POSTSYNAPTIC MECHANISMS OF EXCITOTOXICITY: INVOLVEMENT
OF POSTSYNAPTIC DENSITY PROTEINS, RADICALS, AND OXIDANT
J. P. FORDERaAND M. TYMIANSKIa,b,c*
aDivision of Fundamental Neurobiology, University Health Network,
Toronto, Ontario, Canada M5T 2S8
bDepartment of Physiology, University of Toronto, Toronto, Ontario,
Canada M5S 1A8
cDepartment of Neurosurgery, University of Toronto, Toronto, Ontario,
Canada M5G 1LG
Abstract—Traditional models of neuronal excitotoxicity fo-
cused on the overactivation of receptors such as the ionotropic
N-methyl-D-aspartate (NMDA)-subtype glutamate receptor. Re-
cent developments have shifted focus to downstream neuro-
toxic signaling molecules with exciting implications to specific
strategies for treating excitotoxic disorders. This review out-
lines these developments and introduces newly emerging evi-
dence implicating the involvement of the melastatin subfamily
in anoxic neuronal death. Both of these converge on the pro-
duction of reactive oxygen species (ROS), including superox-
Published by Elsevier Ltd. All rights reserved.
Key words: NMDAR, peroxynitrite, TRPM7, anoxia, neuropro-
Overview of postsynaptic mechanisms of excitotoxicity and the
role of NMDARS
Ionotropic NMDA-subtype glutamate receptor
Subcellular localization of NMDARS and their effect on
PSD proteins in excitotoxicity
Organizational role of PSD-95 in NMDAR signaling
Free radicals and oxidant molecules in excitotoxicity
The role of transient receptor potential channels in anoxic
Excitotoxicity following cerebral ischemia involves an over-
activation of receptors associated with the postsynaptic
densities (PSDs) of target neurons. This receptor activa-
tion initiates a cascade of specific second messengers
resulting in increased intracellular calcium, free radical
production and eventual cell death. Traditionally, the re-
ceptors most intimately connected to excitotoxicity are the
ionotropic glutamate receptors; the most intensely studied
is the N-methyl-D-aspartate (NMDA)-subtype. Recent fo-
cus has shifted to studies elucidating the involvement of
downstream neurotoxic signaling molecules linked to PSD
proteins that interact with the N-methyl-D-aspartate recep-
tor (NMDAR) resulting in exciting implications to highly
specific strategies for treating excitotoxic disorders. Other
newly emerging evidence implicates the involvement of
transient receptor potential channels, especially those of
the melastatin subfamily (TRPM), particularly TRPM7 and
TRPM2, which may initiate non-excitotoxic processes via
induction of oxidative stress and oxygen free radicals. This
report outlines postsynaptic mechanisms of excitotoxicity
with specific focus on the involvement of PSD proteins and
TRPM channels, free radicals and oxidant molecules.
OVERVIEW OF POSTSYNAPTIC MECHANISMS
OF EXCITOTOXICITY AND THE ROLE OF
The traditional calcium hypothesis of excitotoxicity proposes
influx of calcium into the intracellular space due to excessive
excitatory stimulation by glutamate, causing cell membrane
depolarization which leads to activation of ionic channels and
a reduced capacity of ionic pumps and exchangers respon-
sible for maintenance of cellular ionic homeostasis, leading to
entry into neurons have been ascribed to voltage-gated cal-
cium channels (VGCCs) and glutamate-sensitive channels. It
is believed that while physiological levels of glutamate are
effective as a major excitatory neurotransmitter in the CNS,
excessive glutamate release following ischemia leads to ex-
citotoxicity and results in neurodegeneration. This model of
excitotoxicity has been expanded to include other ion chan-
nels and signaling pathways initiated through macromole-
cules found in PSD. We will briefly outline the postsynaptic
responses to glutamate and then focus on NMDARs before
expanding into a discussion on the PSD proteins involved in
*Correspondence to: M. Tymianski, Toronto Western Hospital Re-
search Institute, Suite 4W-435, 399 Bathurst Street, Toronto, Ontario,
Canada M5T 2S8. Tel: ?1-416-603-5896; fax: ?1-416-603-5298.
E-mail address: email@example.com (M. Tymianski).
Abbreviations: GST, glutathione S-transferase; NMDA, N-methyl-D-
aspartate; NMDAR, N-methyl-D-aspartate receptor; nNOS, neuronal
nitric oxide synthase; NO, nitric oxide; NR, N-methyl-D-aspartate re-
ceptor subunit; PSD, postsynaptic density; PSD-95, postsynaptic den-
sity-95 protein; RNAi, RNA interference; RNS, reactive nitrogen spe-
cies; ROS, reactive oxygen species; TRPM, transient receptor poten-
tial channels of the melastatin family.
Neuroscience 158 (2009) 293–300
0306-4522/09 © 2009 IBRO. Published by Elsevier Ltd. All rights reserved.
IONOTROPIC NMDA-SUBTYPE GLUTAMATE
N-methyl-D-aspartate receptors (NMDARs) mediate cal-
cium influx and are involved in trophic developmental pro-
cesses as well as activity-dependent resetting of synaptic
strength. The basic properties of NMDARs include: rela-
tively slow kinetics, high calcium permeability, voltage-
dependent block by magnesium, glycine co-activator, poly-
amine activation, zinc inhibition and large single channel
Functional NMDARs are heteromeric combinations of
N-methyl-D-aspartate receptor subunit (NR) 1 together
with one of four possible NR2 subtypes (NR2A–NR2D)
and, in some cases, NR3 subunits (McBain and Mayer,
1994). NMDAR subtype-specific signaling may differen-
tially govern the direction of synaptic plasticity and play
differential roles in pathological conditions. Specific NR2
subtypes appear to play pivotal roles in stroke (Liu et al.,
2007; Liu et al., 2004).
The differential effects of NMDAR NR2 subtypes have
been intensely investigated in a number of areas to include
developmental plasticity (Cline, 2001; Niell et al., 2004;
Lee et al., 2005; Ewald et al., 2008), epilepsy (Gashi et al.,
2007), Huntington’s disease (Li et al., 2003; Benn et al.,
2007; Fan et al., 2008) and ischemia (Li et al., 2007; Chen et
al., 2008; Gascon et al., 2008). For example, using a four-
vessel occlusion model of transient global ischemia in rats,
Chen et al. (2008) found that blocking NR2A-containing
NMDARs enhanced neuronal death and abolished the induc-
tion of ischemic tolerance, whereas inhibiting NR2B-contain-
ing NMDARs attenuated ischemic cell death and enhanced
These differential effects may be explained, in part, by
the evidence that different NR2 subtypes confer distinct
pharmacological and electrophysiological properties on
the receptors as well as coupling with different signaling
mechanisms. For example, in mature cultured neurons,
NR2A-containing NMDARs promote trafficking of GluR1
while NR2B-containing NMDARs inhibit such trafficking
(Kim et al., 2005). Furthermore, these subtypes have been
found to differentially contribute to the activation of the
downstream MAP kinase signaling pathway (Kim et al.,
2005). NR2A and NR2B have different calcium ion influx
as a consequence of different biophysical properties that
result in fast decay times for NR2A and significantly slower
decay times for NR2B-containing receptors (Monyer et al.,
1994; Sobczyk et al., 2005). For example, Chen et al.
(2008) demonstrated ischemia-induced upregulation of
CREB-targeted genes with NR2A specific blockade reduc-
ing the phosphorylation of CREB induced by precondition-
ing. Irving et al. (2000) report a differential activation of
MAPK/ERK and p38/SAPK pathways in neurons and glia
following focal cerebral ischemia.
SUBCELLULAR LOCALIZATION OF NMDARS
AND THEIR EFFECT ON
In mature neurons, NR2A is preferentially located at syn-
aptic sites while NR2B is enriched in extrasynaptic sites
(Stocca and Vicini, 1998; Tovar and Westbrook, 1999).
The subcellular localization of NMDARs also affects the
nature of NMDAR signaling. Evidence suggests that syn-
aptic NMDAR activity is extremely important for neuronal
survival while the extrasynaptic NMDARs are coupled to
cell-death pathways (Hardingham and Bading, 2003; Pa-
padia et al., 2005). This may be mediated by extrasynaptic
NMDARs opposing synaptic NMDARs by triggering CREB
shutoff and cell death pathways (Pokorska et al., 2003).
The synaptic NMDARs form large and dynamic signaling
complexes in the postsynaptic membrane (Husi et al.,
2000). Indeed, Benn et al. (2007) found complex alter-
ations of glutamate receptor subunit protein localization,
not an increase in transcription of the subunits, in a
YAC128 transgenic mouse model of Huntington’s disease.
PSD PROTEINS IN EXCITOTOXICITY
NMDARs have been shown to be organized into multipro-
tein signaling complexes within a specialized structure
located beneath the postsynaptic membrane aligned with
active zones of presynaptic terminals with the CNS, known
as the PSD (Aarts et al., 2003a,b; Sheng and Hoogenraad,
2007). The PSD is densely packed with membrane-bound,
scaffolding and cytoskeletal proteins. PSDs are involved in
several functions including: cell-to-cell adhesion, regula-
tion of receptor clustering and modulation of receptor func-
tion. Excitatory CNS synapses are especially enriched in
type I PSDs while type II PSDs are characteristic of inhib-
itory GABA synapses (Landis and Reese, 1974). The PSD
at glutamatergic synapses is a dynamic entity and its mor-
phology can be influenced by synaptic activity.
The PSD is composed of four major types of mole-
cules: membrane-bound, cytoskeletal, and scaffolding pro-
teins as well as modulatory enzymes. The most abundant
membrane-bound proteins found in the PSD are ionotropic
glutamate receptors, whereas the abundant cytoskeletal
elements, including actin, fodrin, tubulin and neurofila-
ments are important in localizing and clustering the PSD
receptors and signal complexes (Kennedy, 1998). Scaf-
folding proteins bring the various PSD components into as-
sociation and include specrin, actinin, AKAP 79 and proteins
containing combinations of various postsynaptic density-95
protein (PSD-95)/discs large (DLG)/zona occludens-1 (ZO-1)
(PDZ) domains. PDZ domains are modular, ?90 residue
protein–protein interaction domains. Regulation of PSD com-
ponents and second messengers involves phosphorylation
events carried out by a number of kinases to include mem-
bers of the Src family of non-receptor protein tyrosine ki-
nases, calcium/calmodulin kinase II (CaMKII), protein kinase
C (PKC), ERK2-type mitogen activated kinase and cal-
cineurin. Other key enzymes are also found in the PSD that
significantly affect the glutamate receptor signal pathway. For
example, neuronal nitric oxide synthase (nNOS) is activated
J. P. Forder and M. Tymianski / Neuroscience 158 (2009) 293–300294
by NMDA calcium influx and can modulate NMDAR signaling
(Bredt, 1996). While these PSD proteins may transmit
NMDAR activity into the cell, they also play a part in govern-
ing the signaling properties of the NMDARs, initiating an
interaction of its intracellular domains with cytoskeletal and
signal transduction molecules in the PSD (Fig. 1). The roles
of synaptic versus extrasynaptic NMDARs in cell death and
survival are critically related to their interactions with the
submembrane scaffolding proteins with which they interact,
NR2A and NR2B subunits differ in their interactions with
different PSD proteins (Sans et al., 2000; Krapivinsky et al.,
ORGANIZATIONAL ROLE OF PSD-95 IN
Of the proteins involved in the PSD, PSD-95 plays a prom-
inent organizational role by coupling the NR2 subunits of
NMDARs to intracellular proteins and signaling enzymes
(Brenman et al., 1996; Sheng, 2001; Aarts et al., 2002,
2003a; Aarts and Tymianski, 2004; Arundine et al., 2004;
Cui et al., 2007). PSD-95 contains three PDZ domains, of
which the first two (PDZ1 and PDZ2) interact with the C
termini of NMDAR NR2 subunits linking them to down-
stream neurotoxic signaling molecules such as nNOS.
NMDARs have long been known to mediate ischemic
brain injury (Simon et al., 1984). But blocking them is a
failed strategy in stroke and can be deleterious to animals
and humans (Fix et al., 1993; Morris et al., 1999; Davis et
al., 2000). We have previously tested an alternative strategy to
block excitotoxicity by addressing excitotoxic signaling down-
stream from NMDARs without blocking NMDARs or inhibiting
excitatory neurotransmission. In one attempt, PSD-95 ex-
pression in cortical neurons was suppressed using antisense
oligodeoxynucleotides. This reduced neuronal NMDAR-acti-
vated nitric oxide (NO) production and excitotoxicity without
inhibiting NMDAR ionic currents and calcium signaling (Sat-
tler et al., 1999). However, mutation or suppression of
PSD-95 expression is therapeutically impractical, and
therefore disrupting the interactions of NMDAR NR2B sub-
units with PSD-95 has also been pursued. The interactions
of preformed NR2–PSD-95 complexes were achieved in
cultured neurons, and in experimental animals by using
peptides that mimicked the NR2 c-termini interaction with
the PDZ domains of PSD-95, This was achieved by cou-
pling the nine c-terminal residues of the NR2B NMDAR
subunit to the 11 residue Tat protein transduction domain
to compose a peptide termed Tat-NR2B9c. Disrupting
NMDAR–PSD-95 interactions with this peptide reduced
the excitotoxic vulnerability of neurons as previously ob-
served in PSD-95-deficient cells. In addition, it rendered
neurons of rats resistant to focal cerebral ischemia in vivo
(Aarts et al., 2002).
The human genome contains hundreds of PDZ do-
main-containing proteins (Giallourakis et al., 2006) and all
NR2 subunit c-termini possess a promiscuous type I PDZ
interaction motif (T/SXV). Consequently NMDARs may
bind many other cellular PDZ proteins, any of which could
mediate neurotoxic signaling independently of PSD-95.
Also, Tat-NR2B9c may mediate either neuroprotection or
undesirable side effects by perturbing NR subunit-PDZ
interactions other than with PSD-95. Recently Cui et al.,
2007 successfully elucidated the specificity of action of the
Tat-NR2B9c peptide by cloning all publicly known human
PDZ domains into expression vectors, producing them as
recombinant glutathione S-transferase (GST):PDZ fusion
proteins. Using an ELISA-based competition assay, inter-
action profiles were evaluated including: NMDAR NR1
subunits; NR2A-NR2D subunits; nNOS; and Tat-NR2B9c.
The analyses revealed all previously published and some
Fig. 1. Interactions of NMDARs with PSD proteins (taken from Aarts et al., 2003b). The wide array of proteins found within the PSD interacts to form
the glutamatergic signal transduction machinery. Such interactions govern the activity-dependent and -independent receptor targeting and trafficking
that is important for synaptic plasticity and excitotoxic signaling.
J. P. Forder and M. Tymianski / Neuroscience 158 (2009) 293–300 295
new interactions of NMDARs, nNOS, and Tat-NR2B9c
with PDZ domain-containing proteins. To determine which
of these interactions are involved in excitotoxic processes,
RNA interference (RNAi) was used to suppress the ex-
Fig. 2. Clustered heat map of PDZ-ligand interactions (from Cui et al., 2007). Columns represent peptides corresponding to PDZ ligand C termini. Rows
represent GST-PDZ fusion proteins containing the indicated human PDZ domains. The column headings provide the absolute c-terminal four aa of the PL
peptides and the names of the proteins from which they are derived. The intensity of red color represents the observed colorimetric signal derived from four
or more PDZ–PL binding experiments at 10 ?M unless noted in the column header. For PLs with maximum binding signal ?1 A450unit, the data in that
column were normalized to set the maximum observed binding to 1. The right half of the figure is a continuation of the heat map from the bottom of the left half.
than one domain is listed, the GST construct contains multiple domains. The results shown encompass a total of ?4100 unique potential PDZ–PL interactions. All
using 1?r as the distance metric, where r is the Pearson correlation coefficient, and the relative expression levels are displayed.
J. P. Forder and M. Tymianski / Neuroscience 158 (2009) 293–300 296
pression of each key protein that interacts with Tat-
NR2B9c. Cui et al. tested for excitotoxicity involvement
and demonstrated that Tat-NR2B9c, a neuroprotective
peptide, acts exclusively through its interactions with
PSD-95 and nNOS, and not any of the other PDZ-domain
containing proteins. The data obtained also provided an
extensive database for ?4100 interactions between syn-
aptic proteins/receptors and cellular PDZ proteins among
which are revealed novel interactions of key proteins in-
volved in cellular signaling. Results indicate a surprising
specificity of neurotoxic signaling through PDZ proteins
The role of PSD-95 in stroke and effects of its selective
inhibition without inhibiting excitatory neurotransmission
were explored by (Sun et al., 2008). Infarct size was per-
manently reduced and significant neurobehavioral im-
provements seen in unfasted Sprague–Dawley rats in
which a single i.v. injection of PSD-95 inhibitors (peptides
recapitulating NR2 c-termini) were administered 1 h or 3 h
after stroke onset. This study included experiments in
which the experimental animal’s brain temperature was
permitted to rise to about 40 °C, simulating severe fever.
Despite this hyperthermia, the PSD-95 inhibitors reduced
infarct size. Previous studies in which PSD-95 was dis-
rupted genetically (Migaud et al., 1998), or with antisense
oligonucleotides (Sattler et al., 1999), or with inhibitors
(Aarts et al., 2002) did not affect NMDAR function or
normal excitatory neurotransmission. In mutant mice lack-
ing PSD-95, long-term potentiation is enhanced and yet
synaptic NMDA-receptor currents, subunit expression, lo-
calization and synaptic morphology are all unaffected (Mi-
gaud et al., 1998). It would appear that PSD-95 inhibitors
are the first pharmacological compounds that show effi-
cacy in a post-treatment paradigm even in severe hyper-
thermia. They are also the first compounds that, while
addressing excitotoxic mechanisms, produce permanent
neuroprotection when administered hours after the isch-
emic insult and thus justify a continuing evaluation of this
class of compounds for the treatment of stroke.
FREE RADICALS AND OXIDANT MOLECULES
The important role of reactive oxygen species (ROS) and
reactive nitrogen species (RNS) in cell damage during
ischemia is emphasized by the fact that treatment with free
radical scavengers can be effective in experimental focal
cerebral ischemia. A growing body of literature is emerging
implicating free radicals and oxidant molecules in excito-
toxic cell damage.
While NO is well known as an important signaling
molecule, it has also been well established both in vivo and
in vitro as an important player in excitotoxicity and is known
to be generated following NMDAR activation (Dawson et
al., 1991; Huang and Gean, 1994; Iadecola et al., 1997). It
has been reported that primary brain cultures treated with
NOS inhibitors or cultures from mice with targeted disrup-
tion of nNOS are resistant to NMDA neurotoxicity (Dawson
et al., 1991). In addition to NO, calcium can trigger the
production of oxygen free radicals such as superoxide
1994). Indeed, superoxide production has been shown to
be elevated during ischemia due to mitochondrial damage.
The combination of NO and superoxide results in the for-
mation of highly toxic peroxynitrite (ONOO?) (Fig. 3). Per-
oxynitrite is a highly reactive molecule that can react with
sulfhydryl groups and zinc-thiolate moieties and can oxi-
dize lipids, proteins and DNA (Radi et al., 1991; Darley-
Usmar et al., 1992; Crow and Beckman, 1995a,b). For
example, nitrosylation of GAPDH, an essential mitochon-
drial protein, results in respiratory dysfunction and leads to
cell death (Kiss and Szabo, 2005). Peroxynitrite is also
involved in tyrosine nitration of a large variety of proteins
and can interfere with key phosphorylation events (Buch-
czyk et al., 2000).
?) downstream of phospholipase A2 (Chanock et al.,
THE ROLE OF TRANSIENT RECEPTOR
POTENTIAL CHANNELS IN ANOXIC
Intracellular calcium is a universally important second
messenger influencing a great number of cell functions
and provides a very important regulatory role in both cell
survival and proliferation as well as in apoptotic and ne-
crotic cell death (Orrenius et al., 1996). Work by Apati et al.
(2003) provides evidence that opposing effects of intracel-
lular calcium depend on the duration and amplitude of the
calcium signal. TRPM channels are nonspecific cation-
permeable channels (Fig. 4). Evidence has been accumu-
lating regarding the involvement of the specific TRPM2
and TRPM7 channels in excitotoxicity. Aarts and Tymian-
ski (2005) implicated TRPM2 and TRPM7 in the delayed
calcium deregulation that occurs following the initial spike
in intracellular calcium levels during excitotoxic conditions.
Indeed various reports indicate that TRPM2 can be acti-
vated by H2O2, arachidonic acid, ROS, RNS, and intracel-
lular calcium (Aarts and Tymianski, 2005; MacDonald et
al., 2006; Kaneko et al., 2006; Wehage et al., 2002; Hara
et al., 2002). Further, TRPM2 mRNA has been found to be
upregulated in rats following middle cerebral artery occlu-
Fig. 3. Lethal positive feedback loop of free radical production gen-
erated by TRPM7 activation in ischemia (from Aarts and Tymianski,
2005). NMDAR activation during ischemia injury results in Ca2?entry
that stimulates production of NO and release of superoxide (O*) from
mitochondria. NO and O* then combine to form the highly reactive
species peroxynitrite (ONOO). These free radicals in turn activate
TRPM7, resulting in further Ca2?influx and production of oxygen and
J. P. Forder and M. Tymianski / Neuroscience 158 (2009) 293–300 297
sion (MCAO) with evidence leading to the speculation that
it plays a role in the microglial response to ischemia (Fon-
fria et al., 2006). Knocking down TRPM2 through the use
of RNAi, resulted in a protective response against H2O2
toxicity in HEX cells (Kaneko et al., 2006) and against
oxidative stress in hematopoietic cells (Zhang et al., 2006).
Evidence for the involvement of TRPM7 in anoxic neu-
ronal death is mounting. Aarts et al. (2003a) suggest that
TRPM7 provides a means by which neurons detect changes
in the extracellular concentration of divalents and is a major
contributor to the nonselective cation current that is associ-
ated with calcium overloading and subsequent neuronal
death. Blockade of TRPM7 channels in vitro can prevent
calcium influx and decrease cell death following oxygen-
glucose deprivation (OGD) and TRPM7 can be activated by
ROS and RNS as well as PIP2 (phosphatidylinositol bisphos-
phate) (Aarts et al., 2003a). TRPM7 channels may act as
calcium sensors. Wei et al. (2007) further propose that
TRPM7 contributes to neuronal cell death during ischemia-
reperfusion injury in both the initial stages of an ischemic
attack and during reperfusion. During the initial stages, when
the extracellular divalent concentrations are reduced, it is the
permeability of the TRPM7 channels to the monovalent cat-
ions that leads to TRPM7 contribution to the initial sodium
loading phase and associated toxicity. During reperfusion,
the extracellular concentrations of divalent cations are re-
nitrogen species production which further facilitates TRPM7
current. It is suggested that as a calcium sensor, TRPM7
channels will add to and exacerbate neuronal injury mediated
by other mechanisms (Wei et al., 2007).
Stroke, epilepsy, neurotrauma, and chronic neurodegen-
erative conditions are of major public health significance,
but the mechanisms leading to CNS damage in them are
unclear. Traditionally, the receptor most intimately con-
nected to excitotoxicity is the ionotropic NMDA glutamate
receptor. However, recent focus has shifted to studies
elucidating the involvement of downstream neurotoxic sig-
naling molecules linked to PSD proteins that interact with the
NMDAR and to other receptors such as TRPM channels that
are intimately involved in anoxic and excitotoxic neuronal
damage. The results indicate exciting implications to highly
specific strategies for treating excitotoxic disorders. Both of
these converge on the production of ROS, including super-
oxide, NO and the oxidant peroxynitrite.
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Fig. 4. Organization of TRPM7 subunit (from Aarts and Tymianski, 2005). Like other transient receptor potential (TRP) family members, TRPM7
contains six putative transmembrane domains with intracellular N- and c-termini, a pore loop (P) that regulates ion selectivity, and a consensus TRP
domain (green). TRPM7 and the closely related TRPM8 are unique among TRP subunits in that they contain a c-terminal ?-kinase (red).
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(Accepted 15 October 2008)
(Available online 1 November 2008)
J. P. Forder and M. Tymianski / Neuroscience 158 (2009) 293–300300