunit expression is upregulated

in the forebrain but not in cerebral cortex, presumably as an

adaptive response. This may also play a role in the development

of alcohol dependency, since the NMDA antagonist ifenprodil,

which is selective for NR2B, suppresses alcohol withdrawal

symptoms (Narita et al., 2000).

4. NMDA-mediated regulation of MAPK signaling

pathways

In the forthcoming sections, an extensive elaboration on the

biochemical interactions between MAPKs and the NMDARs is

developed.

4.1. NMDA-mediated regulation of G-coupled receptor

proteins

4.1.1. NMDA, CaMK and MAPK regulation

The first indication of a role for NMDA in regulating an

upstream kinase related to the extended MAPK superfamily

was reported a decade ago. Ca2+/calmodulin-dependent protein

kinase II (CaMK) and MAPKERK2 are enriched in neurons and

possess the capacity to become persistently active, or

autonomous, following removal of the activating stimulus.

Since persistent kinase activation may be a mechanism for

information storage, Murphy et al. (1994) have used primary

cultures of cortical neurons to investigate whether kinase

autonomy can be triggered by bursts of spontaneous synaptic

activity. It was reported that both these kinases (CaMK and

MAPKERK2) respond to synaptic stimulation, but differ

markedly in their kinetics of activation and inactivation, as

well as in their sensitivity to NMDAR blockade. While

maximal CaMK activation was observed almost instanta-

neously of synaptic bursting, MAPK activity was unaffected at

this early time and rose to sub-maximal after stimulation

(Murphy et al., 1994). In addition, following blockade of

synaptic stimulation, CaMK activity decreased, while MAPK

activity decayed instantly.

Although MAPK exhibited relatively slow activation, short

periods of synaptic activity could trigger the MAPK activation

process, which persisted in the absence of synaptic stimulation.

Comparison of the effect of NMDAR blockade on synaptic

activation of these kinases revealed that CaMK activity is

preferentially suppressed. Immunocytochemical studies indi-

cated that CaMK is concentrated in dendritic processes in the

vicinity of synapses (Murphy et al., 1994); therefore, synaptic

Ca2+ transients in fine dendritic processes were measured to

assess their sensitivity to NMDAR blockade.

Ca2+ transients in these fine processes were reduced by

NMDAR blockade, possibly accounting for the profound

sensitivity of CaMK to this treatment. Furthermore, the sharp

contrast between the regulation of CaMK and MAPK by

synaptic activity indicates that they may mediate neuronal

responses to different patterns of afferent stimulation

(Murphy et al., 1994). The relatively slow activation and

inactivation of MAPK suggests that it may be able to

integrate information from multiple, infrequent bursts of

synaptic activity.


Fig. 6. A scheme depicting the activation of NMDA receptors and the regula-

tion of MAPK signaling pathways through cAMP- and PKA/PKC-sensitive

mechanisms. Downstream from G-protein coupled receptors (GPCRs) cAMP

activates PKA, which phosphorylates NMDA receptor. PKA phosphorylates

NMDA receptor subunits, thereby altering receptor conformation to enhance

sensitivity to glutamate. Alternatively, cAMP activates MEK1/2 in a PKA-

independent manner (dashed arrow). NMDA receptor activation results in an

increase in Ca2+ influx, which, in turn, activates MAPKs signaling-related

cascades. MK-801 blocks the NMDA receptor in the phosphorylated state.

Activation of MAPKERK via the upstream MEK1/2, which is regulated by

cAMP/PKA, is potentially blocked by PD-98059. MAPKp38 inhibition by SB-

203580 blocks the phosphorylation of Elk-1 and CREB, which regulates the

phosphorylation of the downstream cofactor, c-Fos.

4.1.2. NMDA, AMPA and MAPK regulation

A crucial mechanism for NMDA regulation has also been

reported to involve an upstream G-coupled receptor protein.

AMPA receptor channels are known to play important roles in

plasticity, neurotransmission and neuro-toxicity in the CNS

(Wang and Durkin, 1995). Of note, AMPA, but not NMDAR,

receptor signaling in rat cortical neurons was found to involve a

G-protein coupled to a protein kinase cascade. While both

NMDA and AMPA activatedMAPKERK2 in neurons, consistent

with the aforementioned observations, only AMPA-induced

MAPK was inhibited by pertussis toxin (PTX).

4.1.3. NMDA, CREB and MAPK regulation

In addition, AMPA, but not NMDA, caused an association of

a G-protein b subunit with a Ras, Raf kinase and MAP/ERK

kinase (MEK)-1 complex (Wang and Durkin, 1995). The

evidence indicates that AMPA, but not NMDA, triggers MAPK

activation via a novel mechanism in which G-protein bg dimers

released from Ga bind to a Ras protein complex causing the

activation of Ras, Raf kinase, MEK-1, and subsequently

MAPK.

Similarly, using primary cultures of mouse striatal neurons,

Perkinton et al. (1999) addressed whether AMPA receptors can

activate the MAPK cascade. It was observed that in the

presence of cyclothiazide, AMPA caused a robust and direct (no

involvement of NMDARs or L-type voltage-sensitive Ca2+

channels) Ca2+-dependent activation of MAPKs. This activa-

tion was blocked by GYKI-53655, a non-competitive selective

antagonist of AMPA receptors.

Probing the mechanism of this activation revealed an

essential role for phosphatidylinositol 3-kinase (PI3-kinase),

and the involvement of a PTX-sensitive G-protein, a Src family

protein tyrosine kinase, and CaMK. Interestingly, KA activated

MAPK in a PI3-kinase-dependent manner. AMPA receptor-

evoked neuronal death and arachidonic acid mobilization did

not appear to involve signaling through the MAPK pathway.

However, AMPA receptor stimulation led to a Ca2+-dependent

phosphorylation of the nuclear transcription factor cAMP-

responsive element binding protein (CREB), which could be

prevented by inhibitors of MEK or PI3-kinase (Perkinton et al.,

1999, 2002). This indicated that Ca2+ permeable AMPA

receptors transduce signals from the cell surface to the nucleus

of neurons through a PI3-kinase-dependent activation of

MAPK.

To further explore the role of Ras in the activation ofMAPKs

and CREB in hippocampal neurons, Iida et al. (2001) inhibited

Ras function by over-expressing a Ras Gap1(m), or dominant

negative Ras by means of adenovirus vectors. Gap1(m)

expression suppressed MAPK activation in response to NMDA,

Ca2+ ionophore, membrane depolarization, forskolin and

BDNF; dominant negative Ras also showed similar effects.

On the other hand, Rap1GAP did not significantly inhibit the

forskolin-induced activation of MAPK.

In contrast to MAPK activation, the inactivation of Ras

activity did not inhibit NMDA-induced CREB phosphoryla-

tion, whereas BDNF-induced CREB phosphorylation was

inhibited almost completely, demonstrating that Ras transduces

signals elicited by a broad range of stimuli to MAPK in

hippocampal neurons, and further suggesting that CREB

phosphorylation depends on multiple pathways (Iida et al.,

2001).

A hypothetical schematic depicting the activation of

NMDARs and the regulation of MAPK signaling pathways

through cAMP- and PKA/PKC-sensitive mechanisms is shown

in Fig. 6.

4.1.4. NMDA, KA and MAPK regulation

A direct effect of NMDA and related glutamate receptors on

MAPK activation was further explored (Hayashi et al., 1999).

The effect of L-glutamate and its structural analogues, NMDA,

KA and AMPA, on the activation of MAPKERK2 was examined

in cultured chick radial glia cells, namely retinal Muller cells

and cerebellar Bergmann cells (Lopez-Colome and Ortega,

1997).

Glutamate, NMDA, AMPA and KA evoked a dose- and

time-dependent increase in MAPK activity. In addition, AMPA

and KA responses were blocked by CNQX, whereas NMDA

responses were sensitive to 3-[(RS)-2-carboxypiperazin-4-yl)]-

propyl-1-phosphonate (CPP), indicating that the increase in

MAPK activity is mediated by AMPA/low affinity KA and

NMDA subtypes of GluRs (Lopez-Colome and Ortega, 1997).

Stress-activated protein kinase (SAPK; MAPKJNK) and

MAPKERK may in some circumstances serve opposing

functions with respect to cell survival. However, SAPK and

MAPKERK can also be coordinately activated in neurons in

response to glutamate stimulation of the NMDARs. To explore

the mechanisms of these MAPK activations, Schwarzschild


Fig. 7. Comparison of receptor interactions, including NMDA, with upstream,

membrane-related cofactors and downstream components of the MAPK signal-

ing pathways.

et al. (1999) compared the ionic mechanisms mediating

MAPKJNK and MAPKERK activations by glutamate. In primary

cultures of striatal neurons, glutamatergic activation of

MAPKERK and one of its transcription factor targets, CREB,

as indicated above, showed Ca2+ dependence typical of the

NMDAR-mediated responses. In contrast, [Ca2+]e was not

required for glutamatergic, NMDAR-mediated activation of

MAPKJNK and phosphorylation of its substrate, c-Jun.

Increasing extracellular Ca2+, moreover, enhanced MAP-

KERK activation but reversed MAPKJNK activation, further

distinguishing the Ca2+ dependencies of these two NMDAR-

mediated effects. Of note, reducing extracellular Na+ prevented

the glutamatergic activation of MAPKJNK but only partially

blocked that of MAPKERK (Schwarzschild et al., 1999). Taken

together, these contrasting ionic dependencies suggest a

mechanism by which NMDAR activation may, under distinct

conditions, differentially regulate neuronal MAPKs and their

divergent functions.

Furthermore, it was recently shown that the NMDA-

mediated activation of MAPKs could be bi-directional. For

instance, NMDAR activation of MAPKERK was examined in

primary cortical cultures. Tetrodotoxin, NMDAR antagonists,

or reduced extracellular Ca2+ decreased basal levels of

phospho-MAPKERK2, indicating that activity-dependent acti-

vation of NMDARs maintained a high level of basal

MAPKERK2 activation. This activity-dependent induction of

MAPKERK2 was blocked by PTX, and inhibition of CaMK and

PI3-kinase, but not by inhibition of PKC.

The addition of a Ca2+ ionophore or NMDA decreased

phospho-MAPKERK2 in the presence of extracellular Ca2+ but

enhanced phospho-MAPKERK2 in [Ca2+]e (Chandler et al.,

2001). The reduction in basal phospho-MAPKERK2 by NMDA

was also reflected as a decrease in phospho-CREB. Moreover,

inhibition of tyrosine phosphatases and serine/threonine

phosphatases protein phosphatase 1 (PP1), PP2A and PP2B

did not prevent the inhibitory effect of NMDA. In the presence

of tetrodotoxin, NMDA produced a bell-shaped dose–response

curve with stimulation of MAPKERK2 and reduced stimulation

at supra-physiological concentrations of NMDA. Furthermore,

NMDA stimulation of MAPKERK2 was completely blocked by

PTX and inhibitors of PI3-kinase, and was partially blocked by

a CaMK inhibitor, suggesting that NMDARs can bi-direction-

ally control MAPKERK signaling (Fuller et al., 2001).

Comparison of receptor interactions, including NMDA, with

upstream, membrane-related cofactors and downstream com-

ponents of the MAPK signaling pathways is depicted in Fig. 7.

4.2. NMDA, MAPKs and apoptosis-related pathways

4.2.1. Insulin-like growth factors and NMDA-mediated

excitotoxicity

Ryu et al. (1999) examined the effects of two insulin-like

growth factors, insulin and insulin-like growth factor-I (IGF-I),

against apoptosis, excitotoxicity and free radical neurotoxicity

in cortical cell cultures. Like IGF-I, insulin attenuated serum

deprivation-induced neuronal apoptosis. The anti-apoptosis

effect of insulin against serum deprivation disappeared by

addition of a broad protein kinase inhibitor, staurosporine, but

not by calphostin C, a selective PKC inhibitor.

Furthermore, addition of PD-98059 blocked insulin-induced

activation of MAPKERK1/2 without altering the neuroprotective

effect of insulin. Cortical neurons underwent activation of PI3-

kinase after exposure to insulin. Inclusion of Wortmannin, or

LY-294002, selective inhibitors of PI3-kinase, reversed the

insulin effect against apoptosis. In contrast to the anti-apoptosis

effect, neither insulin nor IGF-I inhibited excitotoxic neuronal

necrosis following continuous exposure to NMDA or KA (Ryu

et al., 1999).

Surprisingly, concurrent inclusion of insulin or IGF-I

aggravated free radical-induced neuronal necrosis following

continuous exposure to Fe2+ or L-buthionine-(S,R)-sulfoximine

(BSO), an irreversible inhibitor of g-glutamylcysteine synthe-

tase (g-GCS), the rate-limiting enzyme in the biosynthesis of

glutathione (GSH), an antioxidant thiol (Haddad and Land,

2000a, 2002; Haddad et al., 2000, 2001b; Haddad, 2001b,

2002a). Wortmannin, or LY-294002, also reversed this

potentiating effect of insulin, suggesting that IGFs act as

anti-apoptosis factor and prooxidant depending upon the

activation of PI3-kinase (Ryu et al., 1999).

4.2.2. Egr-1 and NMDA-mediated excitotoxicity

Egr-1, an apoptotic cofactor, is one of the immediate early

transcription factors that are induced after brain insults. Using

mouse cortical cultures, Park and Koh (1999) examined the

ionic mechanism of Egr-1 induction and its role in neuronal

death. Although Zn2+, NMDA, or ionomycin induced

comparable neuronal death in cortical culture, only Zn2+


increased Egr-1 expression, which was attenuated by blocking

Zn2+ influx. It is intriguing that brief exposure to Zn2+ induced

sustained MAPKERK activation.

In addition, PD-098059 blocked MAPKERK1/2 activation,

Egr-1 induction, and neuronal death by Zn2+, demonstrating

that Zn2+, rather than Ca2+, induces lasting Egr-1 expression in

cortical culture by activating MAPKERK1/2, which is part of a

cascade that may play an active role in Zn2+ neurotoxicity. It is

proposed that translocation of endogenous Zn2+ may be the key

mechanism of Egr-1 induction and neuronal death in brain

ischemia (Park and Koh, 1999).

4.2.3. MAPKs and NMDA-mediated neurotoxicity

The glutamate concentration effective for inducing apopto-

tic-like cell death was correlated with that for inducing

MAPKERK1/2 diphosphorylation. The MK-801 antagonist or

the elimination of extracellular Ca2+ with EGTA prevented

MAPKERK1/2 phosphorylation and the apoptotic-like cell death.

PD-98059, moreover, completely inhibited MAPKERK1/2

phosphorylation and partially inhibited the apoptotic-like cell

death, suggesting that, largely via the NMDAR-mediated influx

of extracellular Ca2+, MAPKERK1/2 were rapidly and transiently

activated, and were involved in glutamate-induced apoptotic-

like death in rat cortical neurons (Jiang et al., 2000a,b).

In concert with the aforementioned, it has been reported that

U0126 prevents glutamate-induced death in neuronally

differentiated P19 cells (Grant et al., 2001). In addition,

differentiated P19 cells expressed z1, e1 and e2 subunits of the

NMDAR. These P19 cells also exhibited specific NMDAR

binding and intracellular Ca2+ responses to glutamate that were

blocked by MK-801, but not U0126.

Moreover, glutamate treatment of differentiated P19 cells

triggered a rapid and sustained induction in MAPKERK2

phosphorylation, which was blocked by U0126. Pre-treatment

of P19 cells with U0126, but not other classes of protein kinase

inhibitors, protected against glutamate-induced cell death

(Grant et al., 2001). Post-treatment with U0126 also protected

against glutamate toxicity, suggesting that the MAPKERK

pathway may be a critical downstream signaling pathway in

glutamate receptor-activated toxicity.

4.2.4. Neutrophins, MAPKs and NMDA-mediated

neurotoxicity

Accumulating evidence suggests that the neurotrophin

receptors, Trks and p75, play distinct roles in regulating cell

survival and death, with Trks mediating cell survival, and p75

inducing cell death (Kume et al., 2000). In neuronal cultures

from rat cerebral cortex, NGF exerts neuroprotective actions

via p75. Incubating cultures with NGF, for instance, protected

cortical neurons from delayed cytotoxicity induced by brief

exposure to glutamate (Kume et al., 2000).

In addition, delayed neurotoxicity induced by a Ca2+

ionophore, ionomycin, or NO donors, such as S-nitrosocysteine

(SNOC) and 3-morpholinosydnonimine (SIN-1), was also

attenuated by pre-treatment with NGF. RT-PCR analysis

revealed the presence of p75 and TrkB transcripts in cortical

cultures, but did not detect transcripts of TrkA, a high-affinity

receptor for NGF. Of note, BDNF, but not NGF, induced

tyrosine phosphorylation of Trks, indicating that NGF does not

activate Trks in cortical neurons (Kume et al., 2000).

Concurrent application of anti-p75 neutralizing antibody

reduced the neuroprotective effect of NGF, but resulted in only

a modest reduction of that of BDNF. BDNF-induced

neuroprotection, furthermore, but not NGF-induced neuropro-

tection, was inhibited by a protein synthesis inhibitor

cycloheximide. Distinct signaling pathways mobilized by

NGF and BDNF were also revealed in that NGF but not

BDNF stimulated significant production of ceramides, whereas

BDNF, but not NGF, caused persistent activation of MAPKs

(Kume et al., 2000). These results indicate that, although NGF

and BDNF both protect cortical neurons from excitotoxicity,

the mechanisms involved in their effects are different and

demonstrate the principle involvement of p75 in cytoprotective

actions of neurotrophins.

The glial cell line-derived neurotrophic factor (GDNF) is

first characterized for its trophic activity on dopaminergic

neurons. Recent data suggested that GDNF couldmodulate the

neuronal death induced by ischemia. In this regard, it was

demonstrated that both neurons and astrocytes express the

mRNA and the protein for GDNF and its receptor complex

(GFRa-1 and c-Ret) (Nicole et al., 2001). Moreover, the

application of recombinant human GDNF (rhGDNF) to

cortical neurons and astrocytes induced the activation of the

MAPK pathway, as visualized by an increase in the

phosphorylated forms of MAPKERK. Thereafter, it was

demonstrated that GDNF failed to prevent apoptotic neuronal

death but selectively attenuated slowly triggered NMDA-

induced excitotoxic neuronal death via a direct effect on

cortical neurons.

To further characterize the neuroprotective mechanisms of

GDNF against NMDA-mediated neuronal death, pre-treatment

with GDNF reduced NMDA-induced Ca2+ influx. This effect

likely resulted from a reduction of the NMDAR activity rather

than an enhanced buffering or extrusion capacity for Ca2+. In

addition, MAPKERK activation was necessary for GDNF-

mediated reduction of the NMDA-induced Ca2+ response

(Nicole et al., 2001). This suggested a potential pathway by

which the activation of MAPK induced by GDNF modulates

NMDAR activity, a mechanism that could be responsible for

the neuroprotective effect of GDNF in acute brain injury.

4.2.5. Caspases, MAPKs and NMDA-mediated

neurotoxicity

The possibility that MAPKp38 and caspase-3 would be

activated for execution of apoptosis and excitotoxicity, the two

major types of neuronal death underlying hypoxicischemic and

neurodegenerative diseases, was further explored. Mouse

cortical cell cultures, for example, underwent widespread

neuronal apoptosis following exposure to calyculin A, a

selective inhibitor of Ser/Thr phosphatase I and IIA (Ko et al.,

2000). Activity of MAPKp38 was also increased following

exposure to calyculin A. Addition of PD-169316, a selective

MAPKp38 inhibitor, partially attenuated calyculin A neuro-

toxicity (Ko et al., 2000).


Moreover, activity of caspase-3-like proteases was

increased in cortical cell cultures exposed to calyculin A, as

shown by cleavage of DEVD-p-nitroanilide and phosphory-

lated Tau. Proteolysis of Tau, in addition, was completely

blocked by addition of N-benzyloxycarbonyl-Val-Ala-Asp-

fluoromethyl ketone (z-VAD-fmk), a broad-spectrum inhibitor

of caspases, but incompletely by PD-169316. Calyculin A

neurotoxicity was partially sensitive to z-VAD-fmk (Ko et al.,

2000).

Notably, co-treatment with PD-169316 and z-VAD-fmk

showed additive neuroprotection against calyculin A. How-

ever, neither PD-169316 nor z-VAD-fmk showed a beneficial

effect against excitotoxic neuronal necrosis induced by

exposure to NMDA (Ko et al., 2000). Thus, caspase-3-like

proteases and MAPKp38 likely contribute to calyculin A-

induced neuronal apoptosis, but not NMDA-induced neuronal

necrosis.

4.2.6. IEG (pip), MAPKs and NMDA-mediated

neurotoxicity

The IEG pip-92 has been identified in serum-stimulated

BALB/c 3T3 fibroblasts, activated T lymphocytes treated with

cycloheximide and fibroblast growth factor-stimulated hippo-

campal cells during neuronal differentiation. It was demon-

strated that pip-92 is expressed in the mouse brain after a single

intraperitoneal injection of NMDA (Chung et al., 2000).

The region-specific activation of pip-92 in the CNS was

observed after NMDA injection and high levels of pip-92mRNA

were detected in the hippocampal dentate gyrus and piriform

cortex regions (Chung et al., 2000). In addition, the activation of

pip-92 by NMDAwas mediated by the activation of MAPKJNK

and MAPKp38, but not MAPKERK, in the mouse hippocampus

and immortalized rat hippocampal progenitor cells, indicating

that pip-92 is likely to play an important role in neuronal cell

death induced by excitotoxic NMDA injury in the CNS.

4.2.7. PKC, MAPKs and NMDA-mediated neurotoxicity

In the mammalian retina, retinal ganglion cells (RGCs)

purportedly die by apoptosis during development. Additionally,

transection of the optic nerve close to the eye bulb may cause

apoptotic cell death of RGCs in adulthood (Kikuchi et al.,

2000). After axotomy, activated MAPKp38 was visualized by

immunocytochemistry in the nuclei of RGCs. Phosphorylated

MAPKp38 was shortly detected after axotomy, reached a

maximum and then decreased.

To investigate possible roles of MAPKp38 in RGC death, SB-

203580 was administered intravitreally at the time of axotomy

and repeated thereafter. SB-203580 increased the number of

surviving RGCs. Furthermore, MK-801 not only showed

protective effects against RGC apoptosis but also attenuated

MAPKp38 activation (Kikuchi et al., 2000; Santos et al., 2001),

thus implying that MAPKp38 is in the signaling pathway to

RGC apoptosis mediated by glutamate neurotoxicity through

NMDARs after damage to the optic nerve.

Excessive activation of NMDARs leads to cell death in

human embryonic kidney-293 (HEK) cells, which have been

transfected with recombinant NMDARs. To evaluate the role of

PKC activation in NMDA-mediated toxicity, Wagey et al.

(2001) have analyzed the survival of transfected HEK cells

using trypan blue exclusion. It was found that NMDA-mediated

death of HEK cells transfected with NR1/NR2A subunits was

increased by exposure to phorbol esters and reduced by PKC

downregulation.

The region of NR2A that provides the PKC-induced

enhancement of cell death was localized to a discrete segment

of the C-terminus. The use of isoform-specific PKC inhibitors

showed that Ca2+- and lipid-dependent PKC isoforms (cPKCs),

specifically PKCb1, were responsible for the increase in cell

death when phorbol esters were applied prior to NMDA in these

cells. In addition, PKC activity measured by an in vitro kinase

assay was also increased in NR1A/NR2A-transfected HEK

cells following NMDA stimulation (Wagey et al., 2001),

suggesting that PKC acts on the C-terminus of NR2A to

accentuate cell death in NR1/NR2A-transfected cells, and

demonstrating that this effect is mediated by cPKC isoforms.

Therefore, the elevation of cellular PKC activity may increase

neurotoxicity mediated by the NMDAR activation.

4.3. NMDA, MAPKs and long-term potentiation

4.3.1. General aspects of NMDA–MAPK interactions in

the regulation of LTP

Although classically studied as regulators of cell prolifera-

tion and differentiation, MAPKs are highly expressed in post-

mitotic neurons of the adult nervous system (Watanabe et al.,

2002; Opazo et al., 2003). MAPKs have been implicated in

multiple responses to extracellular stimuli in the CNS. For

instance, Baron et al. (1996) showed that MAPK activity is

enhanced after a KCl pulse. This activation correlated with an

increased tyrosine phosphorylation of MAPKERK2.

In addition, MAPKERK2 activity was enhanced, reached a

maximum, and returned to basal level. The activation was

completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione

(CNQX), thus showing the involvement of the AMPA receptor.

Of interest, partial inhibition of MAPK activation by 2-amino-

5-phosphonovalerate (APV) also showed the involvement of

the NMDAR (Baron et al., 1996). Because the KCl pulse used

induced long-term potentiation (LTP) in rat hippocampal slice,

MAPK is proposed to be involved in neuronal transduction

events leading to LTP.

In concert, English and Sweatt (1996) have investigated the

potential role of MAPKs in the regulation of synaptic plasticity

in mature neurons. In particular, the regulation of MAPKERK1/2

was studied in hippocampal LTP, a system widely studied as a

model for the cellular basis of learning and memory. It was

found that MAPKERK2, but not MAPKERK1, was activated in

area CA1 following direct stimulation of two required

components of the LTP induction cascades: PKC and the

NMDAR (English and Sweatt, 1996).

It was also demonstrated that MAPKERK2, but not

MAPKERK1, was activated in area CA1 in response to LTP-

inducing high frequency stimulation and that this activation

required NMDAR stimulation (English and Sweatt, 1996).

These observations indicate that MAPKERK2 can be regulated


in an activity-dependent manner in the hippocampus and

identify it as a potential component of the LTP induction

cascades in area CA1, suggesting that MAPKERK2 might be an

important regulator of synaptic plasticity in post-mitotic

neurons.

In support of the role for NMDA in MAPK-related LTP,

PD-098059, a selective inhibitor of the upstream MAPK

cascade that regulates MAPKERK, as noted earlier on,

blocked MAPK activation in response to direct stimulation of

the NMDAR as well as to LTP-inducing stimuli (English and

Sweatt, 1997). Furthermore, inhibition of the MAPK cascade

markedly attenuated the induction of LTP. PD-098059,

however, had no effect on the expression of established LTP

and the MAPK cascade was not persistently activated during

LTP expression, thereby providing evidence for a role for the

MAPK cascade in the activity-dependent modification of

synaptic connections between neurons in the adult mamma-

lian nervous system.

Similarly, the effect of PD-98059 was investigated on three

types of LTP in the medial perforant path of the rat dentate

gyrus in vitro: LTP induced by (i) high-frequency stimulation

(HFS-LTP), (ii) application of the K+ channel blocker,

tetraethyl-ammonium chloride (TEA-LTP) and (iii) application

of the mGluR agonist (S)-dihydrophenylglycine (S-DHPG)

(DHPG-LTP) (Coogan et al., 1999).

Bath perfusion of PD-98059 inhibited HFS-LTP; PD-98059

had no effect on the isolated NMDA excitatory postsynaptic

potential (NMDA-EPSP) or on the maintenance phase of HFS-

LTP. Moreover, PD-98059 did not affect paired-pulse depres-

sion (PPD; inter-stimulus intervals) of synaptic transmission as

is typically observed in the medial perforant path of the dentate

gyrus (Coogan et al., 1999).

Bath application of S-DHPG gave rise to a potentiation of

the EPSPs slope. Pre-treatment of slices with PD-98059

inhibited the S-DHPG-LTP. The TEA-LTPwas found to be both

2-amino-5-phosphonopentanoic acid (2-AP5) and nifedipine

independent. However, the T type voltage-dependent Ca2+

channel blocker, NiCl2, completely inhibited the observed

potentiation. Of particular interest, the mGluR receptor

antagonist a-methyl-4-carboxy-phenyl glycine (MCPG) and

PD-98059 caused a complete block of the TEA-LTP, suggesting

the involvement of MAPKERK1/2 in the induction and

expression of both an NMDA-dependent and two forms of

NMDA-independent LTP in the dentate gyrus (Coogan et al.,

1999) (see Table 2).

In another report, Dudek and Fields (2001) determined

whether the pattern and intensity of synaptic activity could

differentially regulate MAPK phosphorylation via selective

activation of different modes of Ca2+ influx into CA1 pyramidal

neurons. LTP-inducing stimulation [theta-burst] was effective

in inducing intense staining in both dendritic and somatic

compartments of CA1 neurons. Phosphorylation of MAPK was

also induced, however, with stimulation frequencies not

typically effective in inducing LTP. Moreover, intensity and

extent of staining was better correlated with the spread of

population spikes across the CA1 sub-field than with frequency

(Dudek and Fields, 2001).

Experiments using inhibitors of the NMDARs and voltage-

sensitive Ca2+ channels (VSCCs) revealed that, although

MAPK is activated after both TBS and stimulation, the relative

contribution of Ca2+ through L-type Ca2+ channels differs.

Blockade of NMDARs alone was sufficient to prevent MAPK

phosphorylation in response to stimulation, whereas inhibitors

of both NMDARs and VSCCs were necessary for inhibition of

the TBS-induced staining (Dudek and Fields, 2001). Thus, the

intensity and frequency of synaptic input to CA1 hippocampal

neurons are critically involved in determining the path by which

second-messenger cascades are induced to activate MAPK.

NMDA-independent MAPK mechanisms have been

reported in LTP. For instance, Kanterewicz et al. (2000)

studied the role of MAPKERK in three forms of NMDAR-

independent LTP: LTP induced by very high-frequency

stimulation, LTP induced by TEA and mossy fiber (MF)

LTP (MF-LTP). The authors reported that MAPKERK was

activated in area CA1 after the induction of both LTP and TEA-

LTP, and that this activation required the influx of Ca2+ through

voltage-gated Ca2+ channels.

Furthermore, inhibition of the MAPKERK signaling cascade

with either PD-098059 or U0126 prevented the induction of

both LTP and TEA-LTP in area CA1. In contrast, neither PD-

098059 nor U0126 prevented MF-LTP in area CA3 induced by

either brief or long trains of high-frequency stimulation. U0126

also did not prevent forskolin-induced potentiation in area CA3.

However, incubation of slices with forskolin, an activator of the

cAMP-dependent PKA cascade, did result in increases in active

MAPKERK and CREB phosphorylation in area CA3 (Kanter-

ewicz et al., 2000).

The forskolin-induced increase in active MAPKERK, in

addition, was inhibited by U0126, whereas the increase in

CREB phosphorylation was not, which suggests that in area

CA3 the PKA cascade is not coupled to CREB phosphorylation

via MAPKERK (Kanterewicz et al., 2000). Overall, these

observations indicate that activation of MAPKERK is necessary

for NMDAR-independent LTP in area CA1 but not in area CA3,

and suggest a divergence in the signaling cascades underlying

NMDAR-independent LTP in these hippocampal sub-regions.

4.3.2. Genetic control of NMDA–MAPK interactions in the

regulation of LTP

Maintenance of LTP requires de novo gene expression.

Matsuo et al. (2000) reported the direct isolation, using PCR-

differential display, of genes whose expression level was altered

after induction of long-lasting LTP in the hippocampus of freely

moving and awake rats. Differential display using 480 primer

combinations revealed 17 cDNA bands that showed a

reproducible change in expression level. These cDNAs

represented at least 10 different genes (termed RM1–10), all

of which showed upregulation after LTP induction and a return

to basal expression levels.

Three of these genes were known only from expressed

sequence tags (RM1–3), two were known genes whose

upregulation by LTP has not been described (GADD153/

CHOP and ler5), and five were known genes whose

upregulation by LTP has already been reported (MAPK


Table 2

NMDA receptor nomenclature, agonists, antagonists and channel blockers

Currently accepted name

Glutamate site NMDA glycine site Other site

Structural information

NR1 (920 aa human) – –

NR2A (1464 aa human)

NR2B (1484 aa human)

NR2C (1233 aa human)

NR2D (1329 aa rat)

NR3A (1115 aa rat)

Subtype selective agonists

N-methyl-D-aspartic acid Glycine –

Quinolinic acid D-Serine

R(+)-HA-966 (partial)

Subtype selective antagonists

D(–)-AP-5 7-Chlorokynurenic Ro 25-6981 (NR2B)

D(–)-AP-7 5,7-Dichlorokynurenic acid Ro 8-4304 (NR2B)

CGS19755 MNQX CP 101,606 (NR2B)

CGP37849 L-689,560 Ifenprodil (NR2B)

CPP, (�)-, D- L-701,324

D-CPPene GV 150526

EAA-090

Channel blockers

MK-801 (Dizocilpine) – –

Phencyclidine (PCP)

CNS-1102 (Cerestat)

Ketamine

Channel permeability

Intrinsic ion channel (Na+/K+/Ca2+) – –

Radioligands of choice

[3H]-CPP [3H]-5,7-dichlorokynurenate [3H]-MK-801 (channel)

[3H]-L-glutamate [3H]-L-689,560 [3H]-Ro 25-6981(NR2B)

Abbreviations: AP-5, 2-amino-5-phosphonopentanoic acid; AP-7, 2-amino-7-phosphonoheptanoic acid; D-CCPene, D-3-(2-carboxypiperazin-4-yl)-propyl-1-phos-

phonene; CGP37849, D,L-(E)-2-amino-4-methylphosphono-3-pentanoic acid; CGS19755, 4-phosphonomethyl-2-piperidinecarboxylic acid (Selfotel); CP 101,606,

(1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol; CPP, 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid; EAA-090, [2-(8,9-dioxo-

2,6-diazabicyclo[5.2.0]non-1(7)-en-2-yl)-ethyl]phosphonic acid; GV 150526, 3-[2-(phenylamino)carbonyl]ethenyl-4,6-dichloroindole-2-carboxylic acid; L-

689,560, (�)-4-(trans)-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline; L-701,324, 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-

2(H)-quinolinone; MNQX, 5,7-dinitro-1,4-dihydro-2,3-quinoxalinedione; NMDA, N-methyl-D-aspartic acid; Ro 25-6981, R-(R,S)-a-(4-hydroxyphenyl)-b-methyl-

4-(phenylmethyl)-1-piperidine propanol; Ro 8-4304, 4-{3-[4-(4-fluorophenyl)-3,6-dihydro-2H-pyridin-1-yl]-2-hydroxypropoxy}-benzamide.

phosphatase, NGFI-A/zif268, vesl-1S/homer-1a, Ag2 and

krox-20). The expression profiles of genes in the two former

categories with respect to NMDAR dependency, tissue

specificity, and developmental regulation using northern

blotting and semiquantitative RT-PCR was further character-

ized. For example, the upregulation of all five of these genes

was NMDAR-dependent and correlated with the persistence of

LTP, suggesting that these genes may play functional roles in

prolonged LTP maintenance (Matsuo et al., 2000).

Mice heterozygous for a null mutation of the K-Ras gene (K-

Ras+/�) show normal hippocampal MAPK activation, LTP, and

contextual conditioning. However, a dose of a MEK inhibitor,

ineffective in wild-type controls, blocked MAPK activation,

LTP and contextual learning in K-Ras+/� mutants (Ohno et al.,

2001). This indicates that K-Ras/MEK/MAPK signaling is

critical in synaptic and behavioral plasticity.

A sub-threshold dose of NMDAR antagonists triggered,

furthermore, a contextual learning deficit in mice heterozygous

for a point mutation (T286A) in the aCaMKII gene, but not in

K-Ras+/� mutants, demonstrating the specificity of the

synergistic interaction between the MEK inhibitor and the

K-Ras+/� mutation (Ohno et al., 2001). This pharmacogenetic

approach combines the high temporal specificity that pharma-

cological manipulations offer, with the molecular specificity of

genetic disruptions.

4.4. NMDA, MAPKs and long-term depression

There is growing evidence that activation of either protein

kinases or protein phosphatases determines the type of

plasticity observed after different patterns of hippocampal

stimulation (Rush et al., 2002). Because activation of

MAPKERK has been shown to be necessary for LTP, Norman

et al. (2000) investigated the regulation of MAPKERK in long-

term depression (LTD) in the adult hippocampus in vivo. It was

found that MAPKERK immunoreactivity was decreased


following the induction of LTD and that this decrease required

NMDAR activation.

The LTD-associated decrease in MAPKERK immunoreac-

tivity could be simulated in vitro via incubation of either

purified MAPKERK2 or hippocampal homogenates with either

protein phosphatase 1 or protein phosphatase 2A. The protein

phosphatase-dependent decrease in MAPKERK immunoreac-

tivity was inhibited by microcystin. Furthermore, intrahippo-

campal administration of the protein phosphatase inhibitor

okadaic acid blocked the LTD-associated decrease in MAP-

KERK2, but not MAPKERK1, immunoreactivity (Norman et al.,

2000). Collectively, these data demonstrate that protein

phosphatases can decrease ERK immunoreactivity and that

such a decrease occurs with MAPKERK2 during LTD. These

observations provide the first demonstration of a biochemical

alteration of MAPKERK in LTD.

In support, the MAPKERK cascade was recruited during LTD

of synaptic strength in area CA1 of the adult hippocampus in

vivo, and selectively impacts on phosphorylation of the nuclear

transcription factor Elk-1 (Thiels et al., 2002). Using a

combination of in vivo electrophysiology, biochemistry,

pharmacology and immuno-histochemistry, it was found that:

(i) MAPKERK phosphorylation, including phosphorylation of

nuclear ERK and ERK phosphotransferase activity were

increased markedly, albeit transiently, after the induction of

NMDAR-dependent LTD at the commissural input to area CA1

pyramidal cells in the hippocampus of anesthetized adult rats;

(ii) LTD-inducing paired-pulse stimulation failed to produce

lasting LTD in the presence of the MAPKERK kinase inhibitor

SL327, which suggests that ERK activation is necessary for the

persistence of LTD and (iii) MAPKERK activation during LTD

resulted in increased phosphorylation of Elk-1 but not of CREB

(Thiels et al., 2002).

These findings indicate that the ERK cascade transduces

signals from the synapse to the nucleus during LTD in

hippocampal area CA1 in vivo, as it does during long-term

potentiation in area CA1, but that the pattern of coupling of the

ERK cascade to transcriptional regulators differs between the

two forms of synaptic plasticity.

4.5. NMDA, MAPKs and short- and long-term memory

It is widely accepted that the formation of long-term

memory (LTM) requires neuronal gene expression, protein

synthesis and the remodeling of synaptic contacts. From

mollusk to mammals, the cAMP/PKA/CREB signaling path-

way has been shown to play a pivotal role in the establishment

of LTM (see Vitolo et al., 2002).

More recently, the MAPK cascade has been also involved in

memory processing. In this regard, Cammarota et al. (2000)

provided evidence for the participation of hippocampal PKA/

CREB andMAPK/Elk-1 pathways, via activation of NMDARs,

in memory formation of a one-trial avoidance learning in rats.

Learning of this task is associated with an activation of

MAPKERK1/2, CREB and Elk-1, along with an increase in the

levels of the catalytic subunit of PKA and Fos protein in

nuclear-enriched hippocampal fractions. These changes were

blocked by the immediate post-training intra-hippocampal

infusion of APV, a selective blocker of NMDARs, which

renders the animals amnesic for this task. Moreover, no changes

were found in control-shocked animals (Cammarota et al.,

2000). Thus, inhibitory avoidance training in the rat is

associated with an increase in the protein product of an IEG,

c-Fos, which occurs concomitantly with the activation of

nuclear MAPK, CREB and Elk-1. NMDARs appear to be a

necessary upstream step for the activation of these intracellular

cascades during learning.

Different hippocampal molecular requirements for short-

and long-term retrieval of one-trial avoidance learning were

also reported. Prior to the retention test, through indwelling

cannulae placed in the CA1 region of the dorsal hippocampus,

animals received infusions of: saline, a vehicle, the NMDAR

blocker, 2-AP5, the AMPA/KA receptor blocker, CNQX, the

metabotropic receptor antagonist, MCPG, the inhibitor of

CaMK (KN62), the inhibitor of PKA, Rp-cAMPs, the stimulant

of the same enzyme, Sp-cAMPs or PD-098059 (Izquierdo et al.,

2000a,b).

All these drugs, at the same doses, had been found to affect

short- and long-term memory formation of this task. Retrieval

after training (short-term memory; STM) was blocked by

CNQX and MCPG, and was unaffected by all the other drugs.

In contrast, retrieval measured later on was blocked by MCPG,

Rp-cAMPs and PD-098059, enhanced by Sp-cAMPs and

unaffected by CNQX, AP5 or KN62 (Izquierdo et al., 2000a,b).

The results indicate that, in CA1, glutamate metabotropic

receptors are necessary for the retrieval of both short- and long-

term memory; AMPA/KA receptors are necessary for short-

term but not long-term memory retrieval, and NMDARs are

uninvolved in retrieval. Both the PKA and MAPK signaling

pathways are required for the retrieval of long-term but not

short-term memory.

In the behaving rat, the consumption of a substance of an

unfamiliar taste was shown to activate MAPKERK1/2 in the

insular cortex, which contains the taste cortex. In contrast,

consumption of a familiar taste has no effect. Furthermore,

activation of MAPKERK1/2, culminating in modulation of gene

expression, is obligatory for the encoding of long-term, but not

short-term, memory of the new taste.

Which neurotransmitter and neuromodulatory systems are

involved in the activation of MAPKERK1/2 by the unfamiliar

taste and in the long-term encoding of the new taste

information? Berman et al. (2000) showed, by the use of local

microinjections of pharmacological agents to the insular cortex

in the behaving rat, that multiple neurotransmitters and

neuromodulators are required for encoding of taste memory

in cortex. However, these systems vary in the specificity of their

role in memory acquisition and in their contribution to the

activation of MAPKERK1/2.

NMDARs, metabotropic glutamate receptors, muscarinic

and b-adrenergic and dopaminergic receptors, all contribute to

the acquisition of the new taste memory but not to its retrieval.

Among these, only NMDA and muscarinic receptors specifi-

cally mediate taste-dependent activation of MAPKERK1/2,

whereas the b-adrenergic function is independent of MAP-


KERK1/2 and dopaminergic receptors regulate also the basal

level of MAPKERK1/2 activation (Berman et al., 2000).

4.6. NMDA, MAPKs and electroconvulsive shock

Electroconvulsive shock (ECS) activates MAPKs in the rat

hippocampus via a distinct signaling pathway. The NMDAR

antagonist, MK-801, reduced ECS-induced phosphorylation of

MAPKp38 and its upstream kinaseMKK6.MK-801 also reduced

the phosphorylation of MAPKERK1/2 and MEK1. Moreover, the

reduction in the phosphorylation of MAPKp38 and MKK6 was

greater than that of MAPKERK1/2 and MEK1 (Ahn et al., 2000).

This suggests that ECS activates MAPKp38 and MAPKERK1/2

partly through an NMDAR-mediated signaling system in the rat

hippocampus and that NMDAR mediated signaling is more

responsible for the activation of the MKK6–MAPKp38 pathway

than the MEK1–MAPKERK pathway.

4.7. NMDA, MAPKs and oxidative stress: role of oxidants

and inflammatory mediators

4.7.1. Role for hydrogen peroxide (H2O2)

H2O2 is a potent oxidant stimulator of signal-responsive

phospholipase A2 (PLA2) in vascular smooth muscle and

cultured endothelial cells. In this respect, Samanta et al. (1998)

investigated whether H2O2 plays a similar regulatory role in

neurons. H2O2 did not stimulate a release of arachidonic acid

(AA) from cultured neurons when applied alone but strongly

enhanced the liberation of arachidonic acid evoked by

maximally effective concentrations of either glutamate,

NMDA, the muscarinic receptor agonist carbachol, the Na+-

channel opener veratridine or the Ca2+ ionophore ionomycin

(Samanta et al., 1998).

The potentiating effects of H2O2 were strongly inhibited in

the presence of the PLA2 inhibitor mepacrine, suggesting that

the site of action was within the signal responsive AA cascade.

Moreover, the enhancing effect of H2O2 was not reversed by

PKC inhibitors (chelerythrine chloride or GF-109203X) nor

was it mimicked by phorbol ester treatment. H2O2 alone

strongly enhanced the levels of immuno-detectable activated

MAPKERK1/2 in a Ca2+-dependent manner and this effect was

additive with increases in the levels of activated MAPK evoked

by glutamate (Samanta et al., 1998). Of note, the enhanced

release of AA, however, was not clearly reversed by PD-98059,

although this treatment effectively abolished H2O2 activation of

MAPK, thereby indicating that MAPK activation and Ca2+-

dependent AA release are regulated by oxidative stress.

In concert, primary cortical neurones exposed to an

oxidative insult in the form of H2O2 showed a concentra-

tion-dependent increase in oxidative stress followed by a

delayed NMDAR-dependent cell death (Crossthwaite et al.,

2002). Using phospho-specific antibodies, stimulation of

neurones with H2O2 produced a concentration-dependent

phosphorylation of MAPKERK1/2 and Akt/PKB that was partly

dependent on extracellular Ca2+ and PI3-kinase.

Supra-physiologic concentrations of H2O2 also stimulated

a phosphorylation of MAPKJNK, which was totally dependent

on extracellular Ca2+ but not PI3-kinase. In addition, H2O2-

induced phosphorylation of MAPKERK1/2, Akt/PKB or

MAPKJNK were unaffected by the NMDA channel blocker

MK-801 (Crossthwaite et al., 2002).

Blocking MAPKERK1/2 activation with U0126 enhanced

H2O2-induced neurotoxicity and inhibited H2O2-mediated

phosphorylation of CREB, suggesting that MAPKERK1/2

signals survival under these conditions; H2O2-stimulated a

phosphorylation of c-Jun as well. It is likely, therefore, that

subjecting neurones to moderate oxidative-stress recruits pro-

survival signals to CREB but during severe oxidative stress pro-

death signals through MAPKJNK and c-Jun are dominant

(Crossthwaite et al., 2002).

Insulin receptor-substrate-1 (IRS-1) is a docking protein for

several tyrosine kinase receptors. Upon tyrosine phosphoryla-

tion, IRS-1 binds to signaling molecules that express Src

homology 2 (SH-2) binding domains, including PI3-kinase,

phosphotyrosine phosphatase SHP-2 (Syp), Nck, Crk and Grb-

2. H2O2 induces tyrosine phosphorylation of key signaling

mediators presumably by inhibition of tyrosine phosphatases.

In many cell types, the activation of MAPKs and other protein

kinases by H2O2 leads to transcriptional activation. Recently,

the effect of H2O2 on IRS-1 tyrosine phosphorylation in

primary cultured rat cerebellar granule neurons was investi-

gated. H2O2 stimulated the rapid tyrosine phosphorylation of

IRS-1 MAPKERK1/2 and induced its association with PI3-

kinase. H2O2-induced IRS-1 phosphorylation was rapidly

reversible, whereas MAPK phosphorylation persisted (Hallak

et al., 2001).

NMDA reversed H2O2-mediated tyrosine phosphorylation

of IRS-1 and its association with PI3-kinase. The depho-

sphorylation of IRS-1 by NMDA was Ca2+-dependent and

was inhibited by the calcineurin inhibitor cyclosporine. In

addition, calmodulin-dependent tyrosine phosphatase activ-

ity of calcineurin was observed in vitro using both immuno-

precipitated and recombinant tyrosine-phosphorylated IRS-1

as substrates. These data highlight the role of multiple

phosphatases in the regulation of IRS-1 tyrosine phosphor-

ylation and identify a novel functional property of

calcineurin (Hallak et al., 2001).

4.7.2. Role for superoxide anion (O2��)

Experimental studies in piglets have shown that the

generation of oxygen free radicals (O2��) following traumatic

brain injury and hypoxia/ischemia contribute to the reversal of

NMDA-induced pial artery dilation to vasoconstriction (Philip

and Armstead, 2003; Armstead, 2003).

Exposure of the cerebral cortex to a xanthine oxidase

generating system (OX) reversed NMDA dilation to

vasoconstriction but such impairment was partially prevented

by genistein, U0126 and SB-203580. However, SB-203580

prevented NMDA dilator impairment significantly less than

U0126 (similar results were obtained for glutamate). These

observations indicate that PTK and MAPK activation by the

presence of O2�� contributes to the impairment of NMDA

dilation, and that there is a differential role for MAPKERK

and MAPKp38 activation in impairment of NMDA dilation by


O2�� in the brain (Philip and Armstead, 2003; Armstead,

2003).

4.7.3. Role for nitric oxide (NO)

In cerebral cortical neurons, Ca2+ influx through NMDARs

activates Ras and its downstream effector, MAPKERK, via NO

generation by Ca2+-dependent neuronal NO synthase (Yun

et al., 1999). NO was proposed as a key link between NMDA-

mediated increases in cytoplasmic Ca2+ and activity-dependent

long-term changes, such as differentiation, survival and

synaptic plasticity.

MAP-2 is a neuronal phosphoprotein that modulates

microtubule stability and spatial organization of signal

transduction pathways. The functions of MAP-2 are modulated

by phosphorylation. In this regard, the modulation of MAP-2

phosphorylation using NMDA and the signal transduction

pathways mediating this modulation in primary cultures of rat

cerebellar neurons was unraveled. For instance, NMDA

induced a rapid increase in MAP-2 phosphorylation, which

was not prevented by KN-62, indicating that it is not mediated

by activation of Ca-calmodulin-dependent protein kinase

(Llansola et al., 2001).

NMDA-induced phosphorylation of MAP-2 was inhibited

by the NO synthase inhibitors nitroarginine and 7-nitroindazole

and by PD-098059, but was only slightly reduced by calphostin

C or U-73122, inhibitors of PKC and of phospholipase C,

respectively. This indicates that the main pathway mediating

NMDA-induced phosphorylation of MAP-2 is activation of NO

synthase and subsequent activation of MAPK (Llansola et al.,

2001). In addition, the activation of NMDARs induces an

activation of MAPK, which is prevented by nitroarginine.

The NO-generating agent (+/�)-S-nitroso-N-acetylpenicil-

lamine (SNAP) also induced activation of MAPK and increased

phosphorylation ofMAP-2; other NO-generating agents (NOC-

18 and NOR-3) also increased MAP-2 phosphorylation

(Llansola et al., 2001). The interplay between NMDARs-

associated signal transduction pathways and MAP-2 may be

involved in the modulation of neuronal responses to

extracellular signals and in the regulation of neuronal function.

4.7.4. Role for ammonia

Increased brain ammonia has been implicated in the

pathogenesis of the neuropsychiatric symptoms of liver failure

collectively known as hepatic encephalopathy, in the neuronal

cell loss and ensuing mental retardation that is characteristic of

inherited urea cycle enzymopathies as well as in epilepsy and

some neurodegenerative disorders (Felipo and Butterworth,

2002; Monfort et al., 2002; Seiler, 2002).

Acute administration of large doses of ammonia leads to the

rapid death of animals. Of particular interest, excessive

activation of NMDARs plays a role in mediating ammonia-

induced mortality (Monfort et al., 2002). Acute intoxication

with large doses of ammonia leads to the activation of

NMDARs in brain in vivo. Moreover, excessive activation of

NMDARs is responsible for ammonia-induced death of

animals, which is prevented by different antagonists of

NMDARs.

Furthermore, the activation of NMDARs is also responsible

for the following effects of acute ammonia intoxication: (i)

depletion of brain ATP, which, in turn, leads to release of

glutamate; (ii) activation of calcineurin and dephosphorylation

and activation of Na+/K+-ATPase in brain, thus increasing ATP

consumption; (iii) impairment of mitochondrial function and

Ca2+ homeostasis at different levels, thus decreasing ATP

synthesis; (iv) activation of calpain that degrades the

microtubule-associated protein MAP-2, thus altering the

microtubular network and (v) increased formation of NO

formation, which, in turn, reduces the activity of glutamine

synthetase, thus reducing the elimination of ammonia in brain

(Monfort et al., 2002).

Ammonia induced protein tyrosine nitration in cultured rat

astrocytes, which is sensitive to MK-801. A similar pattern of

nitrated proteins is produced by NMDA. Ammonia-induced

tyrosine nitration depends on a rise in [Ca2+]i, IkB degradation,

and iNOS induction, which are prevented by MK-801 and the

intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-

N,N,N0,N0-tetraacetic acid (BAPTA-AM) (Schliess et al., 2002).

Moreover, the increase in tyrosine nitration is blunted by L-

NMMA, 1400 W, uric acid, Cu/Zn-superoxide dismutase

(SOD)/catalase (CAT) treatment and methionine-sulfoximine,

which indicate the involvement of reactive nitrogen species

(RNS) and intracellular glutamine accumulation. Such RNS,

additionally, mediate ammonia-induced phosphorylation of the

MAPKERK1/2 and MAPKp38.

Among the proteins, which are tyrosine-nitrated by

ammonia, glyceraldehyde-3-phosphate dehydrogenase, the

peripheral-type benzodiazepine receptor, Erk-1 and glutamine

synthetase are identified. Ammonia-induced nitration of

glutamine synthetase is associated with a loss of enzymatic

activity. Astroglial protein tyrosine nitration is found in brains

from rats after acute ammonia-intoxication or after portacaval

anastomosis, indicating an in vivo relevance (Schliess et al.,

2002). The production of RNS and protein tyrosine nitration

may alter astrocyte function and contribute to ammonia

neurotoxicity.

4.7.5. Role for cytokines and inflammatory-related

mediators

Cytokines are soluble mediators of inter- and intracellular

communications (Haddad et al., 2001a,c, 2002; Haddad,

2002b). These small glycoproteins contribute to a chemical

signaling language that regulate development, tissue repair,

wound healing, homeostasis, inflammation, and specific/non-

specific immune response (Lu et al., 1997; Sheng et al., 2001;

Haddad et al., 2001a,b; Haddad, 2001b, 2002b).

Minocycline, a tetracycline derivative with anti-inflamma-

tory effects, inhibits IL-1b-converting enzyme (caspase-1) and

iNOS upregulation in animal models of ischemic stroke and

Huntington’s disease and is therapeutic in these disease animal

models (Tikka and Koistinaho, 2001).

It was reported that minocycline protects neurons in mixed

spinal cord cultures against NMDA excitotoxicity. NMDA

treatment alone, for example, induced microglial proliferation,

which preceded neuronal death and administration of extra


microglial cells on top of these cultures enhanced the NMDA

neurotoxicity; minocycline inhibited all these responses to

NMDA (Tikka and Koistinaho, 2001). Minocycline also

prevented the NMDA-induced proliferation of microglial cells

and the increased release of IL-1b and NO in pure microglia

cultures.

Furthermore, minocycline inhibited the NMDA-induced

activation of MAPKp38 in microglial cells and a specific

MAPKp38 inhibitor, but not a MAPKERK1/2 inhibitor, reduced

the NMDA toxicity (Tikka and Koistinaho, 2001), suggesting

that microglial activation contributes to NMDA excitotoxicity

and that minocycline, a tetracycline derivative, represents a

potential therapeutic agent for brain diseases.

4.8. NMDA, MAPKs and the role of regulatory

transcription factors and cofactors

A single second messenger, Ca2+, controls gene expression

triggered by neuronal activity and the spatial properties of Ca2+

signals determine the type of transcriptional response. Nuclear

Ca2+ is a central regulator of transcription, though synaptic

activity may elicit Ca2+ transients that are confined to a space

near the site of entry (Hardingham et al., 2001).

It was shown that a Ca2+ pool in the immediate vicinity of

synaptic NMDARs is the on switch for MAPKERK1/2-mediated

synapse-to-nucleus signaling; this signal propagates to the

nucleus independently of global increases in calcium con-

centration, stimulates SRE-dependent gene expression and

prolongs the transcriptionally active state of CREB following

brief synaptic stimuli (Hardingham et al., 2001).

The striatum is a brain region involved in motor control and

in diverse forms of implicit memory. It is also involved in the

pathogenesis of many significant human disorders, including

drug addiction, that are thought to involve adaptive changes in

gene expression (Sgambato et al., 2003). It has been shown that

the cyclin L, ania-6, is expressed as at least two splice forms,

which are differentially regulated in striatal neurons by

different neurotransmitters.

Of note, ania-6 transcription is mostly regulated via CREB,

but that signaling pathways that converge on CREB at the

transcriptional level produce different effects on splicing and

neuronal gene expression. Glutamate induced a long ania-6

mRNA that encodes a truncated form of the cyclin. This effect

depended on the activation of NMDARs but was independent of

CaMK and MAPKERK (Sgambato et al., 2003).

Forskolin or BDNF induced a short ania-6 mRNA, that

encodes the full-length cyclin, and this induction depended on

ERK. However, KCl-mediated induction of ania-6 short

mRNA, which required activation of L-type calcium channels,

was independent of ERK but depended on CaMK, suggesting

that different neuronal signals can differentially regulate

splicing and that different intracellular pathways can be

recruited to yield a given splice variant (Sgambato et al., 2003).

NMDARs in the mammalian brain play a central role in

synaptic plasticity underlying refinement of neuronal connec-

tions during development, or processes like LTP, learning and

memory. On the other hand, over-activation of glutamate

receptors leading to neuro-degeneration has been implicated in

major areas of brain pathology (Nong et al., 2004). Any

sustained effect of a transient NMDAR activation is likely to

involve signaling to the nucleus and coordinated changes in

gene expression.

Classically, a set of immediate-early genes is induced first;

some of them are themselves transcription factors that control

expression of other target genes. Fos, Jun and Egr (Krox)

transcription factors, for instance, are responsive to NMDA or

non-NMDA (AMPA/KA) ionotropic receptor agonists in vivo

or in neuronal cultures in vitro (Platenik et al., 2000). In

addition, the mechanism of induction of a model immediate-

early gene c-Fos in response to Ca2+ influx through activated

NMDARs or voltage-sensitive calcium channels is possible.

Both modes of Ca2+ entry induce c-Fos via activation of

multiple signaling pathways that converge on constitutive

transcription factors CREB, SRF and a ternary complex factor

(TCF), such as Elk-1.

In contrast to the traditional view of the NMDAR as a

ligand-gated Ca2+ channel, whose activation leads to Ca2+

influx and activation of CaMK, recent evidence highlights

involvement of the RasMAPK pathway in the NMDA signaling

to the nucleus (Platenik et al., 2000).

Nuclear factor-kB (NF-kB), an oxygen and redox-sensitive/

responsive inflammatory transcription factor (Haddad and

Land, 2000a,b; Haddad et al., 2000, 2001c; Haddad, 2004a),

has been implicated in the synaptic plasticity and neurotoxicity

mediated by ionotropic glutamate receptors. KA, but not

AMPA or NMDA, was found, for example, to activate NF-kB in

superfused slices of rat striatum (Cruise et al., 2000). A similar

activation was produced by the Ca2+ ionophore A23187. The

NF-kB activation by KA was not observed in the absence of

extracellular Ca2+, and was blocked by PD-98059, but not by

the MAPKp38 inhibitor, SB-203580, demonstrating that striatal

KA receptors are coupled to NF-kB activation via Ca2+ influx

and MAPKERK kinase activation.

4.9. NMDA, MAPKs and developmental processes

Developmental changes in the signaling properties of

NMDARs have been proposed to underlie the loss of plasticity

that accompanies brain maturation. Ca2+ influx through

postsynaptic NMDARs can stimulate neuronal gene expression

via signaling pathways, such as the Ras–MAPK pathway and

the transcription factor CREB. In this respect, Sala et al. (2000)

analyzed MAPKERK1/2 and CREB activation in response to

NMDAR stimulation during the development of hippocampal

neurons in culture. At all stages of development NMDA

stimulation induced a rapid phosphorylation of CREB on Ser-

133.

However, the time course of decline in phospho-CREB

changed dramatically with neuronal maturation. At 7d in vitro

(7 DIV) phospho-CREB remained elevated after strong NMDA

stimulation, whereas at 14 DIV phospho-CREB rose only

transiently and fell back to below basal levels. Moreover, at 14

DIV, but not at 7 DIV, NMDAR stimulation induced a

dephosphorylation of CREB that previously had been


phosphorylated by KCl depolarization or forskolin, suggesting

an NMDAR-dependent activation of a CREB phosphatase (Sala

et al., 2000).

There was no developmental change in the time course of

phospho-CREB induction that followed KCl depolarization or

PKA activation, nor was there a developmental change in the

time course of phospho-MAPKERK1/2 induced by NMDAR

activation (Sala et al., 2000). During neuronal maturation,

NMDAR activation becomes linked specifically to protein

phosphatases that act on Ser-133 of CREB. Such a devel-

opmentally regulated switch in themode ofNMDARcoupling to

intracellular signaling pathwaysmay contribute to the changes in

neural plasticity observed during brain development.

4.10. NMDA, MAPKs and pain/hyperalgesia mechanisms

Ji et al. (1999) investigated the involvement of MAPKERK

within spinal neurons in producing pain hypersensitivity.

Within a minute of an intense noxious peripheral or C-fiber

electrical stimulus, many phospho-ERK-positive neurons were

observed, most predominantly in lamina I and II of the

ipsilateral dorsal horn; this staining was intensity and NMDAR

dependent. Low-intensity stimuli or A-fiber input had no effect,

however.

Furthermore, inhibition of MAPKERK reduced the second

phase of formalin-induced pain behavior, a measure of spinal

neuron sensitization. MAPKERK signaling within the spinal

cord seems, therefore, to be involved in generating pain

hypersensitivity. Because of its rapid activation, this effect

probably involves regulation of neuronal excitability without

changes in transcription (Ji et al., 1999).

Recently, Svensson et al. (2003) indicated that the activation

of spinal NMDAR initiated activation of the MAPKp38

pathway, leading to spinal release of prostaglandins and

hyperalgesia. Accordingly, inhibition by SD-282, a selective

MAPKp38 blocker, attenuated both NMDA-evoked release of

PGE2 and thermal hyperalgesia. Furthermore, NMDA injection

led to increased phospho-MAPKp38 immunoreactivity in

superficial (I and II) dorsal laminae.

Co-labeling studies revealed co-localization of activated

MAPKp38 predominantly with microglia but also with a small

subpopulation of neurons. Taken together, these observations

revealed a role for MAPKp38 in NMDA-induced PGE2 release

and hyperalgesia, and that microglia are involved in spinal

nociceptive processing (Svensson et al., 2003).

The central glutamatergic system has been implicated in the

pathogenesis of neuropathic pain, and a highly active central

glutamatergic system regulates the uptake of endogenous

glutamate (Sung et al., 2003). It was demonstrated that the

expression and uptake activity of spinal GTs changed after

chronic constriction nerve injury (CCI) and contributed to

neuropathic pain behaviors in rats. CCI induced an initial GT

upregulation within the ipsilateral spinal cord dorsal horn,

which was followed by a GT downregulation. Furthermore,

intrathecal administration of the tyrosine kinase receptor

inhibitor K252a and PD-98059 reduced and nearly abolished

the initial GT upregulation in CCI rats, respectively.

Of note, prevention of the CCI-induced GT upregulation by

PD-98059 resulted in exacerbated thermal hyperalgesia and

mechanical allodynia reversible by MK-801, indicating that the

initial GT upregulation hampered the development of neuro-

pathic pain behaviors (Sung et al., 2003). Moreover, CCI

significantly reduced glutamate uptake activity of spinal GTs,

which was prevented by riluzole (a positive GT activity

regulator) given intrathecally.

Consistently, riluzole attenuated and gradually reversed

neuropathic pain behaviors, suggesting that changes in the

expression and glutamate uptake activity of spinal GTs may

play a critical role in both the induction and maintenance of

neuropathic pain after nerve injury via the regulation of

regional glutamate homeostasis, a new mechanism relevant to

the pathogenesis of neuropathic pain (Sung et al., 2003).

4.11. NMDA, MAPKs and behavioral mechanisms

A central feature of drugs of abuse is to induce gene

expression in discrete brain structures that are critically

involved in behavioral responses related to addictive processes.

Although MAPKERK has been implicated in several neurobio-

logical processes, including neuronal plasticity, its role in drug

addiction remains obscure. A recent study was designed to

analyze the activation ofMAPKERK by cocaine, its involvement

in cocaine-induced early and long-term behavioral effects, as

well as in gene expression (Valjent et al., 2000). Immunocy-

tochemistry analysis showed that acute cocaine administration

activates MAPKERK throughout the striatum, rapidly but

transiently. This activation was blocked when SCH-23390, a

specific dopamine (DA)-D1 antagonist, but not raclopride, a

DA-D2 antagonist, was injected before cocaine. Glutamate

receptors of NMDA subtypes also participated in MAPKERK

activation, as shown after injection of the NMDAR antagonist

MK-801.

Furthermore, the systemic injection of SL-327, a selective

inhibitor of the MAPKERK kinase MEK, before cocaine,

abolished the cocaine-induced MAPKERK activation and

decreased cocaine-induced hyper-locomotion, indicating a role

of this pathway in events underlying early behavioral

responses. Moreover, the rewarding effects of cocaine were

abolished by SL-327 in the place-conditioning paradigm.

Because SL-327 antagonized cocaine-induced c-Fos expres-

sion and Elk-1 hyper-phosphorylation, it is suggested that the

MAPKERK signaling cascade is also involved in the prime burst

of gene expression underlying long-term behavioral changes

induced by cocaine (Valjent et al., 2000). Altogether, these

results revealed a potential mechanism to explain behavioral

responses of cocaine related to its addictive properties.

NMDARs also mediate long-lasting changes in synapse

strength via downstream signaling pathways. In a recent novel

study, Husi et al. (2000) reported proteomic characterization

with mass spectrometry and immunoblotting of NMDAR

multi-protein complexes (NRC) isolated frommouse brain. The

NRC comprised 77 proteins organized into receptor, adaptor,

signaling, cytoskeletal and novel proteins, of which 30 are

implicated from binding studies and another 19 participate in


NMDAR signaling. NMDA and metabotropic glutamate

receptor subtypes were linked to cadherins and L1 cell-

adhesion molecules in complexes lacking AMPA receptors.

These neurotransmitter-adhesion receptor complexes were

bound to kinases, phosphatases, GTPase-activating proteins

and Ras with effectors including MAPK pathway components.

Activity-dependent genes also encoded several proteins.

Genetic or pharmacological interference with 15 NRC proteins

impairs learning and with 22 proteins alters synaptic plasticity

in rodents (Husi et al., 2000). Mutations in three human genes

(NF1, Rsk-2 and L1) are associated with learning impairments,

indicating the NRC also participates in human cognition.

Molecular signaling pathways in the cerebral cortex are

required for retrieval of one-trial avoidance learning in rats.

Rats were implanted bilaterally with cannulae in the CA1

region of the dorsal hippocampus, the entorhinal cortex,

anterior cingulate cortex, posterior parietal cortex or the

basolateral complex of the amygdala (Barros et al., 2000;

Izquierdo et al., 2001). The animals were trained in one-trial

step-down inhibitory avoidance. Retrieval test performancewas

blocked by DNQX, MCPG, Rp-cAMPs and PD-098059 and

enhanced by Sp-cAMPs infused into CA1 or the entorhinal

cortex.

The drugs had similar effects when infused into the

parietal or anterior cingulate cortex, except that in these two

areas AP5 also blocked retrieval, and in the cingulate cortex

DNQX had no effect. Furthermore, infusions into the

basolateral amygdala were ineffective except for DNQX,

which hindered retrieval. None of the treatments that affected

retrieval had any influence on performance in an open field or

in a plus maze; therefore, their effect on retention testing

cannot be attributed to an influence on locomotion,

exploration or anxiety (Barros et al., 2000; Izquierdo

et al., 2001). The results indicate that the four cortical

regions studied participate actively in, and are necessary for,

retrieval of the one-trial avoidance task. They require

metabotropic and/or NMDARs, PKA and MAPK activity.

In contrast, the basolateral amygdala appears to participate

only through maintenance of its regular excitatory transmis-

sion mediated by glutamate AMPA receptors.

Intra-amygdala infusions of NMDAR antagonists have been

reported to block the extinction of conditioned fear (Lu et al.,

2001). Of note, MAPKs can be activated by NMDAR

stimulation and is involved in excitatory fear conditioning.

Lu et al. (2001) evaluated the role of MAPK within the

basolateral amygdala in the extinction of conditioned fear. Rats

received 10 light-shock pairings; fear was assessed by eliciting

the acoustic startle reflex in the presence of the conditioned

stimulus (CS) (CS-noise trials) and also in its absence (noise-

alone trials).

Rats subsequently received an intra-amygdala or intra-

hippocampal infusion of either DMSO or PD-98059 followed

by presentations of the light CS without shock (extinction

training). Afterwards, they were again tested for fear-

potentiated startle. PD-98059 infusions into the basolateral

amygdala but not the hippocampus significantly reduced

extinction, which was otherwise evident in DMSO-infused

rats. Control experiments indicated that the effect of intra-

amygdala PD-98059 could not be attributed to lasting

damage to the amygdala or to state dependency (Lu et al.,

2001). These results suggest that a MAPK-dependent

signaling cascade within or very near the basolateral

amygdala plays an important role in the extinction of

conditioned fear.

Two functionally different MKPs were investigated to

clarify their roles in behavioral sensitization to methamphe-

tamine (METH) (Takaki et al., 2001). MKP-1 mRNA levels

increased substantially in a range of brain regions, including

several cortices, the striatum and thalamus after acute METH

administration. After chronic METH administration its

increase was less pronounced in the frontal cortex. MKP-1

protein levels also increased after acute or chronic METH

administration. MKP-3 mRNA levels increased in several

cortices, the striatum and hippocampus after acute METH

administration, but only in the hippocampus CA1 after chronic

METH administration.

Pre-treatment with the D(1) dopamine receptor antagonist,

SCH-23390, attenuated the METH-induced increase of

MKP-1 and MKP-3 mRNA in every brain region, while

pre-treatment with MK-801 attenuated it in some regions

(Takaki et al., 2001). These findings suggest that in METH-

induced sensitization, MKP-1 and MKP-3 play important

roles in the neural plastic modification in widespread brain

regions in the earlier induction process, but in the later

maintenance process, they do so only in restricted brain

regions, such as MKP-1 in the frontal cortices and MKP-3 in

the hippocampus.

4.12. NMDA, MAPKs and obesity

The obese gene product leptin is an important signaling

protein that regulates food intake and body weight via

activation of the hypothalamic leptin receptor (Ob-Rb)

(Shanley et al., 2001). However, there is growing evidence

that Ob-Rb is also expressed in CNS regions, not directly

associated with energy homeostasis. In the hippocampus, an

area of the brain involved in learning and memory, it was

found that leptin facilitates the induction of synaptic

plasticity.

Leptin converts short-term potentiation of synaptic

transmission induced by primed burst stimulation of the

Schaffer collateral commissural pathway into long-term

potentiation. The mechanism underlying this effect involves

facilitation of NMDAR function because leptin rapidly

enhances NMDA-induced increases in intracellular Ca2+

levels and facilitates NMDA, but not AMPA, receptor-

mediated synaptic transmission. The signaling mechanism

underlying these effects involves activation of PI3-kinase,

MAPK and Src tyrosine kinases (Shanley et al., 2001). These

data indicate that a novel action of leptin in the CNS is to

facilitate hippocampal synaptic plasticity via enhanced

NMDAR-mediated Ca2+ influx. Impairment of this process

may contribute to the cognitive deficits associated with

diabetes mellitus.


4.13. NMDA, MAPKs and pathophysiology: potential

interactions in the evolution of disease/injury

Perturbation of normal survival mechanisms may play a role

in a large number of disease processes. Glutamate neurotoxi-

city, particularly when mediated by the NMDARs, has been

hypothesized to underlie several types of acute brain injury,

including stroke. Several neurological insults linked to

excessive release of glutamate and neuronal death result in

tyrosine kinase activation, including MAPKs.

For instance, removal of the endogenous blockade by Mg2+

of the NMDAR in cultured hippocampal neurons triggers a self

perpetuating cycle of excitotoxicity, which has relatively slow

onset and is critically dependent on NMDARs and activation of

voltage-gated Na+ channels (Skaper et al., 2001). These injury

conditions led to a rapid phosphorylation of MAPKERK1/2 that

was blocked by MEK inhibitors. In addition, MEK inhibition

was associated with protection against synaptically mediated

excitotoxicity. Interestingly, hippocampal neurons precondi-

tioned by a sublethal exposure to Mg2+-free conditions were

rendered resistant to injury induced by a subsequently longer

exposure to this insult; the preconditioning effect was MAPK

dependent.

Brain reperfusion may be of particular importance in the

etiology of periventricular leukomalacia, of which the common

findings are gliosis and ventricular dilatation. To investigate the

mechanism of this pathogenesis, a metabolic inhibition (MI)

model using cyanide plus deoxyglucose treatment of cultured

glia isolated from fetal rat brain was used and examined the

activity of MAPKERK during MI and also during the recovery

from MI (Masuhara et al., 2000). MAPKERK activation was

stimulated duringMI and the recovery fromMI; the time course

and extent of activation of ERK during MI and the recovery

from MI, however, were distinctly different. Activation of

MAPKERK, in addition, was stimulated and declined thereafter.

Activation of MAPKERK was sustained during the recovery

phase fromMI and the extent of the activation was much greater

than that during MI. Pre-treatment with EGTA to eliminate

extracellular Ca2+, or with APV, an NMDAR antagonist, to

inhibit Ca2+ influx through the NMDAR, attenuated the

activation of MAPKERK. Moreover, pre-treatment with PMA to

downregulate PKC abolished the activation of MAPKERK. PD-

98059 attenuated the cell proliferation induced by MI followed

by recovery from MI (Masuhara et al., 2000). These results

suggest that MAPKERK is involved in gliosis during the

recovery phase from MI and may play a role in the etiology of

periventricular leukomalacia.

Estrogen replacement therapy (ERT) in women is associated

with improvement of cognitive deficits and reduced incidence

of Alzheimer’s disease (Osterlund et al., 2000; Bi and Sze,

2002; Katzman, 2004;Mulnard et al., 2004; Li and Shen, 2005).

Estrogen is purported to be a neuroprotective agent against

NMDA- and KA-mediated neurotoxicity, an effect mediated by

tyrosine kinase/MAPK pathways (Bi et al., 2000). Estrogen

also stimulates tyrosine phosphorylation of NMDARs via an

Src tyrosine kinase/MAPK pathway. Furthermore, estrogen-

mediated enhancement of LTP in hippocampal slices is

mediated by activation of an Src tyrosine kinase pathway.

Thus, estrogen, by activating a Src tyrosine kinase and the

MAPKERK signaling pathway, both enhance NMDAR function

and LTP and retains neuroprotective properties against

excitotoxicity. These findings clearly warrant further evaluation

of the usefulness of estrogenic compounds for the treatment of

Alzheimer’s disease and other neurodegenerative diseases.

Conjugated equine estrogens (CEE) are the most widely

prescribed pharmaceutical ERT for postmenopausal women in

the United States and are the ERT of the Women’s Health

Initiative. Previous studies have demonstrated that CEE exerts

neurotrophic and neuroprotective effects in neurons involved in

learning and memory, and which are affected in Alzheimer’s

disease (Nilsen et al., 2002). It was also demonstrated that CEE

potentiated the rise in intracellular Ca2+ following exposure to

physiological concentrations of glutamate. In contrast, the

reverse effect occurred in the presence of excitotoxic levels of

glutamate exposure, where CEE attenuated the rise in Ca2+.

Potentiation of the glutamate response was mediated by the

NMDAR, as the NMDAR antagonist MK-801 blocked the

CEE-induced potentiation, whereas the L-type Ca2+ channel

blocker nifedipine did not (Table 2).

Further, the CEE-potentiated glutamate response was

mediated by a Src tyrosine kinase, as the tyrosine kinase

inhibitor PP2 blocked the potentiation induced by CEE and

neurons treated with CEE displayed increased phosphorylated

tyrosine. The inhibition by CEE of Ca2+ rise in the presence of

excitotoxic levels of glutamate was mediated by MAPK, as the

protective effect of CEE was blocked by inhibiting MAPK

activation with PD-98059, providing potential mechanisms to

explain the cognitive enhancing and neuroprotective effects

exerted by ERT (Nilsen et al., 2002).

It has been demonstrated that there is elevation of the

MAPKERK pathway in the cerebellum from patients with

schizophrenia, an illness that may involve dysfunction of the

NMDAR. Since the NMDA antagonist, phencyclidine (PCP),

produces schizophrenic-like symptoms in humans and abnor-

mal behavior in animals, the effects of chronic PCP

administration in time- and dose-dependent manner on

MAPKERK, MAPKp38 and MAPKJNK were investigated in

rat brain. Osmotic pumps for PCP and saline were implanted

subcutaneously in rats. There was no change at 3 days, but a

significant increase in the phosphorylation of MAPKERK1/2 and

MEK in the cerebellum (Kyosseva et al., 2001).

A dose-dependent elevation in the phosphorylation of

MAPKERK1/2 was observed only in the cerebellum but not in

brainstem, frontal cortex or hippocampus. In addition, the

activities of MAPKJNK and MAPKp38 were unchanged in all

investigated brain regions including cerebellum, demonstrating

that chronic infusion of PCP in rats produces a differential and

brain region-specific activation of MAPKs, suggesting a role

for the MAPKERK signaling pathway in PCP abuse and perhaps

in schizophrenia (Kyosseva et al., 2001).

A marked decrease in the activity of mitochondrial complex

II (succinate dehydrogenase, SD) has been found in the brains

of Huntington’s disease patients. The possibility that SD

inhibitors might produce their toxic action by increasing


corticostriatal glutamatergic transmission was subsequently

investigated (Centonze et al., 2001). It was reported that SD

inhibitors produce a durable augmentation of NMDA-mediated

corticostriatal excitation (DANCE) in striatal spiny neurons,

but not in striatal cholinergic interneurons. In addition, DANCE

involves increased [Ca2+]i, activation of MAPKERK and is

critically dependent upon endogenous DA acting via D2-like

receptors. This pathological form of corticostriatal synaptic

plasticity might play a key role in the regional and cell-type

specific neuronal death observed in Huntington’s disease.

The antibiotic minocycline mediates neuroprotection in

experimental models of neuro-degeneration; it inhibits the

activity of caspase-1, caspase-3, inducible NOS (iNOS) and

MAPKp38 (Zhu et al., 2002). Although minocycline does not

directly inhibit these enzymes, the effects may result from

interference with upstream mechanisms resulting in their

secondary activation. Because the above-mentioned factors are

important in amyotrophic lateral sclerosis (ALS), Zhu et al.

(2002) tested minocycline in mice with ALS. The reported that

minocycline delays disease onset and extends survival in ALS

mice.

Given the broad efficacy of minocycline, understanding its

mechanisms of action is of great importance. It was found that

minocycline inhibits mitochondrial permeability-transition-

mediated cytochrome c release. Minocycline-mediated inhibi-

tion of cytochrome c release was demonstrated in vivo, in cells

and in isolated mitochondria. Understanding the mechanism of

action of minocycline will assist in the development and testing

of more powerful and effective analogues. Because of the safety

record of minocycline and its ability to penetrate the blood-

brain barrier, this drug may be a novel therapy for ALS (Zhu

et al., 2002).

Cell signaling commanding death or survival in human

epileptic hippocampus is difficult to trace because of the long

interval between the beginning of symptoms and the sampling

of damaged cerebral tissue for neuropathological examination.

Intraperitoneal injection of the glutamate analogue KA is a

useful tool to analyze the effects of seizures and the excitotoxic

damage in the rodent hippocampus (Ferrer, 2002).

KA acts on NMDA and KA receptors, whereas it has little

impact on AMPA receptors. Neurons of the hilus and CA3

neurons are primary targets of KA, although parvalbumin

containing GABAergic neurons are less vulnerable than

glutamatergic neurons. Immediate responses to KA are Hsp

70 mRNA induction and HSP 70/72 protein expression, as well

as c-Fos and c-Jun mRNA, and c-Fos and c-Jun protein

expression in the hippocampus. Yet increased c-Fos and c-Jun

expression is not a predictor of cell death or cell survival. In

contrast, the tissue plasminogen activator (tPA) and the

membrane Fas/Fas L signaling pathway probably have a role

in facilitating cell death following KA injection (Ferrer, 2002).

The involvement of other pathways remains controversial.

Increased expression of the pro-apoptotic Bax together with

decreased Bcl-2 suggests Bax-mediated apoptosis. Activation

of the mitochondrial pathway includes leakage of cytochrome c

to the cytosol and activation of the caspase cascade leading to

apoptosis. However, other studies have emphasized the limited

expression of caspase 3, the main executioner of apoptosis, and

the relevance of necrosis as the main form of cell death

following KA excitotoxicity (Ferrer, 2002).

Phosphorylation-dependent activation of several kinases,

including MAPKp38 and MAPKJNK and their substrates has

been found in KA treated animals. Decreased CREBp

expression is associated with cell death whereas increased

ATF 2P and Elk 1P are associated with cell survival. Trophic

factors probably do not play a significant role during the early

stages of hippocanmpal damage but they are important in the

remodeling of the granule cells and the sprouting of mossy

fibers to the molecular layer of the dentate gyrus. This abnormal

regeneration, in turn, facilitates seizure recruitment and the

chronic maintenance of convulsions (Ferrer, 2002).

MAPKJNK3, the only neural-specific isoform, has been

shown to play an important role in excitotoxicity and neuronal

injury (hypoxia/reperfusion). To analyze the variation of

MAPKJNK3 activation, levels of phospho-MAPKJNK3 were

measured at various time points of ischemia and selected time

points of reperfusion, respectively.

To unravel the mechanism of MAPKJNK3 activation,

antioxidant NAC, AMPA/KA receptor antagonist DNQX,

NMDAR antagonist ketamine and L-type voltage-gated Ca2+

channel (L-VGCC) antagonist nifedipine were given to the rats

prior to imposition of ischemia. NAC obviously inhibited

MAPKJNK3 activation during the early reperfusion, whereas

DNQX preferably attenuated MAPKJNK3 activation during the

latter reperfusion (Aizenman et al., 2000; Tian et al., 2003). In

addition, Ketamine and nifedipine had no significant effects on

MAPKJNK3 activation during reperfusion. Consequently, ROS

and AMPA/KA receptors were closely associated with

MAPKJNK3 activation following global ischemia.

5. Summary, conclusion and prospects

Collectively, it is apparent that NMDA–MAPK interactions

constitute a revolving neurochemical axis that regulates a

plethora of physiologic functions (Haddad et al., 2002, 2003;

Haddad, 2004b). From receptor signaling to functional

pharmacogenomics, the NMDA–MAPK axis is crucial for

mediating both physiologic and pathophysiologic phenomena

associated with phosphorylation/dephosphorylation mechan-

isms. Recent accumulating evidence points in the direction of

the fact that MAPKs play a crucial role in regulating the

neurochemistry of NMDARs, their physiologic properties and

their potential role in pathophysiology (Yun et al., 1999;

Cammarota et al., 2000; Chattopadhyay and Brown, 2000;

D’Onofrio et al., 2000; Jiang et al., 2000b; Fuller et al., 2001;

Hardingham et al., 2001; Rush et al., 2002).

To recapitulate, this survey discussed: (i) the MAPK

revolutionary role in regulating a plethora of cellular functions;

(ii) the role of MAPK modules in regulating the biochemistry

and physiology of NMDA receptors and related properties; (iii)

the kinetics of MAPK–NMDA interactions and their biologic

properties; (iv) how cellular signaling pathways, related

cofactors (transcription factors) and intracellular/intercellular

conditions within the microenvironment affect NMDA–MAPK


Fig. 8. An overview scheme depicting the complex interactions of NMDA–MAPK pathways (see discussion for further details).

interactions and (v) the role of NMDA–MAPK modules in

pathophysiology and the evolution of disease conditions.

NMDA regulation of MAPKs and visa versa constitutes a

revolving neurochemical axis that opens a new horizon for

screening targets in experimental pharmacology and therapeu-

tics.

The complex interactions governing NMDA–MAPK path-

ways are schematically depicted in Fig. 8.

Acknowledgments

The author would like to appreciate the comments of his

colleagues at the University of California (UCSF, CA, USA)

and the American University of Beirut (AUB, Beirut, Lebanon)

for constructive criticism. Some of the reproduced figures, with

the exceptions of Figs. 1 and 2, were adapted with

modifications, courtesy of authored and co-authored references

therein cited. Dr. John Haddad held the Georges John Livanos

(London) and the NIH (UCSF) fellowships. The author’s own

publications are, in part, financially supported by the

Anonymous Trust (Scotland), the National Institute for

Biological Standards and Control (England), the Tenovus

Trust (Scotland), the UK Medical Research Council (MRC,

London), the Wellcome Trust (London) (Tayside Institute of

Child Health, Ninewells Hospital and Medical School,

University of Dundee, Scotland, UK), and the National

Institutes of Health (Department of Anesthesia and Periopera-

tive Care, University of California at San Francisco, CA, USA).

The author would also like to especially thank Dr. Christian

Fahlman (UCSF) for contributing to writing and evaluating this

manuscript. This manuscript was written at UCSF when Dr.

John Haddad was a research fellow, and amended at the

American University of Beirut and the Lebanese International

University.

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