Biochemical evidence for calpain's involvement in long term synaptic plasticity

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Content BIOCHEMICAL EVIDENCE FOR CALPAIN’S
INVOLVEMENT IN
LONG TERM SYNAPTIC PLASTICITY
by
Xiaoying Lu
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOLOGY)
December 2001
Copyright 2001 Xiaoying Lu
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U N IV E R SITY OF SOUTHERN C A L IF O R N IA
The Graduate School
University Park
L O S A N GELES. CALIFORNIA 90089-1695
This dissertation , w ritten b y
.. ... (rlA -_____
Under th e direction o f bJkL. D issertation
Com m ittee, an d approved b y a d its members,
has been p resen ted to and a ccep ted b y The
Graduate School in p a rtia l fulfillm ent o f
requirem ents fo r th e degree o f
DOCTOR OF PHILOSOPHY
o f Gn d u a t e S t u d i e s
***** .May 1 0 , 2 Q n ?
DISSERTATION COMMITTEE
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DEDICATION
To my husband, Bo Ma, my parents, Yuechu Wang and Wenzhang Lu, and
brother, Ping Lu and his family.
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ACKNOWLEDGMENTS
I am gratefully indebted to my mentor and advisor Dr. Michel Baudry, for his
constant guidance, support and encouragement throughout the course of my
dissertation work. Being both an excellent scientist and a caring individual, he is
patient, helpful and considerate to his students. I also want to thank my committee
members, Dr. Richard Thompson and Dr. Jean-Michel Maarek for their outstanding
advice and support. Besides, Judith Thompson and other members of Thompson lab
have generously shared their equipments and space with me for the past four years
and their help and kindness are greatly appreciated.
I also greatly appreciate the support and help from my colleagues, especially
Dr. Steve Standley, who taught me the basic cellular biological techniques and
helped me start my first project, Dr. Yongqi Rong, who provided tremendous
technical assistance during the last three years of my research, as well as Dr. Ruifen
Bi, Dr. Xiaoning Bi and Dr. Greg Broutman, Jean-Marie Bouteiller, Ruolan Liu and
Ordina Zhang. I would further acknowledge the hard work of Barbara Valastro and
Emile Banayan, who had been working with me briefly during my dissertation
research.
I would like to express my heartfelt thanks to my parents who inspired my
interest in natural science when I was little and who continued to educate, encourage
and guide me through my life. Their endless love is one of the most valuable things
I’ve ever had. Finally, I deeply appreciate the understanding and help of husband, Bo
Ma.
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TABLE OF CONTENTS
Dedication....................................................................................................................ii
Acknowledgments......................................... iii
List of Figures.............................................................................................................vi
Abstract.................................................................................................................... viii
Chapter One: Introduction
I) Hippocampal long-term synaptic plasticity as the cellular
basis of learning and memory..................................................2
II) Role of protein phosphorylation in synaptic plasticity and
learning................................................................................... 8
III) Regulation of receptor trafficking modifies synaptic
efficacy...................................................................................10
IV) Involvement o f calpain in long-term synaptic
plasticity.................................................................................12
V) Specific Aims.........................................................................14
Chapter Two: Preferable degradation of non-phosphorylated form of aCaMK.II by
Calpain
Abstract..............................................................................................16
Introduction........................................................................................17
Materials and Methods...................................................................... 19
Results...............................................................................................22
Discussion......................................................................................... 27
Chapter Three: Degradation of a major postsynaptic protein PSD-95 by calpain
Abstract..............................................................................................30
Introduction........................................................................................ 31
Results................................................................................................32
Discussion..........................................................................................40
iv
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Chapter Four The association between GRIP and AMP A receptor subunit GluR2
was disrupted by as a consequence of calpain activation
Abstract.............................................................................................43
Introduction.......................................................................................44
Materials and Methods.....................................................................46
Results...............................................................................................51
Discussion.........................................................................................60
Chapter Five: Calpain-mediated truncation of rat brain AMPA receptors increases
their Triton X-100 solubility
Abstract.............................................................................................64
Introduction.......................................................................................65
Materials and Methods..................................................................... 66
Results...............................................................................................69
Discussion......................................................................................... 77
Chapter Six: General discussion............................................................................ 82
References................................................................................................................. 93
v
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LIST OF FIGURES
1. In vitro digestion of aCaMKII......................................................................22
2. Activation of calpain following brief NMD A stimulation of
cultured hippocampal slices........................................................................... 24
3. Calpain inhibitor blocks the degradation of spectrin and a-CaMKII
following brief NMD A stimulation of cultured hippocampal slices..............25
4. Calpain preferably degrades non-phosphorylated form of a-CaMKII.......... 26
5. PSD-95 degradation by calpain in rat forebrain synaptic membranes...........33
6. Effect of tyrosine phosphorylation on calpain-mediated truncation of
PSD-95...........................................................................................................36
7. Effect of calcium treatment of frozen-thawed brain sections and
NMDA treatment of organotypic hippocampal cultures on PSD-95............. 38
8. Developmental pattern of PSD-95.................................................................39
9. Calpain-mediated degradation of GRIP in synaptic membranes and
different subcellular fractions.........................................................................52
10. Effects of calcium treatment of frozen-thawed brain sections on GRIP........54
11. Effects of NMDA receptor activation in cultured hippocampal
slices on GRIP................................................................................................ 55
12. Disruption of GluR2-GRIP interaction following calpain-treatment
of synaptic membranes...................................................................................57
13. Effects of NMDA treatment of acute hippocampal slices on
GIuR2-GRIP interaction.................................................................................59
14. Triton X-100 insolubility of GluRl, GluR2/4, GluR2/3, NR2A, NR2B
and aCaMKII, and effect of calcium on Coomassie blue staining
pattern of PSD-enriched fractions...................................................................70
15. Effects of calpain activation on the properties and Triton X-100
solubility of GluRl subunits...........................................................................73
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16. Effects of calpain activation on the properties and Triton X-100
solubility of GluR2 subunits.......................................................................... 74
17. Effects of calpain activation on the properties and Triton X-100
solubility of NR2B subunits.......................................................................... 76
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ABSTRACT
Calcium dependent neutral proteases (calpain) have been demonstrated to
participate in long-term synaptic plasticity. To identify the critical downstream
events, we studied the effects of calpain on several proteins that play important roles
in synaptic function.
Calpain-mediated proteolysis of GRIP and PSD-95 was first studied in vitro
by calpain I digestion of rat forebrain membranes. In both cases, several degradation
products were identified suggesting the existence of multiple cutting sites. Most of
the fragments were later confirmed following in situ calpain activation by brief
NMDA stimulation of cultured hippocampal slices, which indicates that calpain-
mediated proteolysis of GRIP and PSD-95 might occur in vivo. We also found the
PSD-95 breakdown products were abundant in developing hippocampus, and
dramatically declined during the adulthood. Combined with previous reports, we
proposed that calpain degradation of PSD-95 is involved in synaptic remodeling
during development, and possibly in adulthood following appropriate stimulus. To
explore the potential functions of calpain-mediated GRIP degradation, we analyzed
GRIP and GluR2 interaction using co-immunoprecipitation techniques. We found
that calpain activation resulted in the disruption of GRIP-GIuR2 interaction and since
GRIP has been hypothesized as AMPA receptor anchoring protein, such disruption
could facilitate release of the AMPA receptors from the synaptic sites.
To further address the impact of calpain activation on glutamate receptor
trafficking, frozen-thawed rat forebrain sections were first treated with calcium to
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activate endogenous calpain, and PSD proteins were extracted as Triton-insoluble
fractions. Following calpain activation, AMPA receptor subunits GluRl and GluR2
are fragmented and, interestingly, the degradation species of the subunits were absent
from PSDs and recovered in Triton-soluble fractions. Our data thus revealed the
potential function of calpain in glutamate receptor trafficking.
The study on calpain degradation of a-CaMKII suggested that
autophosphorylated form of a-CaMKII is resistant to calpain-meidated proteolysis.
Since it has been previously shown that calpain decreases the activity of non-
phosphorylated form of the kinase, our results thus ague that calpain might function
to decrease the kinase activity.
In summary, our studies demonstrated the regulatory role of calpain on
several important synaptic molecules and provided biochemical evidence for calpain
involvement in synaptic modification.
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CHAPTER 1
INTRODUCION
Throughout history, numerous scholars have been attracted by the intriguing
topic of memory formation. The great philosopher Plato, among the earliest, wrote in
his Theaetetus that memories are formed on the waxy substrate of one’s mind (Plato,
360BC), which was echoed by more contemporary predications that emotionally
exciting experiences are remembered by leaving “a scar on the cerebral tissues”
(James, 1890) (P670). In fact, the understanding of “the waxy substrate” or cerebral
structure was greatly advanced with the introduction of the neuron doctrine in 1891
by the neuroanatomist Wilhelm von Waldeyer (Waldeyer-Hartz, 1891), largely based
on the research of Santiago Ramon y Cajal. Two years later, the neurologist Eugenio
Tanzi (1893) (Tanzi, 1893) proposed the hypothesis that the plastic changes involved
in learning probably take place at the junction between neurons. The junction was
later named “synapse” by Charles Sherrington (Foster and Sherrington, 1897), a term
that has since been adapted by all neuroscientists. Today, a phenomenon called long
term potentiation (LTP) (Bliss and Lomo, 1973) that was first described 30 years ago
at hippocampal synapses, as well as its reversed process, long-term depression
(LTD) (Dudek and Bear, 1992), despite suffering serious challenges from opponents,
remain the best candidates for cellular processes of synaptic changes that underlie
learning and memory in vertebrate brain.
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I) Hippocampal long-term synaptic plasticity as the cellular basis of learning
and memory
Hippocampus, a pair of seahorse shaped structure located deep inside the
temporal lobes, has long been well recognized for its role in learning and memory.
Extensive evidence gathered from both animal data and patient analysis show that
even though it does not seem to store information for long term, because ablation of
hippocampus does not destroy long-term memory; the hippocampus is essential to
process information for long-term storage elsewhere in the brain. Specifically,
damage to the hippocampus in human beings produces a characteristic anterograde
amnesia, which selectively impairs new memory formation but spares the memories
that were already formed prior to the injury (Squire, 1987).
Excitatory synaptic transmission in hippocampus is mediated by glutamate
and glutamate receptors. Three families of ionotropic glutamate receptors, the a-
amino-3-hydroxy-5 methyl-4-isoxazolepropionate (AMPA), N-methyl-D-aspartate
(NMDA), and kainate (KA) receptors, were identified and named after their
preferential synthetic agonists (Monaghan et al., 1989). Both AMPA and NMDA
receptors are thought to be heteromeric assemblies, with 3 real transmembrane-
spanning domains, TM1, TM3 and TM4, plus a reentrant pore loop segment (TM2).
Native AMPA receptors are probably tetrameric or pentameric complexes, consisting
of different combinations of 4 homologous subunits (GluRl through GluR4). All
AMPA receptor subunits are approximately 900 amino acid residues in length with a
calculated molecular weight of around 100 kDa. NMDA receptors, on the other
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hand, consist of two families of homologous subunits (NR1 and NR2A-D). The
individual NR2 subunits, in contrast to NR1, evoke no appreciable
electrophysiological responses after agonist application and combined expression of
NR1 ami NR2 subunits markedly potentiates responses to NMDA or glutamate.
Thus, NR.1 serves as a fundamental subunit necessary for NMDA receptor-channel
complex and forms a heteromeric configuration, most likely pentameric, with
different members of the NR2 subunits. Distinct channel properties of AMPA and
NMDA receptors account for their different functions in synaptic transmission and
plasticity. AMPA receptors in hippocampus are usually calcium impermeable, due to
the existence of edited GluR2 subunits, while NMDA receptors are permeable to
calcium; AMPA receptors are characterized by fast onset and fast decay gating
kinetics while NMDA receptors have slower gating kinetics. Finally, NMDA
receptor channels require glycine as a co-agonist of glutamate and their activity is
blocked by Mg++ at resting membrane potential.
Neurobiologists from several generations have worked industriously in
search of the mechanism(s) by which plastic changes associated with learning and
memory are formed in hippocampus. The contribution o f Timothy Bliss and Teije
Lomo, who discovered a phenomenon of long-lasting synaptic plasticity, has been a
landmark and represents one of the most studied research topics in neuroscience
since its discovery in 1973. These 2 scientists demonstrated that a brief high-
frequency train of stimuli to the perforant path produced an increase in the excitatory
synaptic potentials in the granule cells of the dentate gyrus, which, in the intact
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animal, lasted for days and even weeks (Bliss and Collingridge, 1993; Bliss and
Lomo, 1973). This phenomenon, characterized by long-lasting, activity-dependent
increase in synaptic efficacy, was named LTP (long-term potentiation). Because LTP
shares certain properties with associative learning and because the hippocampus has
been implicated in some forms of memory, LTP is considered a cellular mechanism
underlying certain forms o f learning and memory. In hippocampus, in addition to its
existence in the originally reported synapses between the perforant path fibers and
granule cells in the dentate gyrus, two other synapses, the Schaffer
collateral/commissural synapses in CA1 (Schwartzkroin and Wester, 1975) and the
mossy fibers synapses in CA3 (Alger and Teyler, 1976), also exhibit robust LTP,
although differing in properties (Bliss and Collingridge, 1993).
After two decades of intensive research, a great deal of progress has been
made regarding the mechanisms underlying the induction, expression and
consolidation of LTP. Though recent experiments suggested the involvement of
metabotropic glutamate receptors (mGluRs) in the induction of LTP, the results were
sometimes contradictory, probably due to the lack of specific mGluR antagonists
(Bear and Malenka, 1994; Bliss and Collingridge, 1993). Thus, mGluRs might be
involved in LTP induction, although their precise roles remain far from conclusive.
In contrast, the important role of the NMDA receptors in LTP induction, by virtue of
their Ca++ permeability and voltage-dependent blockade by Mg++, has been well
established. At Schaffer collateral synapses on CA1 pyramidal cells, Ca++
permeating through NMDA receptor channels provides a transient signal, which is
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necessary and in most cases sufficient for LTP induction. This hypothesis was
mainly based on several important experimental findings in the late eighties
(Malenka and Nicoll, 1999). First, AP-5 (or APV), a NMDA receptor antagonist,
reversibly prevents LTP induction. Second, LTP induction is blocked by intracellular
injection of the Car*-+ chelator EGTA (Lynch et al., 1983). Finally, NMDA receptors
are concentrated in dendritic spines where excitatory synapses are formed (Bekkers
and Stevens, 1989), and more recently, using Ca++ imaging, it was found that tetanic
stimulation, which elicits LTP, also elevates intracellular Ca++ within dendrites and
spines (Regehr and Tank, 1990). Even though the question regarding the exact locus
of LTP expression has been heavily debated over the past decade, the prevailing
answer is postsynaptic or a combination of postsynaptic and presynaptic
modifications. The evidence supporting the presynaptic hypothesis first came from
early microdialysis data suggesting that release of neurotransmitter was increased
after LTP (Dolphin et al., 1982). Four years later the same group also found that the
ability of a depolarizing stimulus to release preloaded radiolabelled glutamate was
elevated in potentiated hippocampal tissues (Feasey et al., 1986). Another major
body of evidence in support of a presynaptic locus came from quantal analysis of
synaptic transmission (Bekkers and Stevens, 1990; Bliss and Collingridge, 1993;
Malinow and Tsien, 1990): LTP was found to be associated with a decrease in the
coefficient of variation (CV=standard deviation/mean) of EPSPs recorded from
postsynaptic cells. This is compatible with an increase in transmitter quanta released
from presynaptic terminals. Moreover, by using a minimal stimulation paradigm to
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evaluate the failure rate before and after LTP, a decrease in incidence of failures was
observed shortly after LTP induction. The decrease was then attributed to an
increased probability of neurotransmitter release (Pr), by following the well-studied
synaptic transmission model of the neuromuscular junction. More recently, Stevens
and Wang (Stevens and Wang, 1994) also used a weak stimulation to determine
response failures in CA1 pyramidal cells. They found that the proportion of
transmission failures decreased, and that the amplitude of the non-failure responses
stayed the same after LTP induction. Thus, they concluded that there is an increase
in Pr, while postsynaptic responsiveness remains unchanged, in the expression phase
ofLTP.
A strong argument in favor of a postsynaptic expression of LTP is that LTP is
expressed as an increase in the component of synaptic responses mediated by AMPA
receptors, with little or no change in that mediated by NMDA receptors (Kauer et al.,
1988; Muller and Lynch, 1988), on the assumption that a presynaptic change would
result in a similar increase in both AMPA and NMDA receptor-mediated
components. Other groups have directly demonstrated an increase in sensitivity to
exogenous applied AMPA or quisqualate after LTP induction (Davies et al., 1989),
as well as an increase in the total number of agonist binding sites for AMPA
receptors (Maren et al., 1993). In addition, many experimental observations made
the presynaptic hypothesis unlikely, in particular, the fact that LTP induction does
not affect paired-pulse facilitation (PPF) (McNaughton, 1982), a typical indicator of
presynaptic modifications. More recently, Manabe and Nicoll (Manabe and Nicoll,
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1994) used the NMDA receptor channel antagonist MK801, which only blocks open
channels, to address this issue. The key assumption was that if LTP were due to
increased transmitter release, one would expect to see a greater inhibitory effect of
MK801 on NMDA receptor-mediated currents after LTP. However, the effects of
MK801 remained unchanged before and after LTP, supporting a postsynaptic
expression. Perhaps the landmark in the field was the introduction of the “silent
synapse” hypothesis. In 1989, Bekkers and Stevens (Bekkers and Stevens, 1989), by
studying the synaptic currents in cultured hippocampal neurons, found that some of
the synapses only contain NMDA receptors with no functional AMPA receptors.
This idea was further elaborated by Lynch and Baudry (Lynch and Baudry, 1991)
who proposed the transformation of silent synapses into active synapses as a possible
LTP mechanism. These synapses were later identified in hippocampal slices as a
significant fraction of synapses were silent during basal synaptic transmission at
resting membrane potential, due to the absence of AMPA receptors. “Silent synapse
hypothesis” states that LTP is expressed by the switch of silent synapses to
functional synapses, which can be obtained either by insertion of AMPA receptors or
activation of preexisting non-functional AMPA receptors at the synapse (Lynch and
Baudry, 1991). The hypothesis was put on a firmer basis when in 1995 two groups of
electrophysiologists (Isaac et al., 1995; Liao et al., 1995) independently
demonstrated that silent synapses with no AMPA receptor-mediated currents could
be transformed into AMPA receptor-containing synapses by LTP induction
protocols. These 2 groups showed that a fraction o f hippocampal synapses
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demonstrate no response to minimal presynaptic stimulation at negative holding
potentials but are, however, responsive at positive holding potentials, with the
response being blocked by NMDA receptor antagonists. Given that NMDA receptors
are blocked by Mg++ at negative potentials, these synapses are in fact silent
synapses, presumably containing only NMDA but not AMPA receptors. Following
pairing protocols that induce LTP, they acquired functional AMPA receptors as
evidenced by their responsiveness to minimal test stimulus at negative holding
potentials. In addition, the silent synapse model could reconcile some observations
that were previously interpreted as supporting presynaptic modifications, such as
changes in failures, quantal content or coefficient of variance after LTP induction.
II) Role of protein phosphorylation in synaptic plasticity and learning
Protein phosphorylation and dephosphorylation can produce rapid and robust
modification of AMPA receptor function and several lines of evidence indicated that
protein kinases, in particular calcium/calmodulin dependent protein kinase II
(CaMKII), protein kinase C (PKC) and protein kinase A (Soderling and Derkach,
2000), and various protein phosphatases, such as PP-1, PP2A, and PP2B (Lieberman
and Mody, 1994; Mulkey et al., 1994; Zhuo et al., 1999), play important roles in LTP
and LTD, respectively. Postsynaptic injection of inhibitors of these kinases blocks
LTP induction (Malenka et al., 1989; Malinow et al., 1989; O'Dell et al., 1991; Wang
and Feng, 1992), whereas inhibitors of phosphatases have been demonstrated to
block LTD (Lieberman and Mody, 1994; Mulkey et al., 1994; Zhuo et al., 1999). In
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agreement, several serine/threonine phosphorylation sites are predicted on GluRl, of
which Ser-831 and Ser-845 have been confirmed by mutagenesis studies (Mammen
et al., 1997). Phosphorylation at Ser-831 was shown to be responsible for increased
conductance of the AMPA receptor channel. Moreover, both CaMKII and PKC are
activated in an NMDA receptor-dependent manner following LTP induction (Barria
et al„ 1997; Klann et al., 1998; Sacktor et al., 1993). That CaMKII plays an
important role in LTP is strongly supported by the finding that synaptic transmission
is enhanced and LTP is occluded by postsynaptic application of constitutively active
CaMKII in CAl cells (Lledo et al., 1995). On the other hand, bath application of
NMDA induces LTD and concurrent dephosphorylation of Ser-845 of GluRl
subunits of AMPA receptors in hippocampal slices (Lee et al., 1998). Although there
is still some inconsistency regarding which serine residues are involved in LTP and
LTD induction, the current experimental data demonstrated that
phosphorylation/dephosphorylation is the common signal transduction mechanism
that is shared by these two forms of synaptic plasticity. It is generally assumed that
small amount of calcium influx resulting from weak NMDA receptor stimulation
activates phosphatases, leading to the depression of synaptic responses, whereas
large increase in intracellular calcium concentration after LTP induction activates
protein kinases, leading to synaptic potentiation (Bear and Malenka, 1994; Lisman,
1989). More recently, Cormier et al. (Cormier et al., 2001) used glutamate
iontophoresis method to induce plastic changes in rat CAl pyramidal synapses and
simultaneously measured intracellular calcium. They found that relatively low
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calcium levels (180-500 nM) typically led to LTD of synaptic transmission and
higher levels (>600 nM) often led to LTP. However, phosphorylation is a reversible
process and can only produce transient modifications of receptor properties that are
not compatible with the long lasting duration of the changes in synaptic efficacy.
Furthermore, phosphorylation and dephosphorylation cannot easily account for the
morphological changes occurring in LTP.
In parallel to the above-described electrophysiological data, agents that
inhibit CaMKII or PKC were found to prevent memory formation in animals, either
short-term or long-term (Rosenzweig, 1992; Serrano et al., 1994).
Ill) Regulation of receptor trafficking modifies synaptic efficacy
Changes in the number of postsynaptic AMPA receptors clustered at synaptic
sites intuitively constitute an important mechanism that could mediate long-term
plasticity. The number of receptors at a given synapse is the result of a dynamic
equilibrium between the insertion and removal rates of receptors (Turrigiano et al..
1998; Turrigiano and Nelson, 1998). Ligand binding study showed that LTP
induction produced an increase in AMPA binding to synaptic membranes, due to an
increase in the maximal number of binding sites but not to changes in affinity
(Maren et al., 1993). More recently, it was demonstrated that, in hippocampal
neurons, AMPA receptors are rapidly delivered to dendritic spines following NMDA
receptor stimulation (Shi et al., 1999). In hippocampus, manipulations that block
endocytosis were shown to occlude LTD (Carroll et al., 1999; Carroll et al.. 1999;
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Luscher et al., 1999). Several studies using pulse chase and biotinylation methods
demonstrated that the turnover rates of AMPA receptors are activity-dependent
(O'Brien etal., 1998).
It is now clear that mature AMPA and NMDA receptors reside in PSDs
(postsynaptic densities), where they are suitably positioned to bind to the
neurotransmitter glutamate released from presynaptic terminals. A significant
fraction of receptors can be retrieved in PSD (postsynaptic density) preparations,
which correspond to the detergent insoluble component of synaptic membranes
(Cotman et al., 1974). PSDs were first characterized with electron microscopy as a
dense, detergent insoluble fibrous structure that adheres to the postsynaptic
membrane. A prominent feature of PSDs is the presence of an extensive meshwork
of spectrin and actin filaments (Matus et al., 1982). In addition, various linker
proteins, scaffolding proteins, and signaling molecules were also found in PSDs.
Therefore, the architecture of PSDs is important for maintaining synaptic
morphology, mediating postsynaptic responses and regulating the efficacy of
synaptic transmission. Recently, much progress has been made in identifying the
proteins that anchor the AMPA and NMDA receptors in the PSD. In particular, the
C-terminal tails of GluR2 subunits of AMPA receptors were found to bind GRIP,
PICK1, and NSF (Dong et al., 1997; Nishimune et al., 1998; Osten et al., 1998; Song
et al., 1998; Xia et al.. 1999), whereas NR2 subunits of NMDA receptors bind to
MAGUKs (membrane associated guanylate kinase family) proteins, such as PSD-95,
PSD-93, SAP-102 and probably spectrin (Kimetal., 1996; Komauetal.. 1995;
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Muller et al., 1996; Wechsler and Teichberg, 1998). To remove the receptors from
PSDs, i.e., to initiate the internalization process, thus requires the disruption of these
anchoring mechanisms. The glutamate receptors, at least AMPA receptors, are
probably internalized via the common and well-studied endocytotic pathway
involving clathrin-coated vesicles (Carroll et al., 1999).
The mechanisms mediating the insertion of receptors are not yet well studied.
Several years ago, Lynch and Baudry (Lynch and Baudry, 1984) proposed that
degradation of a cytoskeleton protein, spectrin, by the calcium-dependent protease,
calpain, triggered by calcium influx through NMDA receptors during LTP induction,
could promote the insertion of functional AMPA receptors into PSDs (Lynch and
Baudry, 1984). Recent evidence suggests that aCaMKII and a recently identified
AMPA receptor interacting protein, NSF (N-ethylmaleimide-sensitive fusion
protein), could be involved in the AMPA receptor insertion process (Hayashi et al.,
2000; Nishimune et al., 1998; Passafaro et al., 2001; Song et al., 1998).
IV) Involvement of calpain in long-term synaptic plasticity
Calpains, a family of calcium dependent neutral proteases, represent a well-
characterized non-lysosomal proteolytic system of mammalian cells, of which 2
major isoenzymes, p-calpain and m-calpain, are ubiquitous and obtained their name
from their differential calcium requirements for activation (Carafoli and Molinari,
1998; Sorimachi et al., 1997). A number of studies have demonstrated that calpain is
widely distributed in neurons, bot h in the cytosol and in synapses. Autolysis and
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membrane association have been proposed to be the mechanisms for calpain
activation in cellular environment Both processes are also believed to dramatically
reduce the calcium requirement of calpain. The endogenous calpain inhibitor,
calpastatin, is a cytosolic protein; therefore, calpain activation by membrane
association will also sequester calpain away from inhibition mechanisms. In contrast
to most proteases, the proteolytic activity of calpain has been considered to play
regulatory rather than degradative roles. In the central nervous system, these
enzymes have often been implicated as participants in various pathologies associated
with altered protein mechanism and/or altered calcium homeostasis, including
ischemic injury to neuron, cell death, and degenerative disorders (Croall and
DeMartino, 1991).
The involvement of calpain in long-term synaptic plasticity was first
proposed by Lynch and Baudry (Lynch and Baudry, 1984) based on the observation
that calcium rapidly and irreversibly increases the number of receptors for glutamate
in forebrain synaptic membranes and such effect was likely mediated by calpain.
since the increase in glutamate binding was blocked by calpain inhibitors (Baudry et
al., 1981). Evidence in support for such a claim has been accumulated over the years.
Calcium influx through NMDA receptors resulting from high frequency stimulation
could activate calpain, as evidenced by the increase in spectrin breakdown products
in stimulated postsynaptic dendritic spines (Vanderklish et al., 1995). Calpain
inhibitors block LTP in hippocampal slices (Denny et al., 1990; Staubli et al., 1988).
Rats with genetic deficiency in calpastatin, the endogenous calpain inhibitor, showed
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enhanced hippocampal long-term potentiation (Muller et al., 1995). Meanwhile,
despite the demonstrated involvement of calpain in synaptic modifications, the
detailed biochemical pathways that participate in this process have not been fully
worked out. Electron microscopic study has shown that the action of calpain caused
a partial unraveling of the PSD (Dosemeci and Reese, 1995). Opening up of the PSD
structure may facilitate receptor trafficking and expose previously occluded
functional sites. Proteolysis of critical signaling molecules could be another
important mechanism to bias the direction of synaptic modification, either
potentiation or depression. Indeed, calpain-mediated proteolytic modifications of
PKC greatly enhance the catalytic activity of the enzyme, and thus could contribute
to synaptic potentiation (Kishimoto et al., 1989; Sessoms et al., 1992; Tanaka et al..
1991). Recently, several novel substrates of calpain, including AMPA and NMDA
receptor subunits, have been identified in our laboratory (Bi et al., 1996; Bi et al.,
1997; Bi et al., 1998). Since regulation of receptor trafficking has been proven to be
a powerful mechanism to modify synaptic strength, it would be interesting to further
study the functional relevance of calpain-mediated degradation of glutamate
receptors in terms of receptor endocytosis and exocytosis and to test whether
glutamate receptor anchoring molecules could be targets of calpain.
V) Specifc Aims
Even though calpain has long been demonstrated to play important role in
long-term synaptic plasticity, the critical cellular targets that are responsible for such
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changes have not been identified. The goals of my research were: i) to characterize
calpain-mediated degradation of a-CaMKII, the critical signaling molecule in
synaptic plasticity and learning and memory; ii) to identify new calpain substrates
that are potentially important in synaptic transmission, in particular to test whether
the recently identified glutamate anchoring proteins are calpain targets, and iii) to
clarify the functional consequences of calpain-mediated degradation of the above-
mentioned substrates with regard to synaptic plasticity.
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CHAPTER 2
PREFERABLE DEGRADATION OF NON-PHOSPHORYLATED FORM OF
aCaMKH BY CALPAIN
ABSTRACT
Ca2 + /calmodulin-dependent protein kinase II (CaMKII) is a mediator for long-term
synaptic plasticity. CaMKII catalyzed GluRl phosphorylation and synaptic delivery
of AMPA receptors constitute important expression mechanisms of long-term
synaptic potentiation. CaMKII activates through autophosphorylation at Thr286 and
it has been demonstrated that proteolysis of non-phosphorylated CaMKII by calpain
decreases the enzyme activity. Using in vitro digestion of rat forebrain membranes
and NMDA stimulation of organotypic hippocampal cultures, we found that calpain
digestion of a-CaMKII produces a 33 kDa fragment. In addition,
immunoprecipitation data show that calpain preferably degraded non-phosphorylated
form while sparing the phosphorylated form of a-CaMKII, suggesting that effect of
calpain-mediated truncation of a-CaMKII would likely cause a reduction in the
catalytic activity of the enzyme. Our data indicate that one of the calpain's
contributions to synaptic modifications occurs through the regulation of CaMKII
activity.
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INTRODUCTION
Excitatory synaptic transmission in central nervous system is mainly
mediated by AMP A and NMDA subtype of ionotropic glutamate receptors and long
term changes in the efficacy o f transmission at excitatory synapses have been
proposed to represent cellular mechanisms of learning and memory (Malenka and
Nicoll, 1999). Despite some controversy, it is generally accepted that calcium
permeable NMDA receptors mediate the induction of certain forms of long-term
potentiation (LTP) and long-term depression (LTD) of synaptic transmission while
the expression of LTP/LTD is mainly mediated by changes in AMPA receptors
(Malenka and Nicoll, 1999). Two major systems activated by calcium permeating
through NMDA receptor channels have been proposed to play key roles in LTP and
LTD, i.e., CaMKII (Ca2 + /calmodulin-dependent protein kinase II) and calpain
(Barria et al., 1997; Carafoli and Molinari, 1998; Chan and Mattson, 1999; Denny et
al., 1990; Lynch and Baudry, 1984; Malenka et al., 1989).
CaMKII is highly expressed in neurons and enriched in the postsynaptic
densities, the postsynaptic machinery for signal transduction, synaptic plasticity and
maintenance of dendritic architecture (Goldenring et al., 1984; Kennedy, 1998;
Suzuki et al., 1994).Even though the initial activation of CaMKII requires a rise in
intracellular calcium, subsequent autophosphorylation at Thr286 renders CaMKII
constitutively active even at resting calcium concentration (Barria et al., 1997;
Fukunaga et al., 1995). The critical role of the a isoforms of CaMKII in
synaptic plasticity and learning and memory has been established by studies
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indicating that transgenic mice lacking functional a-CaMKII exhibit impaired LTP
and spatial learning deficiency (Bach et al., 1995; Silva et al., 1992; Silva et al.,
1992). The potentiation effect seems to be the result of kinase activity since CaMKII
phosphorylates GluRl subunit of AMPA receptors and such phosphorylation greatly
enhances AMPA receptor-mediated synaptic currents (Barria et al., 1997; Lledo et
al., 1995; Mammen et al., 1997; McGlade-McCulloh et al., 1993). In addition, recent
data have shown that CaMKII can mediate the insertion of GluRl subunits to the
synapse even when the CaMKII phosphorylation site on GluRl was eliminated,
suggesting that multiple potentiation mechanisms are probably involved (Hayashi et
al., 2000; Passafaro et al., 2001).
Calpains represent a family of calcium-activated neutral proteases and have
been shown to be necessary for LTP formation (del Cerro et al., 1994; Denny et al.,
1990; Lynch, 1998; Oliver et al., 1989; Vanderklish et al., 1996). Calpain has been
found in postsynaptic structures (Domanska-Janik et al., 1999; Perlmutter et al.,
1988; Siman et al., 1983) and NMDA receptor stimulation results in calpain
activation in dendritic spines (Vanderklish et al., 2000). In addition to its traditional
target of spectrin, several other important synaptic proteins, such as several subunits
of AMPA and NMDA ionotropic glutamate receptors and their anchoring molecules,
GRIP and PSD-95, have been added to the list (Bi et al., 1997; Bi et al., 1998; Lu et
al., 2000; Lu et al., 2001). However the interactions between these two calcium
sensitive systems, calpain and CaMKII, have not been carefully studied.
Nonetheless, calpain has been reported to regulate the catalytic activity of CaMKII
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and calpain-mediated proteolysis drastically decreases the activity of the non-
phosphorylated form of CaMKII (Kwiatkowski and King, 1989). Here we report that
a-CaMKII is proteolysed by calpain and brief NMDA receptor stimulation results in
calpain-mediated degradation of CaMKII in cultured hippocampal slices. In addition,
calpain preferably degrades the non-phosphorylated form of the kinase, which may
contribute to the down-regulation of the kinase activity.
MATERIALS AND METHODS
/. Calpain disestion o f synaptic membranes
Synaptic membranes were prepared from Sprague-Dawley rat forebrain
according to Massicotte et al. (Massicotte et al., 1990) as previously described (Bi et
al., 1996; Lu et al., 2000), and saved at -70 °C until the day of use. Synaptic
membranes were thawed and about 300 pg proteins were incubated in Tris-acetate
buffer with or without 2 mM calcium and calpain I (2.4 units/ml) at 37 °C for 30
min. The resulting aliquots were immediately processed for SDS-PAGE and Western
blot. For immunoprecipitation experiments, the digestion was performed using larger
amounts of synaptic membranes (equivalent to 0.6 mg proteins) and in the presence
o f400 nM okadaic acid.
2. Activation o f endogenous calpain in oreanotvpic hippocampal cultures
Organotypic cultures of hippocampal slices were prepared using the technique of
1 9
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Stoppini et al. (Stoppini et al., 1991) as described previously (Bruce et al., 1995;
Gellerman et al., 1997). In brief, hippocampi were harvested in sterile conditions
from 7 day old Sprague-Dawley rat brains in chilled minimum essential medium
(MEM) (GIBCO no. 61100061) containing 25 mM HEPES, 10 mM Tris-base, 10
mM D-glucose and 3 mM MgCb and placed on a Teflon stage of a Mcllwain tissue
chopper. Transverse slices (400 pm thick) were cut and incubated at 35 °C with a 5%
COr-enriched atmosphere. After 14 days in vitro, hippocampal slice cultures were
incubated with growth medium without serum for 24 hrs. The cultures were exposed
to high calcium medium (4 mM CaCl2 ) with or without 300 pM NMDA or 50 pM
calpain inhibitor III for 5 min and the slices were rapidly washed and maintained in
serum free medium. For cultures treated with calpain inhibitor III during NMDA
stimulation, 50 pM calpain inhibitor III was included in incubation medium
thereafter. The slices were then collected at indicated time points and processed for
Western blot.
S. Immmmoprecipitation
Proteins from calpain-treated synaptic membranes (100 pi, equivalent to 0.6 mg
proteins) were solubilized by adding 1% SDS (final concentration) and boiled for 5
min. Following addition of 400 pi of ice-cold lysis buffer [0.1 M sodium phosphate
buffered saline (PBS), pH = 7.2, 2mM EDTA, 2% Triton], extracts were centrifuged
at 24,000 g for 30 min and the supernatant was used for immunoprecipitation. One
hundred pi of a 1:1 protein A slurry was added to 450 pi solubilized supernatant
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along with 10 tig anti-aCaMKU antibodies (Chemicon International Inc., Temecula,
CA). The mixture was gently rotated at 4 °C overnight After washing the protein A
beads with lysis buffer twice and one final wash with PBS, 80 til sample buffer (see
below) were added to the beads and the proteins were eluted by boiling for 5 min
4. SDS-PAGE and Western blots
Aliquots from different fractions were boiled at 100 °C for 5 min in sample
buffer [2% SDS, 50 mM Tris-HCl (pH 6.8), 10% 2-mercaptoethanol, 10% glycerol,
and 0.1% bromophenol blue]. SDS-PAGE was performed according to the method of
Laemmli(Laemmli, 1970). Proteins were then transferred onto nitrocellulose
membranes as described by Towbin et et al. (Towbin et al., 1979). Nitrocellulose
membranes were first blocked with 3% gelatin in Tris-buffered saline (TBS) at room
temperature for 1 h and incubated overnight at 4 °C with primary antibodies against
a-CaMKII (1:5000 dilution) or phospho-a-CaMKII (BIOMOL, Plymouth Meeting,
PA, US; 1:2000 dilution) in 1% gelatin prepared in TBS with 0.5% Tween-20
(TI BS). After 2 washes with TTBS for 5 min, membranes were incubated with
alkaline phosphatase-conjugated anti-rabbit (1:2000) or anti-mouse (1:2000) IgG for
2 h at room temperature; antigens were then visualized with nitroblue tetrazolium
and 5-bromo-4-chlor-3-indolyphosphate toluidine salt. Blots were scanned and
quantitatively analyzed by densitometry with ImageQuant™ software.
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RESULTS
1. Exogenous calpain cleaves a-CaMKII in vitro.
The in vitro digestion experiment was carried out by incubating synaptic
membranes with purified calpain I (2.4 U/ml) in the presence of 2 mM C a^at 37 °C
for 30 min (Fig. 1). In control condition, Western blots stained with anti-a-CaMKII
antibodies showed a major band migrating with an apparent molecular weight of 50
kDa, consistent with the predicted molecular weight of native a-CaMKII and in
agreement with previous reports (Suzuki et al., 1994; Yamagata and Obata, 1998).
Following incubation with calpain, the immunoreactivity of the 50 kDa band
decreased and three smaller species appeared with calculated molecular weights of
36,33, 31 kDa respectively.
Ctrl Calp
S O kDa
36 kDa
£ 33 kDa
^ 3 1 kDa
Fig* 1 In vitro digestion of aCaMKlI.
Rat forebrain synaptic membranes were treated with purified calpain I (2.4
U/ml) plus calcium (2mM) at 37 °C for 30 min. The resulting aliquots were
immediately processed for SDS-PAGE and Western blot using antibodies against
aCaMKII (1:5000 dilution). Molecular weights of the native aCaMKII and
breakdown products are labeled on the right.
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2. Calpain activation after brief exposure to NMDA In hippocampal slice
culture
NMDA treatment of organotypic cultures of hippocampal slices has been
shown to activate calpain, and has often been used to investigate mechanism* of
synaptic plasticity (Musleh et al., 1997; Vanderklish et al., 1995). Many features of
the organization and function of the hippocampus are well preserved in such
preparation, thus allowing for the effects of calpain activation to be studied under
conditions closely matching the in vivo environment. Since the induction of LTP and
LTD happens in minutes, we decided to test the effects of brief NMDA receptor
stimulation. Organotypic hippocampal slice cultures were prepared from postnatal
day 7 rats; after 14 days in vitro, the culture was briefly exposed to 300 pM NMDA
for 5 min. The slices were collected at various time points after NMDA treatment
and processed for Western blots with anti-spectrin or anti-a-CaMKII antibodies (Fig.
2). Immunoblots revealed that calpain was indeed activated by short term NMDA
receptor stimulation as evidenced by the degradation of both spectrin and a-CaMKII
and the blockade of such proteolysis by the cell permeable calpain inhibitor, calpain
inhibitor III (Fig. 2, 3). NMDA stimulated hydrolysis of spectrin was significant as
early as 1 hr after NMDA treatment and continually proceeded until 24 hrs after
NMDA stimulation, the earliest and latest time points tested. Specific degradation
produced two fragments of 155 and 145 kDa, respectively, and was mediated by
calpain since it was blocked by the cell permeable calpain inhibitor, calpain inhibitor
III (Fig. 3). The degradation of a-CaMKJI was also detected after 1 hr and similarly
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B
• lh 3h lOh 24 h
♦ 2 4 0 kDa
0 lh Jh lOh 24 h
« ISO kDa ’ *** * 0 W 0 **-.i
‘ •'145 kDa
50 kDa
* 35 kDa
♦ 3 3 kDa
Fig. 2 Activation of calpain following brief NMDA stimulation of cultured
hippocampal slices.
Organotypic hippocampal cultures were prepared from 7 day old Sprague-
Dawley rat pups and maintained in vitro for 14 days. After incubation in serum free
medium for 24 h, the cultures were exposed to 300 jiM NMDA in high calcium
medium (4mM) for 5 min and then maintained in serum free medium. Cultured
slices were collected at different time points and processed for SDS-PAGE and
Western blots using antibodies against spectrin (A) or a-CaMKII (B). The molecular
weights of the native species and breakdown products are indicated on the right, h,
hour(s).
continued up to 24 hrs; interestingly, the formation of the 33 kDa breakdown
product that appeared at a later time point than the 35 kDa is more effectively
blocked by calpain inhibitor III (Fig.3). Thus it seems that even though purified
calpain I digestion of synaptic membranes revealed three distinct cutting sites, it is
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likely that calpain in fact preferably degrades at one cutting site that produces 33
kDa fragment in the in vivo condition.
1 hr 3 hr
Anti-
Spectrin
■' r 3
— *ryt * *
I®1 ' jAi W* "
240 kDa
150 kDa
145 kDa
Anti- ----- 50kDa
a -C a M K II ___ ___ 35 kDa
' 33 kDa
1 2 3 1
Fig. 3 Calpain inhibitor blocks the degradation of spectrin and a-CaMKII following
brief NMDA stimulation of cultured hippocampal slices.
Cultured hippocampal slices were exposed to 300 pM NMDA for 5 min with
or without calpain inhibitor III (50 pM). Cultures were collected 1 or 3 hr after
NMDA stimulation and processed for Western blots using antibodies against spectrin
or a-CaMKII. Molecular weight of native species and breakdown products were
indicated on the right side. 1 : control; 2: NMDA treated; 3: NMDA plus calpain
inhibitor III.
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A WBw/ B
aCaMKII P- aCaMKII
IPw /aC aM K II aCaM KII P-aCaMKII
Fig. 4 Calpain preferably degrades non-phosphorvlated form of a-CaMKII.
Rat forebrain synaptic membranes were treated with purified calpain I (2.4
U/ml) and calcium (2mM) at 37 °C for 30 min and were then solubilized with 1%
SDS. Proteins precipited with a-CaMKII antibodies were resolved with SDS-PAGE
and stained with anti-a-CaMKII (aCaMKII) or anti-phospho-a-CaMKII (P-a-
CaMKII) antibodies. A: representative Western blots. B: quantification of Western
blots showing percent of undegraded total a-CaMKII and phospho-a-CaMKII.
Results are means±S.D. of 2 experiments.
3. Effect of phosphorylation on calpain-mediated degradation of a-
CaMKII
It has previously been shown that, in response to synaptic activity, a-CaMKII
is activated via auto-phosphorylation at Thr286; in many cases protein
phosphorylation regulates the extent of calpain-mediated proteolysis of target
proteins, such as in case of ionotropic glutamate receptors (Bi et al., 1998; Bi et al.,
2000). Therefore we tested whether auto-phosphorylation protects a-CaMKII from
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calpain-mediated degradation. Synaptic membranes were first digested by purified
calpain I in the present of okadaic acid to prevent dephosphorylation during the
experimental procedure. a-CaMKII proteins were then precipitated with anti-a-
CaMKII antibodies and the immunocomplex was resolved by SDS gel
electrophoresis. Western blots stained with anti-a-CaMKII showed a 28% digestion
of total a-CaMKII protein as evidenced by the decrease in the 50 kDa native species
and the appearance of the truncated smaller species in calpain-digested versus
control samples. In contrast, under the same conditions, there was no significant
degradation of the phosphorylated form of a-CaMKII (Fig 4), which demonstrates
that auto-phosphorylation protects the enzyme from calpain-mediated proteolysis.
DISCUSSION
The major neuronal isoform of CaMKII, a-CaMKII, is very rapidly activated
by autophosphorylation at Thr286 following an intracellular calcium surge. The
activated form becomes relatively independent of calcium and may prolong
physiological responses to a transient increase in calcium, and thus could serve as a
molecular switch to “memorize” past experiences (Lisman and Zhabotinsky, 2001;
Stevens et al., 1994; Yamagata and Obata, 1998). Several other mechanisms have
also been reported to regulate CaMKII activity, including PP1, PP2A and mitogen-
activated protein kinase (MAPK) (Bennecib et al., 2001; Colbran, 1992;
Giovanniniet al., 2001; Lisman and Zhabotinsky, 2001). In this study we show that
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a-CaMKII can be cleaved by calpain both in vitro and in situ, and this partial
proteolysis may represent a novel regulatory pathway for the enzyme.
In hippocampal slice cultures, a brief NMDA treatment activates calpain, as
evidenced by both spectrin and a-CaMKII degradation and the proteolytic process
continued for 24 hours after the end of NMDA treatment. Though in the initial in
vitro experiments, calpain activation by autolysis seemed to require a calcium
concentration that is far beyond the physiological range, more recent experiments
have shown that lipid component or substrate binding can dramatically lower the
calcium requirement for calpain activation (Inomata et al., 1989). The prolonged
calpain activity observed in our experiment could be the combined results of
sustained effects of active calpain and disturbed calcium homeostasis following
NMDA receptor stimulation. The brief NMDA incubation resulted in the production
of two smaller species of 35 and 33 kDa in hippocampal cultures and the 35 kDa
product seems to be less sensitive to calpain inhibition. In addition, the 35 kDa
product was absent from purified calpain digestion of synaptic membranes and a 35
kDa breakdown product of a-CaMKII has been previously observed in cultures
treated with staurosporine and can be completely blocked by a caspase inhibitor but
only partially blocked by a calpain inhibitor (McGinnis et al., 1998). It is possible
that the same molecule weight species observed in our experiment is actually a
product of caspase-mediated degradation. Given that calpain inhibitor has significant
inhibitory effect on 35 kDa species, it is possible that one function of calpain is to
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regulate caspase activity, a scenario that has been previously suggested (Blomgren et
al., 2001).
Our results also demonstrated that calpain preferably degrades the non-
phosphorylated form of a-CaMKII and spares its active phosphorylated form. It has
been shown that calpain degradation decrease the activity of non-phosphorylated
form of a-CaMKII (Kwiatkowski and King, 1989). Overall, calpain-mediated
proteolysis, thus, would serve to decrease catalytic activity of a-CaMKII, an effect
that seems to be more regulatory and long lasting rather than robust and dominant,
especially when a huge portion o f the kinase has been phosphorylated following
drastic calcium elevation. Indeed, NMDA exposure of hippocampal neurons has
previously been shown to cause suppression of a-CaMKII activity (Chum et al.,
1995).
In addition to being a key mediator of long term synaptic plasticity and
participating in certain forms of learning and memory, a-CaMKII is also involved in
regulation of cell death (Blomgren et al., 2001). Thus, any event that regulates a-
CaMKII would significantly contribute to synaptic modification and cell death. Our
results demonstrate a novel regulatory pathway for a-CaMKII via calpain-mediated
partial proteolysis and indicate that such regulation might favor a synaptic depression
in the in vivo condition.
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CHAPTER 3
DEGRADATION OF A MAJOR POSTSYNAPTIC DENSITY PROTEIN PSD-
95 BY CALPAIN
ABSTRACT
PSD-95 is a major postsynaptic density protein that is degraded as a result of
synaptic activity. We used 4 different methods to test the hypothesis that calpain is
involved in PSD-95 turnover. Treatment of synaptic membranes with purified
calpain resulted in a decrease in immunoreactivity of the native 95 kDa protein and
the appearance of two smaller molecular weight species, migrating at 50 kDa and 36
kDa, respectively. Calcium treatment of ffozen-thawed brain sections produced an
identical digestion pattern, an effect blocked by calpain inhibitors. NMDA treatment
of organotypic hippocampal cultures produced truncation of PSD-95 and
accumulation of the 36 kDa species. Finally, calpain-generated degradation products
of PSD-95 were prominent in neonatal hippocampus, and disappeared with postnatal
development. Our data suggest that PSD-95 is a substrate for calpain, and that
calpain-mediated truncation contributes to PSD-95 turnover.
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INTRODUCTION
PSD-95 is a major postsynaptic protein and belongs to the MAGUK
(membrane associated guanylate kinase) family. Recent studies have suggested that
it plays an important role in the assembly and organization of excitatory postsynaptic
architecture (Craven et al., 1999; Kornau et al., 1997; Sheng, 1996). PSD-95
contains three PDZ motifs, a Src homology domain (SH3) and a C-terminal
guanylate kinase-like (GK) domain which binds to ion channels, specific proteins of
the neuronal cytoskeleton and intracellular signaling enzymes (Brenman et al., 1996;
Kim et al., 1995; Tezuka et al., 1999). In particular, the second PDZ domain interacts
with the C-terminal domain of NR2 subunits of the N-methyl-D-aspartate (NMDA)
receptors as well as with neuronal nitric oxide synthase, thereby contributing to both
synaptic targeting of the receptors and their signaling functions (Brenman et al.,
1996; Sattler et al., 1999). Disruption of PSD-95/NMDA receptor interaction
attenuates NMDA receptor mediated neurotoxicity (Sattler et al., 1999). However,
despite significant advances in the identification of binding partners of PSD-95, little
is known about the turnover of this important postsynaptic organizing molecule.
Calpains are a family of calcium dependent neutral proteases, and 2 major
isoenzymes, ^-calpain and m-calpain, exhibit different calcium requirements for
activation and are ubiquitous in the nervous system (Carafoli and Molinari, 1998;
Sorimachi et al., 1997). Calpains are widely distributed in neurons, both in the
cytosol and in synaptic terminals. Their activities have been implicated in many
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cellular events such as regulation of enzymatic activity, degradation of important
cellular proteins as well as in neuropathological conditions, such as ischemic
neuronal damage (Carafoli and Molinari, 1998; Sorimachi et al., 1997). Furthermore,
two types of ionotropic glutamate receptors, the AMPA and NMDA receptors, are
truncated by calpain (Bi et al., 1997; Bi et al., 1998), and this mechanism could be
involved in modification of synaptic efficacy by altering the number and properties
of glutamate receptors (Lu et al., 2000). Calpain is activated by calcium permeating
through NMDA receptors during intense synaptic activity, and has been implicated
in morphological modifications of synapses taking place during neuronal
development and in adult synaptic plasticity (Vanderklish et al., 1995). It was
therefore of interest to determine whether PSD-95 was a substrate for calpain, and
whether calpain activation was accompanied by calpain-mediated PSD-95
truncation. Moreover, as we previously showed that calpain was constitutively
activated during the postnatal period (Vanderklish et al., 1995), we also determined
changes in PSD-95 during the postnatal period.
RESULTS
The initial characterization of calpain-mediated degradation of PSD-95 was
carried out by incubating rat forebrain synaptic membranes with purified calpain I.
Briefly, synaptic membranes were prepared from adult Sprague-Dawley rats as
previously described (Bi et al., 1997), and kept at - 80 'C. Usually 300 pg of
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synaptic membrane proteins were incubated with calpain I (2.4 units/ml) and calcium
(2 mM) for 30 min at 37 *C. Incubation was terminated by adding equal volume of
SDS sample buffer, and the samples were boiled for S min. The resulting proteins
were resolved by SDS-PAGE, transferred onto nitrocellulose membranes and probed
with anti-PSD-95 antibodies (Upstate, 1:250 dilution).
A ^ ^ B Calpain
C S * V V V ^
♦ s o
♦ 3 6
Fig. 5 PSD-95 degradation bv calpain in rat forebrain svnaptic membranes.
Rat synaptic membranes were incubated with purified calpain I at 37 °C for
30 min. Aliquots were subjected to Western blots and stained with anti-PSD-95
antibody. Equal amounts of proteins were loaded on each lane. (A) Degradation of
PSD-95 into 2 smaller molecular weight species, in the absence or presence of 100
pM calpain inhibitor III (CalpI III) and 100 pM leupeptin (Leu). The sizes of native
and truncated forms of PSD-95 are indicated on the right in kilodaltons. (B)
Degradation of PSD-95 with increasing calpain concentrations. Results are
representative of 5 experiments.
33
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Western blots indicated that the antibodies recognized a doublet band, at a
molecular weight of about 95 kDa, under control conditions (Fig. 5). Calpain
treatment resulted in a decrease in native PSD-95 protein, and in the formation of
two prominent truncation products, two doublet bands migrating at 50 kDa and 36
kDa, respectively. This effect was completely blocked by calpain inhibitors, either
calpain inhibitor III (100 pM) or leupeptin (100 pM) (Fig. 5). Quantification of the
blots revealed a 50 % decrease in the amount of native PSD-95 under these
conditions, and this decrease matched the total increase in the amount of the two
degradation products. As total immunoreactivity was not modified, it is unlikely that
the 2 degradation products represent the formation of proteins unrelated to PSD-95.
Increasing calpain concentration did not increase the maximal degradation of PSD-
95, which remained at 50 % (Fig. 5).
Protein phosphorylation has been reported to protect ionotropic glutamate
receptors from calpain-mediated proteolysis (Bi et al., 1998). As PSD-95 is
physically linked to the tyrosine kinase, Fyn, in postsynaptic densities (Tezuka et al.,
1999), we examined whether native PSD-95 was tyrosine phosphorylated and the
effect of tyrosine phosphorylation on calpain-mediated truncation of PSD-95. PSD-
95 proteins were immunoprecipitated from rat synaptic membranes using anti-PSD-
95 antibodies (5pl/300pg protein), and the precipitated proteins processed for
Western blots and stained with either PSD-95 or anti-phosphotyrosine antibodies
(Upstate; 1:1000). A fraction of PSD-95 proteins appears to be tyrosine
34
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j
!
phosphorylated under basal conditions (Fig. 6). When calpain digestion of synaptic
membranes was performed in the presence of orthovanadate (2 mM), a tyrosine
phosphatase inhibitor, PSD-95 degradation pattern was unchanged (Fig. 6), nor was
it affected by preincubating synaptic membranes with Fyn and ATP (data not
shown).
We then tested whether PSD-95 was degraded as a result of calpain
activation in frozen-thawed rat forebrain sections. Adult Sprague-Dawley rats were
killed by decapitation, their brains rapidly removed and frozen in isopentane at - 20
*C, then kept at - 80 *C . Coronal, 20-pm-thick sections were cut on a cryostat and
thawed-mounted onto chrome/alum gelatin-coated slides. Sections were incubated
with 2 mM calcium in Tris buffer (100 mM Tris base, pH 7.4, 100 pM EGTA) at
room temperature for 30 minutes. Brain sections as well as the incubation buffer
were then collected and processed for Western blots (Fig. 7). The proteolytic pattern
was identical to that obtained with calpain I digestion of synaptic membranes, with
clear identification of the 50 kDa and 36 kDa bands. To further verify that calpains,
but not other proteases, were responsible for the calcium-induced degradation of
PSD-95, a series of protease inhibitors were added to the incubation buffer. Calcium-
dependent degradation of PSD-95 was not affected by the serine protease inhibitor,
aprotinin (50 pM), but was totally blocked by EGTA (2 mM) and two calpain
inhibitors, calpain inhibitor III (100 pM) and leupeptin (100 pM). Thus, calcium-
mediated PSD-95 degradation in frozen-thawed rat brain sections is due to calpain
35
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A
I P : a n ti-P S D -9 5
P ro b e d : a n ti-P S D -9 5 a n ti-P Y
95
100
60
20
Fig. 6 Effect of tyrosine phosphorylation on calpain-mediated truncation of PSD-
91
(A) Rat forebrain synaptic membranes (300 pg) were solubilized by boiling in 1
% SDS for 5 min, followed by centrifugation at 40,000 g for 20 min. PSD-95
proteins were then immunoprecipitated using PSD-95 antibodies. The
immunoprecipitated proteins were subjected to SDS-PAGE and Western blots,
stained with either anti-phosphotyrosine (anti-PY) or anti-PSD-95 antibodies. (B)
The in vitro digestion experiment was performed at the indicated calpain
concentrations in the absence (empty bars) and presence (shaded bars) of 2 mM
Na3V04, a tyrosine phosphatase inhibitor. The amounts of native PSD-95 proteins
under each condition, were expressed as percent of control. Data are means ± S.E.M.
of 3 experiments.
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activation. Interestingly, the truncation products were not observed when only the
tissue sections were processed for immunoblotting, suggesting that, after their
formation, they were released in the incubation medium.
NMDA treatment of organotypic cultures of hippocampal slices has been
shown to activate calpain (Vanderklish et al., 1995). Crganotypic hippocampal
cultures were prepared according to Stoppini et al. (Stoppini et al., 1991). After 14
days in vitro, hippocampal slice cultures were incubated with exposure medium
(serum-free growth medium containing 4 mM CaCh) in the presence or absence of
50 pM NMDA for 3 hours. The cultures were then collected, homogenized and
processed for SDS-PAGE and Western blots. NMDA receptor simulation resulted in
the degradation of PSD-95 protein, with the clear identification of the 36 kDa band
(Fig. 7). The lack of significant accumulation of the 50 kDa band in NMDA-
stimulated slices could be due to a high basal level of calpain activity in young
animals (see below). The dose-response experiment (Fig. 5) also suggests that the 50
kDa truncation product could be further degraded into the 36 kDa species at higher
calpain concentration.
Finally, we determined changes in PSD-95 and calpain-mediated breakdown
products in hippocampus during the postnatal period. Hippocampi were dissected
from Sprague-Dawley rats of different ages, ranging from postnatal day one to 3
month. They were then homogenized in Tris buffer and processed for Western blots.
The expression of PSD-95 displayed a general increase with time after birth (Fig. 8).
37
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B
Ctrl NMDA
♦ 9 5
♦ 9 5
♦ 5 0
♦ 3 6
♦ 3 6
Fig.7 Effect of calcium treatment of frozen-thawed brain sections and NMDA
treatment of organotypic hippocampal cultures on PSD-95.
Frozen-thawed tissue sections (20 pm) were incubated with 2 mM calcium at room
temperature for 30 min, with or without the following protease inhibitors: 100 pM
calpain inhibitor III (CalpI III), 100 pM leupeptin (Leu), 2 mM EGTA, 50 pM
aprotinin (Aprot). Tissue was collected and processed for Western blots using
antibodies against PSD-95. (B) Cultured hippocampal slices were incubated with
NMDA (50 pM) for 3 h, collected immediately and processed for Western blots
using PSD-95 antibodies. Results are representative of 5 experiments.
Specifically, the protein was barely detectable upon birth and showed a marked
increase within the first month of development. The strongest expression was
obtained at 3 month, the last time point tested. In addition to the native PSD-95
38
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proteins, the two calpain-generated degradation products were clearly identified in
young animals (Fig. 8); their levels declined with postnatal development until they
almost completely disappeared in adult rat hippocampus. The ratio of the S O kDa and
36 kDa products over the intact PSD-95 proteins was 22 % and 28 % at postnatal day
7 respectively, compared to 1.3 % and 0.5 % at 1 month.
B
too
75
5s 25
2
a
3
$
*5
*
7 14 28
30
20
10
0
7 14 28
Age (Postnatal day)
Age (Postnatal day)
Fig. 8 Developmental pattern of PSD-95.
Hippocampus homogenates prepared from rat of different ages, ranging from
postnatal day 1 through 100, were analyzed by SDS-PAGE and immunoblotting with
antibodies against PSD-95. (A) Histogram showing the relative amount of PSD-95
expressed as percent of the 3-month value. Levels were measured by densitometric
scanning of Western blots and are means ± S.E.M. of 3 experiments. (B) Right:
representative blot indicating the presence of calpain-generated cleavage products in
young animals. Left: quantification of the blots. The levels of 50 kDa and 36 kDa
degradation products were expressed as percent of the 95 kDa full-length protein at
P7, P14 and P28. Results are means ± S.E.M. of 3 experiments.
39
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DISCUSSION
The results of this study clearly demonstrate that the PSD-9S protein is a
target of calpain in rat brain. Calpain treatment of synaptic membranes decreased the
amount of native PSD-95 and generated two truncation products migrating at 50 kDa
and 36 kDa, respectively. Considering that the antibodies recognize an epitope
located at residues 353-504, this would suggest that the truncation sites are between
PDZ2 and SH3. Furthermore, our data indicate a potential sequential truncation, with
the formation of the 50 kDa species occurring first followed by a second truncation
to generate the 36 kDa species. We have used calcium treatment of frozen-thawed
brain sections as a paradigm to investigate in situ calpain activation (Bi et al., 1998:
Lu et al., 2000). This treatment resulted in the same pattern of degradation of PDS-
95 as that observed with calpain treatment of synaptic membranes. Moreover, the
effects of calcium were completely blocked by calpain inhibitors and not by other
protease inhibitors. This indicates that calpain is located in close proximity to PSD-
95. This fits well with recent data showing calpain activation in dendritic spines
(Vanderklish et al., 2000). More importantly, the truncation is also very likely to
occur in vivo, since NMDA treatment of organotypic hippocampal cultures resulted
in a decrease in the amount of intact PSD-95 protein and a concurrent accumulation
of the 36 kDa species. We previously observed that tyrosine phosphorylation of NR2
subunits of NMDA receptors protects them from calpain-mediated degradation
(Rongetal., 1999). This does not seem to be the case for PSD-95, as tyrosine
40
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phosphorylation did not modify the extent of PSD-95 degradation by calpain.
Interestingly, the maximal truncation of PSD-95 observed in our experiments never
exceeded 50 %, indicating the existence of mechanisms protecting PSD-95 from
calpain-mediated degradation. In particular, we found that heat-denatured (65 'C for
5 min) PSD-95 was no longer degraded by calpain; on the other hand, heat-denatured
NR2A subunits of NMDA receptors retained their sensitivity to calpain (not shown).
This suggests that the conformation rather than the primary sequence of PSD-95
protein determines its calpain sensitivity. It is possible that some unique
conformations acquired or lost as a result of interactions with multiple binding
partners or during self-aggregation, make the protein resistant to calpain digestion.
Alternatively, this result could be due to the existence of different isoforms of PSD-
95, with some isoforms less sensitive to calpain-mediated truncation.
We previously found that another substrate of calpain, spectrin, was
constitutively degraded by calpain during the postnatal period in rat cortical
structures (Najm et al., 1992). We attributed this result to higher levels of calpain in
the early developmental period and to higher levels of spontaneous synaptic activity.
In the present study, PSD-95 degradation products were also identified in the
hippocampus of naive young animals, thus indicating that spectrin and PSD-95 are
degraded at a faster rate in the early stages of postnatal development. PSD-95
degradation drastically decreased during the first postnatal month, a time frame that
matches that of synapse formation in hippocampus. Thus, the period of intense
41
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synaptic formation is associated with a high turnover of PSD-95 and suggests that
calpain-mediated degradation of PSD-95 might be a critical element in synapse
formation. Our results are in good agreement with a recent report examining changes
in PSD-95 during the developmental period (Sans et al., 2000). Recently, Okabe et
al. (Okabe et al., 1999) observed a constant retraction of synapses and turnover of
PSD-95 associated with synaptic activity, suggesting that PSD-95 turnover is a
reliable marker of synaptic turnover. Our results suggest that such turnover of PSD-
95 is mediated by calpain activation. Since PSD-95 is linked to the C-terminal
domains of NMDA receptors, cytoskeleton proteins as well as signaling molecules,
calpain-mediated truncation of PSD-95 is likely to disrupt, if not all, at least some of
the interactions of PSD-95 with numerous binding partners.
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CHAPTER 4
THE ASSOCIATION BETWEEN GRIP AND AMPA RECEPTOR SUBUNIT
GLUR2 WAS DISRUPTED AS A CONSEQUENCE OF CALPAIN
ACTIVATION
A B S T R A C T
Activation o f the calcium-dependent protease calpain has been proposed to be a key
step in synaptic plasticity in hippocampus. However, the exact pathway through
which calpain mediates or modulates changes in synaptic function remains to be
clarified. We report here that glutamate receptor interacting protein (GRIP) is a
substrate of calpain, as calpain-mediated GRIP degradation was demonstrated with 3
different approaches: i) purified calpain I digestion of synaptic membranes, ii)
calcium treatment of frozen-thawed brain sections, and iii) NMDA-stimulated
organotypic hippocampal slice cultures. More importantly, calpain activation
resulted in the disruption of GRIP binding to the GluR2 subunit o f AMPA (a-amino-
3-hydroxy-5-methyIisoxazole-4-propionate) receptors. Since GRIP has been
proposed to function as an AMPA receptor targeting and synaptic stabilizing protein,
as well as a synaptic organizing molecule, calpain-mediated degradation of GRIP
and disruption of AMPA receptor anchoring are likely to play important roles in the
structural and functional reorganizations accompanying synaptic modifications in
LTP and LTD.
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INTRODUCTION
Ionotropic glutamate receptors mediate the majority of excitatory synaptic
transmission in central nervous system. Among them, the N-methyl-D-aspartate
(NMDA) and AMPA receptors play critical roles in the induction and expression of
long term synaptic plasticity in the hippocampus, a proposed cellular basis of
learning and memory (Bliss and Collingridge, 1993; Malenka and Nicoll, 1999). In
the past few years, protein phosphorylation was demonstrated to result in the rapid
potentiation of synaptic transmission at excitatory synapses (Barria et al., 1997;
Carroll et al., 1998; Lu et al., 1998; Malenka and Nicoll, 1999; Yu et al., 1997).
Moreover, recent studies using green fluorescent protein (GFP)-tagged GluRl
transfection of hippocampal neurons have indicated that long-term potentiation
(LTP) involves the rapid insertion of AMPA receptors into postsynaptic membranes,
while long-term depression (LTD) could be due to an opposite process (Carroll et al.,
1999; Shi et al., 1999). In particular, reagents that interfere with exocytosis and
endocytosis were found to block the formation of LTP and LTD, respectively (Lledo
et al., 1995; Luscher et al., 1999; Man et al., 2000). Thus, the hypothesis proposed 15
years ago that LTP was mediated by changes in the number of synaptic AMPA
receptors (Lynch and Baudry, 1984) has received strong experimental support.
Although accumulating evidence indicate that AMPA receptors are recycling
between a synaptic and an intracellular compartment under both basal conditions and
in response to appropriate signals, the detailed mechanisms implicated in synaptic
insertion or removal of AMPA receptors remain largely unknown. Functional AMPA
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receptors do not freely diffuse in postsynaptic membranes but rather are stabilized
within a highly dense and specialized structure, called the postsynaptic density
(PSD), by interactions with a family of anchoring proteins (Craven and Bredt, 1998;
Sheng, 1997; Wyszynski et al., 1999). Thus, receptor relocalization, at least removal
of receptors from PSDs, might require the disruption of their association with
anchoring proteins. The glutamate receptor interacting protein, GRIP, contains
several PDZ domains, is enriched in PSDs, and has been suggested to be such an
anchoring protein (Dong et al., 1997; Dong et al., 1999; Wyszynski et al., 1998;
Wyszynski et al., 1999). GRIP was initially identified in the yeast-two-hybrid system
by its interaction with the C-terminal domain of GluR2 subunits of AMPA receptors
and later found to interact with AMPA receptors in neurons. It was therefore
suggested that GRIP might play a role in AMPA receptor synaptic stabilization.
Furthermore, as PSDs contain numerous interconnected proteins, including ion
channels, scaffolding proteins, signal transduction-related proteins and cytoskeletal
proteins (Kennedy, 1998; Luscher et al., 2000; Ziff, 1997), it is likely that insertion
of AMPA receptors into PSDs requires significant structural modifications. Indeed,
morphological changes in dendritic spines have been consistently observed in long
term synaptic plasticity (Geinisman et al., 1993; Luscher et al., 2000; Schuster et al.,
1990; Toni et al., 1999; Trommald et al., 1996; Weeks et al., 1999).
Calpains represent a family of calcium-activated neutral proteases and have
been shown to be necessary for LTP formation (del Cerro et al., 1994; Denny et al.,
1990; Lynch, 1998; Oliver et al., 1989; Vanderklish et al., 1996). Calpain has been
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found in postsynaptic structures (Domanska-Janik et al., 1999; Perlmutter et al.,
1988; Siman et al., 1983) and NMDA receptor stimulation results in calpain
activation in dendritic spines (Vanderklish et al., 2000), Thus, it is likely that calpain
participates in the process of receptor mobilization and synaptic reorganization,
possibly by modifying the structure of glutamate receptor anchoring molecules.
Here, we report that calpain activation results in the truncation of GRIP, and that
such truncation produces the disruption of GRIP/GluR2 subunit interactions.
MATERIALS AND METHODS
/. Preparation o f synaptic membranes and tissue sections.
Synaptic membrane fractions were prepared from Sprague-Dawley rat forebrain
according to Massicotte et al. (Massicotte et al., 1990). Briefly, rat forebrains were
homogenized in 0.32 M sucrose containing 10 mM Tris, 1 mM EGTA. 1 mM
EDTA, 50 pM leupeptin, 100 pM PMSF and 2 pg/ml aprotinin (pH 7.4), with 10 up
and down strokes in a motor-driven glass-Teflon homogenizer to obtain the whole
homogenate. Pellets of membrane fractions were obtained by centrifugation of
homogenates at 48,000 g for 15 min and resuspension in 0.32 M sucrose.
Membranes were washed with ice-cold distilled water containing 2 mM EGTA and
0.1 mM leupeptin and finally resuspended in 100 mM Tris-acetate buffer, pH 7.4,
containing 0.1 mM EGTA. Aliquots were stored at - 80 °C until the day of use.
Alternatively, subcellular fractionation was performed as described by Whittaker et
al. (Whittaker et al., 1964). In brief, hippocampal homogenates prepared in 0.32 M
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sucrose buffer containing 10 mM Tris, 0.1 mM EGTA, pH 7.4, were first centrifuged
at 1,000 g for 10 min to remove the nuclear fraction (PI). The supernatant (SI) was
recentrifuged at 10,000 g for 20 min to yield the mitochondrial fraction (P2); S2 was
then centrifuged at 100,000 g for 60 min to yield the microsomal fraction (P3) and
the cytosolic fraction, S3. P2 and P3 were resuspended in 0.32 M sucrose containing
10 mM Tris, 0.1 mM EGTA, pH 7.4 and they, as well as the S3 fraction were later
treated with calpain.
Frozen-thawed tissue sections were prepared following rapid freezing of adult
Sprague-Dawley rat forebrains in methyl-butane at - 30 *C . Coronal 20 pm-thick
sections were cut on a crytostat and thaw-mounted onto chrom-alum gelatin coated
slides; they were kept at -70 °C until the day of use (generally < 1 week).
2. Calpain digestion o f synaptic membranes and calcium incubation o f frozen
thawed brain sections
Synaptic membranes or other subcellular fractions (containing about 300 pg
proteins) were incubated in Tris-acetate buffer with or without 2 mM calcium and
calpain I (2.4 units/ml) at 37 °C for 30 min. The resulting aliquots were immediately
processed for SDS-PAGE and Western blot. For immunoprecipitation experiments,
larger amounts of synaptic membranes (300 pi, equivalent to 1.5 mg proteins) and a
lower calpain concentration (0.6 U/ml) were used. Alternatively, adjacent tissue
sections were thawed and incubated in Tris-acetate buffer (100 mM, pH 7.4,
containing 0.1 mM EGTA) with or without 2 mM Ca** at 37 °C for 30 min. After
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incubation, sections were collected and sonicated in 0.32 M sucrose containing 10
mM Tris, 1 mM EGTA, 1 mM EDTA, 50 pM leupeptin, 100 pM PMSF and 2 pg/ml
aprotinin (pH 7.4).
3. Activation o f endogenous calpain in orsanotvpical hippocampal cultures
Organotypic cultures of hippocampal slices were prepared using the technique of
Stoppini et al. (Stoppini et ai., 1991) as described previously (Bruce et al., 1995;
Gellerman et al., 1997). In brief, hippocampi were harvested in sterile conditions
from 7 day old Sprague-Dawley rat brains in chilled minimum essential medium
(MEM) (GIBCO no. 61100061) containing 25 mM HEPES, 10 mM Tris-base, 10
mM D-glucose and 3 mM MgCfe and placed on a Teflon stage of a Mcllwain tissue
chopper. Transverse slices (400 pm thick) were cut and transferred to a humidified
membrane insert (Millicell-CM, Millipore, Bedford, MA, USA; 0.4 mm pore size),
which was placed in a deep-well plate with 1 ml of MEM (GIBCO no. 41200-072)
containing 3 mM glutamine, 30 mM HEPES, 5 mM NaHCCh. 30 mM D-glucose 0.5
mM L-ascorbate, 2 mM CaCb, 2.5 mM MgS0 4, 1 pg insulin, and 20 % horse serum,
pH 7.2. In this manner, explants were supported by the membrane at an air-medium
interface and kept in an incubator at 35 °C with a 5 % C02-enriched atmosphere.
After 14 days in vitro, hippocampal slice cultures were incubated with growth
medium without serum, followed by exposure medium (serum-free growth medium
containing 300 pM NMDA and 4 mM CaCh) in the presence or absence of 100 pM
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calpain inhibitor III for 5 min. The slices were collected 1.5 hrs following NMDA
treatment and processed for Western blot.
4. NMDA treatment o f acute hippocampal slices
Transverse hippocampal slices (400 pm thick) were prepared from 30 day-old
Sprague-Dawley rats in oxygenized ice-old dissection buffer (in mM: sucrose, 212.7;
K .C 1, 2.6; NaH2 P04 , 1.23; NaHC03 , 26; glucose, 10; MgCI2 , 3; and CaCl2 , 1, pH,
7.2) using a McIIwain tissue chopper. The slices were gently transferred in a
chamber containing artificial cerebrospinal fluid (ACSF; composition in mM: NaCl,
124; KC1, 5; NaH2 P04, 1.25; NaHC03 , 26; glucose, 10; MgCI2 , 1.5; and CaCI2 , 2.5)
bubbled with 95 % Ch and 5 % CO2 and were allowed to equilibrate at 30 °C for 1
hour. Twenty pM NMDA was added for 5 min. Slices were collected 15 min after
the washout of NMDA and rapidly homogenized in Tris buffer (Tris, 100 pM;
EDTA, ImM; Aprotinin, 100 |iM; PMSF 100 pM; leupeptin, 50 pM; pH, 7.4) and
processed for immunoprecipitation.
5. Immmunoprecipitation
Proteins from calpain-treated synaptic membranes (300 pi, equivalent to 1.5 mg
proteins) or from NMDA-treated acute slices (300 pi, roughly equal to 1.5 mg
proteins) were solubilized by adding 2 volumes (600 pi) of 1.5 % deoxycholate
(DOC) solution (1.5 % DOC, 50 mM Tris, 2 mM EGTA, 150 pM leupeptin, 150 pM
PMSF, 3 pg/ml aprotinin, pH 9.5) followed by incubation at 35 °C for 30 min.
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Following addition of 0.1 % Triton X-100 (final concentration), extracts were
centrifuged at 24,000 g for 30 min and the supernatant was used for
immunoprecipitation. One hundred and fifty pi of a 1:1 protein A slurry was washed
three times in cold Tris buffer (50 mM Tris, 2mM EGTA, pH7.4), to which 550 pi of
DOC-soIubilized supernatant along with the antibodies of interest were then added (5
pg GluR2 or 10 pg of GRIP). The mixture was gently rotated at 4 °C overnight.
After washing the protein A beads with 50 mM Tris buffer, pH 7.4, (the first two
washes in the presence of 0.1 % Triton X-100), 70 pi sample buffer (see below) were
added to the beads and the proteins were eluted by boiling for 5 min.
6. SDS-PAGE and Western blots
Aliquots from different fractions were boiled at 100 °C for 5 min in sample
buffer [2 % SDS, 50 mM Tris-HCl (pH 6.8), 10 % 2-mercaptoethanol, 10 %
glycerol, and 0.1 % bromophenol blue]. SDS-PAGE was performed according to the
method of Laemmli (Laemmli, 1970). Proteins were then transferred onto
nitrocellulose membranes as described by Towbin et et al. (Towbin et al., 1979).
Nitrocellulose membranes were first blocked with 3 % gelatin in Tris-buffered saline
(TBS) at room temperature for I h and incubated overnight at 4 °C with primary
antibodies against GRIP (1:1500 dilution; see (Wyszynski et al., 1999) for details
regarding the generation and characterization of the antibody), NR1 (1:200 dilution),
a-CaMKJI (1:5000 dilution), or the N-terminal domains of GluR2 (1:1000 dilution).
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These 3 antibodies were purchased from Chemicon International Inc. (Temecula,
CA), in 1 % gelatin prepared in TBS with 0.5 % Tween-20 (TIBS). After 2 washes
with TIBS for 5 min, membranes were incubated with alkaline phosphatase-
conjugated anti-rabbit (1:2000) or anti-mouse (1:2000) IgG for 2 h at room
temperature; antigens were then visualized with nitroblue tetrazolium and 5-bromo-
4-chlor-3-indoIyphosphate toluidine salt. Blots were scanned and quantitatively
analyzed by densitometry with ImageQuant™ software. Student’ s t-Test was used
when needed.
RESULTS
1. Exogenous calpain cleaves GRIP in vitro.
The initial experiment was carried out by incubating synaptic membranes
with purified calpain I (2.4 U/ml) in the presence of 2 mM Ca~at 37 °C for 30 min
(Fig. 1). Western blots stained with anti-GRIP antibodies showed a major band
migrating with an apparent molecular weight of 130 kDa and a few minor bands with
lower molecular weights, in good agreement with previous reports (Dong et al.,
1997; Wyszynski et al., 1998). Following incubation with calpain, the intensity of
the 130 kDa band decreased and several smaller species appeared, demonstrating the
existence of multiple truncation sites for calpain. Most prominent were fragments
with apparent molecular weights of 74, 55, 49 and 45 kDa. Since the overall
immunoreactivity for GRIP remained unchanged between control and calpain-treated
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H P2 P3 Cyt
Fig. 9 Calpain-mediated degradation of GRIP in svnaptic membranes and different
subcellular fractions.
A) Synaptic membranes were incubated with purified calpain I (Calp, 2.4 U/ml) and
calcium (2 mM) at 37 °C for 30 min. Membrane proteins were resolved by SDS-PAGE,
transferred onto nitrocellulose membranes and probed with anti-GRIP antibodies. Molecular
weights of native GRIP as well as of calpain-generated degradation products are indicated on
the right in kDa. B) Adult brains were homogenized and fractionated into P2, P3, and S3
(Cyt) fractions. Aliquots from each fractions were incubated with purified calpain I (2.4
U/ml) and calcium (2 mM) at 37 °C for 30 min. Top, Western blots showing the degradation
of native GRIP species in different fractions. Bottom, quantification of Western blots
showing the percent un-degraded GRIP following calpain incubation in different fractions.
Results are means ± S.E.M. of 3 experiments.
samples, it is unlikely that the breakdown products represent unrelated proteins.
Since limited proteolysis was observed in similar studies of calpain-mediated
degradation of ionotropic glutamate receptors and of the NMDA anchoring protein
PSD-95 (Bi et al., 1998; Lu et al., 2000; Lu et al., 2000), we tested whether partial
proteolysis reflected a lower sensitivity of GRIP to calpain. Incubation of synaptic
membranes with increasing calpain concentrations showed that GRIP was partially
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degraded at low calpain concentrations, and that increasing calpain concentration
failed to further increase the extent of GRIP degradation (data not shown). To
determine whether phosphorylation was involved in protecting GRIP from calpain-
mediated truncation, the in vitro digestion experiment was repeated in the presence
of protein phosphatase inhibitors. Addition of orthovanadate (2 mM), a tyrosine
phosphatase inhibitor, or okadaic acid (400 nM), a serine phosphatase inhibitor,
however, did not decrease the extent or modify the degradation pattern of GRIP (not
shown). We next examined whether different populations of GRIP exhibited
different sensitivity to calpain. Adult rat forebrain was homogenized and differential
centrifugation was used to obtain various subcellular fractions. When whole
homogenate, P2, P3, and S3 fractions were incubated with purified calpain I, GRIP
from the S3 fraction was almost completely degraded, with a truncation of about 85
% as opposed to 25 % for P2 (Fig. 9).
2. GRIP degradation following activation of endogenous calpain.
To study the effect of endogenous calpain activation on GRIP degradation,
frozen-thawed rat forebrain sections were incubated with calcium (2 mM) at room
temperature for 30 min. The sections were then collected, homogenized and aliquots
of homogenates were subjected to SDS-PAGE and Western blotting with anti-GRIP
antibodies. Calcium incubation resulted in the partial truncation of GRIP in a manner
identical to calpain treatment of synaptic membranes, with the appearance of
breakdown products with apparent molecular weights of 74, 55,49 and 45 kDa (Fig.
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10). These effects were blocked by EGTA and 2 calpain inhibitors, leupeptin and
calpain inhibitor III. However, cleavage of GRIP following calcium incubation was
not blocked by the addition of PMSF or aprotinin (serine protease inhibitors), further
confirming that GRIP degradation as a result of calcium incubation was solely due to
endogenous calpain activation.
Fig. 10 Effects of calcium treatment o f frozen-thawed brain sections on GRIP.
Frozen-thawed tissue sections (20 pm) were incubated with 2 mM calcium
at room temperature for 30 min, with or without the following protease inhibitors:
100 pM calpain inhibitor III (CalpI III), 100 pM leupeptin (Leu), 2 mM EGTA, 50
pM aprotinin (Aprot), 100 pM PMSF. Tissue was collected and processed for
Western blots using antibodies against GRIP. A) Representative Western blot. B)
Quantification of immunoreactivity of the native species of GRIP (130 KDa)
expressed as percent of control. Results are means ± S.E.M. of 4 experiments (*p <
0.05, Student’s t-Test).
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NMDA treatment of organotypic cultures of hippocampal slices has been shown to
activate calpain, and has often been used to investigate mechanisms of synaptic
plasticity (Musleh et al., 1997; Vanderklish et al., 1995). Several features of the
organization and function of the hippocampus are well preserved in such preparation,
thus allowing the effect of calpain activation to be studied under conditions closely
matching the in vivo environment Organotypic hippocampal slice cultures were
GRIP
Fig. 11 Effects of NMDA receptor activation in cultured hippocampal slices on
GRIP.
Cultured hippocampal slices were incubated with NMDA (20 |iM), in the
presence or absence of calpain inhibitor III (CalpI III), for 3 h. The tissue was
collected immediately and processed for Western blots using anti-GRIP antibody (A)
or anti-NRl antibodies (B). Results are representative of 5 experiments.
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prepared from postnatal day 7 rats; after 14 days in vitro, the culture was briefly
exposed to 300 pM NMDA 5 min. The slices were collected 1.5 hours after NMDA
treatment and processed for Western blots with anti-GRIP antibodies (Fig. 11).
Quantification of immunoblots revealed a modest yet significant (10 ± 3.4 %, p <
0.05, Student’s t-Test) decrease in immunoreactivity of the native GRIP band and the
concurrent appearance of several breakdown products, in particular, the 55, 49 and
45 kDa fragments. In addition, when the cell permeable calpain inhibitor, calpain
inhibitor III (100 pM), was applied together with NMDA to the culture medium, the
decrease in immunoreactivity of the 130 kDa band and the appearance of the
breakdown products, were greatly reduced, if not completely blocked. In contrast,
NR1, another PSD protein, was not degraded under these conditions (Fig. 11).
3. Calpain-mediated truncation of GRIP disrupts its interaction with
AMPA receptors.
Previous studies using yeast two-hybrid system or co-immunoprecipitation of
rat brain homogenates demonstrated that GRIP is associated with the GIuR2 subunits
of AMPA receptors through PDZ domains 4 and 5 both in vitro and in vivo.
Interestingly, most calpain-generated GRIP degradation products had apparent
molecular weights around 50 kDa. Given that full length GRIP protein contains 1112
amino acids, it is reasonable to predict that some, if not all, of the calpain truncation
sites are located within PDZ domains 4 and 5, suggesting that degradation of GRIP
by calpain might disrupt the binding between GRIP and GluR2. In previous studies,
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we found that GluR2 was also truncated following calpain activation (Lu et al.,
2000). Since GRIP but not GluR2 was hydrolyzed at a low calpain concentration of
0.6 U/ml (Fig. 12), we determined the effects of incubating synaptic membranes with
this concentration on the interactions between GRIP and AMPA receptors. After
incubation with calpain I (0.6 U/ml) at 37 °C for 30 min, synaptic membranes were
GRIP antibodies, Western blots labeled with antibodies against GluR2 and GRIP
showed that under control conditions, a fraction of GRIP was associated with GluR2
WB: Anti-GRIP WB: Anti-duR2
IP W /: GRIP GMt2 IP W /: GfaR2 GRIP
■
IlH
I«G
Ctrl Calp Ctrl Calp
M S
-4- IgG
Ctrl Crip Ctrl Crip
Fig. 12 Disruption of GluR2-GRlP interaction following calpain-treatment of
svnaptic membranes.
Following incubation with Calpain I (0.6 U/ml) at 37 °C for 30 min,
synaptic membranes were solubilized in 1 % deoxycholate and immunoprecipation
(IP) was performed using anti-GRIP or anti-GluR2 antibodies. The precipitated
immunocomplexes were resolved by SDS-PAGE and probed with anti-GRIP (left) or
anti-GluR2 (right) antibodies. Results are representative of 3 experiments.
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(Fig. 12). A similar result was obtained following immunoprecipitation with anti-
GluR2 antibodies, in good agreement with previous reports (Dong et al., 1999;
Wyszynski et al., 1999). Following incubation with calpain, GRIP was partially
hydrolyzed and GRIP antibodies successfully precipitated both the 130 kDa native
species as well as several breakdown products (Fig. 12). As expected, GluR2 was not
significantly degraded under such conditions, as GluR2 antibodies precipitated the
same amount of GluR2 before and after calpain incubation. However, the interaction
between GRIP and GluR2 was dramatically reduced, as very little GRIP co-
immunoprecipitated with GluR2 and none of the GRIP breakdown products were
observed to be associated with GluR2. Furthermore, whereas the levels of native
GRIP were decreased by 35 ± 6 % following incubation with calpain, the levels of
GiuR2 co-immunoprecipitated with GRIP were reduced by 67 ± 23 % (n=3, P <
0.05, Student’s t-Test).
Acute hippocampal slices were incubated with 50 pM NMDA for 5 min, a
manipulation that has been reported to produce a long-lasting increase in synaptic
efficacy in CA1 as well as calpain activation (Broutman and Baudry, 2001). Co-
immunoprecipitation with anti-GRIP or anti-GluR2 antibodies was performed to
determine the extent of GRIP/GluR2 interaction. No significant differences in the
amount of precipitated native GRIP or GluR2 proteins between control and NMDA-
stimulated conditions were observed, suggesting that GRIP or GluR2 degradation, if
it occurs, is very limited following a brief NMDA receptor stimulation. In contrast,
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the interaction between GRIP and GluR2 was decreased by about 40 % (Fig. 13).
This effect was completely blocked by calpain inhibitor III.
WB w/GluR2
Fig. 13 Effects of NMDA treatment of acute hippocampal slices on GluR2-GRIP
interaction.
Following incubation of acute hippocampal slices with NMDA (50 pM) at 30 °C for
5 min, slice homogenates were solubilized in 1 % deoxycholate and
immunoprecipation (IP) was performed using anti-GRIP antibodies. The precipitated
immunocomplexes were resolved by SDS-PAGE and probed with anti-GRIP or anti-
GluR2 antibodies. Top: representative Western blots. Bottom: quantification of the
blots. Results are means ± S.E.M of 3 experiments (*p < 0.05, Student’s t-Test).
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DISCUSSION
The present results indicate that GRIP is a substrate for calpain and that
calpain-mediated cleavage of GRIP is likely to occur in vivo in response to increase
in intracellular calcium concentration and NMDA receptor activation. We used three
strategies to study the effects of calpain activation on GRIP properties. First, GRIP
was significantly degraded by purified calpain I in rat forebrain synaptic membranes.
Second, the same degradation pattern was observed following incubation of frozen-
thawed brain sections with calcium, indicating that activation of endogenous calpain
produces the truncation of GRIP. Finally and more importantly, NMDA treatment of
organotypic hippocampal cultures also lead to calpain-mediated degradation of GRIP
and the formation of truncation products with the same molecular weights as those
observed in the calpain treatment experiment. In every case, the involvement of
calpain was assessed by the addition of specific calpain inhibitors and of non-calpain
protease inhibitors. In every case, GRIP degradation was only partial and limited,
although it was much more extensive in soluble than membrane fractions. The
difference between the different calpain sensitivity of these two populations of GRIP
is not clear at present. A possible explanation could be that the presence of multiple
interactions between GRIP and other proteins in the PSD limits the access of calpain
to GRIP. Alternatively, differences in posttranslational states could account for the
difference.
As previously reported (Dong et al., 1997; Dong et al., 1999; Wyszynski et
al., 1998), GRIP is enriched in synaptic membranes of rat hippocampus and co-
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precipitates with GluR2 subunits of AMPA receptors. The GRIP-GluR2 interaction
was markedly disrupted by calpain activation in synaptic membranes. Interestingly,
although only a relatively small fraction of GRIP (35 %) appeared to be truncated by
calpain, almost all the interaction (> 70 %) between GRIP and GluR2 was disrupted.
Furthermore, we also demonstrated that a brief NMDA application (5 min) in acute
hippocampal slices produced calpain activation and disruption of GRIP-GluR2
interaction. Several possibilities could account for the lack of GRIP degradation
under these conditions. First, it is possible that only a small fraction of GRIP was
degraded, an effect too small to be detected by our assay. Indeed, even following
prolonged NMDA application in organotypical hippocampal cultures. GRIP
degradation was also very limited. Given that the same discrepancy between the
level of GRIP degradation and percent decrease in GRIP-GluR2 binding also
occurred in calpain-treated synaptic membranes, these data suggest that calpain
might preferably truncate the fraction of GRIP associated with GluR2 subunits of
AMPA receptors. Alternatively, other proteins could participate in the interactions
between GRIP and GluR2 and these additional proteins could themselves be calpain
substrates. It might therefore be interesting to test whether the recently described
GRASP (Ye et al., 2000) is also a calpain substrate. In addition, adaptin has been
reported to be associated with GluR2 subunits of AMPA receptors (Man et al., 2000)
and was previously found to be a substrate for calpain (Sato et al., 1995).
The induction of two forms of long term synaptic plasticity, LTD and LTP,
requires NMDA receptor activation, followed by transient elevations of postsynaptic
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calcium concentration (Bliss and Collingridge, 1993; Malenka and Nicoll, 1999).
Downstream effects of elevated calcium include the activation of the calcium-
dependent protease calpain as well as of phosphatases (LTD) or kinases (LTP)
(Barria et al., 1997; del Cerro et al., 1994; Lee et al., 2000; Malenka and Nicoll,
1999; Muslehetal., 1997). Several studies have also shown that calpain activation
was necessary for LTP formation (del Cerro et al., 1994; Denny et al., 1990; Oliver
et al., 1989; Staubli et al., 1988; Vanderklish et al., 1996). However, the precise
pathway by which calpain participates in the expression of long term synaptic
plasticity remains unclear. We previously reported that GluRl and GluR2 subunits of
AMPA receptors were truncated by calpain activation, and that the truncated species
of GluRl and GluR2 were removed from PSDs, possibly for internalization and
further degradation (Lu et al., 2000). We further postulated that this would bias
synaptic strength toward LTD. The present results show that the AMPA receptor
anchoring protein GRIP is also a substrate for calpain and that calpain-mediated
truncation of GRIP, GluR2 or some other associated proteins, disrupts GRIP-GluR2
interactions. On one hand, this could facilitate the removal of the receptors from the
synapses and interrupt the insertion of new receptors into synaptic membranes, since
one possible function of GRIP is to sort and transport AMPA receptors to synaptic
sites (Dong et al., 1999). Such an effect could possibly contribute to LTD expression.
Indeed, it has recently been show that GIuR2-GRIP interaction is required to activate
silent synapses in the spinal cord, probably by recruiting more AMPA receptors to
the synapses, and disruption of GluR2-GRIP interaction was also found to be
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associated with LTD in cerebellum (Li et al., 1999; Matsuda et al., 2000). On the
other hand, given that morphological changes, such as perforated synapses, synaptic
splitting and budding (Luscher et al., 2000; Toni et al., 1999; Trommald et al., 1996)
have been consistently observed in the expression or consolidation phase of LTP and
since GRIP was found to form polymeric complexes as well as to bind to several
PSD proteins (Dong et al., 1997; Torres et al., 1998; Ye et al., 2000), it is likely that
GRIP could function as a synaptic organizing protein. In this way, degradation of
GRIP by calpain could possibly be involved in structural modifications observed in
LTP. In addition, truncation of GRIP as well as of two other PSD proteins, PSD-95
and spectrin, by calpain (del Cerro et al., 1994; Lu et al., 2000; Lynch and Baudry,
1987), could potentially facilitate the insertion of receptors by opening up the PSD
and making the postsynaptic membranes more accessible to secretory vesicles
carrying AMP A receptors. In this regard, the effect would be permissive and some
other triggering signals might be required to initiate the exocytotic machinery.
In conclusion, our results indicate that GRIP is a substrate of calpain and that
calpain activation, by degrading GRIP or some other GRIP-associated protein,
eliminates GRIP/GluR2 interaction. This effect is likely to play an important role in
the structural and functional reorganizations accompanying synaptic modifications
observed in LTP and LTD.
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CHAPTERS
CALPAIN-MEDIATED TRUNCATION OF RAT BRAIN AMPA
RECEPTORS INCREASES THEIR TRITON X-100 SOLUBILITY
ABSTRACT
Previous studies have indicated that calpain activation results in the truncation of the
C-terminal domains of AMPA and NMDA receptor subunits. The present study
determined the distribution of the truncated species of the subunits between Triton-
soluble and -insoluble fractions. Western blots were performed with various
antibodies to quantify the amounts of the various species of GluRl, GluR2, GluR3
and NR2B subunits. The results indicate that calpain activation decreased the amount
of all the intact subunits in Triton-insoluble fractions. Calpain-generated truncated
forms of GluRl and GluR2, but not NR2B, were absent in these fractions, and were
recovered in Triton-soluble fractions. These findings suggest that calpain-mediated
truncation of AMPA but not NMDA receptor C-terminal domains results in
modifications of the interactions of the receptors and postsynaptic densities, and that
this mechanism could be involved in activity-dependent changes in subcellular
distribution.
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INTRODUCTION
Glutamate ionotropic receptors play critical roles in synaptic transmission,
synaptic plasticity and various forms of neuropathology. The 2 major families of
glutamate ionotropic receptors, i.e., the AMPA and the NMDA receptors, are now
well characterized at the gene and structural levels. Significant progress has been
made in identifying proteins responsible for targeting and anchoring the receptors in
postsynaptic structures. Thus, several families of proteins linking the C-terminal
domains of various subunits of AMPA and NMDA receptors to cytoskeletal proteins
have been discovered (Dong et al., 1997; Komau et al., 1995; Nishimune et al.,
1998; Song et al., 1998). Several posttranslational processes regulate the properties
of AMPA and NMDA receptors. Among these, phosphorylation processes have
received considerable attention as phosphorylation/dephosphorylation of the C-
terminal domains of AMPA and NMDA receptors regulates channel properties as
well as interactions of the receptors with targeting/anchoring proteins (Kohr and
Seeburg, 1996; Wang and Salter, 1994; Wechsler and Teichberg, 1998). Little is
known however regarding the processes involved in receptor turnover, and in
particular, in receptor internalization and degradation. Reported values for AMPA
receptor half-life are in the range of 30 to 50 hrs (Archibald et al., 1998; Horikawa
and Nawa, 1998; Mammen et al., 1997). A complicating factor in the case of AMPA
receptors is the existence of a significant pool of non-synaptic receptors (Baude et
al., 1995; Bernard et al., 1997; Mammen et al., 1997). These receptors are presumed
to be present either in extra-synaptic membrane domains or in an intracellular
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vesicular compartment and could represent newly synthesized receptors targeted to
synaptic regions (Hall et al., 1997; Henley, 1995; Standley et al., 1998).
Another posttranslational modification for both AMPA and NMDA receptors
consists in the truncation of the C-terminal domains of various subunits of both types
of receptors (Bi et al., 1997; Bi et al., 1998). This truncation is mediated by the
calcium-dependent protease calpain and we showed it occurred under both
physiological and pathological conditions (Bi et al., 1996; Bi et al., 1998; Musleh et
al., 1997). In the case of AMPA receptors, calpain-mediated truncation of GluRl
subunits results in the formation of a new species of the subunit with an apparent Mr
of about 98 kDa as compared to 105 kDa for the native species. As it is not present
under control conditions, it is likely that this species has a very short half-life, thus
suggesting that truncation of the C-terminal domains of AMPA receptor subunits
represents an initial step towards their degradation. Here we report evidence that
calpain-mediated truncation of the C-terminal domains of subunits for the AMPA but
not the NMDA receptors is associated with the removal of the receptors from
postsynaptic densities (PSDs) and could thus represent an initial step towards the
degradation of the receptors.
MATERIALS AND METHODS
Sprague-Dawley rats (Charles River) were kept under standard laboratory
conditions with a I2-h day and night cycle. All procedures using animals were in
accordance with the NIH guidelines and authorized by the USC Animal Care and
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Use Committee. Adult rats were killed by decapitation following methoxyflurane
anesthesia, their brains rapidly removed and immediately frozen in isopentane
(methylbutane) at -20 °C, then kept at -70 °C until used. Coronal 20 pm-thick
sections were cut on a crytostat and thaw-mounted onto chrom-alum gelatin coated
slides; they were kept at -70 °C until the day of use (generally < I week).
I. Tissue treatment and preparation o f membranes
Adjacent sections were thawed and incubated in Tris-acetate buffer (100 mM,
pH 7.4) containing 0.1 mM EGTA with or without 2 mM Ca** at 37 °C for 30 min.
After incubation, sections were collected and homogenized in 0.32 M sucrose
containing 10 mM Tris, 1 mM EGTA, 1 mM EDTA, 50 pM leupeptin, 100 pM
PMSF and 2 pg/ml aprotinin (pH 7.4), with 10 up and down strokes in a motor-
driven glass-Teflon homogenizer. Subcellular fractionation was performed as
described by Whittaker et al. (Whittaker et al., 1964). In brief, homogenates were
first centrifuged at 1,000 g for 10 min to remove the nuclear fraction (PI). The
supernatant was recentrifuged at 10,000 g for 20 min and the resulting pellet (P2)
was resuspended in 40 mM Tris-HCl, pH 7.4; this fraction was defined as the crude
mitochondrial/synaptic membrane fraction and was later used for Triton extraction or
subjected to SDS-PAGE and Western blotting.
Alternatively, crude synaptic membrane fractions were prepared from adult
forebrain according to Massicotte et al. (1990). Aliquots (containing about 300 pg
proteins) were incubated in Tris-acetate buffer with or without 2 mM calcium and
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calpain I (5 units/ml) at 37 °C for 30 min. Incubations were terminated by
centrifugation at 24,000 g for 20 min and the pellets were resuspended in 40 mM
Tris-HCl, pH 7.4, and used for SDS-PAGE or for Triton X-100 extraction.
2. Preparation o f Triton X-100 insoluble and soluble fractions from membranes
Aliquots of membrane fractions (about 100 pg proteins) were treated with I
ml of Triton extraction buffer (1 % Triton X-100,40 mM Tris, 150 mM NaCl, 1 mM
EDTA, 50 pM Ieupeptin, 100 pM PMSF and 2 pg/ml aprotinin, pH 7.4) on ice for
15 min with intermittent vortexing. The extraction mixture was centrifuged at 16,000
g for 20 min. The resulting pellet and supernatant fractions were defined as PSD-
enriched (Triton-insoluble) and Triton-soluble fractions, respectively. The pellets
were resuspended in Triton extraction buffer without Triton X-100 or directly in
sample buffer (see below). The supernatant was freeze-dried and reconstituted in a
minimal volume of sample buffer to perform SDS-PAGE separation.
3. SDS-PAGE and Western blots
Aliquots from different fractions were boiled at 100 °C for 5 min in sample
buffer [2 % SDS, 50 mM Tris-HCl (pH 6.8), 10 % 2-mercaptoethanol, 10 %
glycerol, and 0.1 % bromophenol blue]. SDS-PAGE was performed according to the
method of Laemmli (Laemmli, 1970). Proteins were then transferred onto
nitrocellulose membranes as described by Towbin (1979). Nitrocellulose membranes
were first blocked with 3 % gelatin in Tris-buffered saline (TBS) at room
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temperature for 1 h and incubated overnight at 4 °C with primary antibodies against
the N-terminal domains of GluRl (generous gift from Dr. R.J. Wenthold, NIH; 1:500
dilution), GluR2 (Pharmingen, San Diego, CA; GluR2/4, 1:1500), the C-terminal
domains of GluR2/3 (1:1500) and NR2B (1:200), or aCaMKII (1:15000) in 1 %
gelatin prepared in TBS with 0.5 % Tween-20 (TI BS) (these 3 antibodies were
purchased from Chemicon International Inc., Temecula, CA). After 2 washes with
TI BS for 5 min, membranes were incubated with alkaline phosphatase-conjugated
anti-rabbit (1:2000) or anti-mouse (1:2000) IgG for 2 h at room temperature;
antigens were then visualized with nitroblue tetrazolium and 5-bromo-4-chlor-3-
indolyphosphate toluidine salt. Blots were scanned and analyzed with ImageQuant™
software. As previously reported (Bi et al., 1997; Bi et al., 1998), this software
allows reliable quantification of peaks that are close to each other.
RESULTS
1. Triton X-100 insolubility of AMPA and NMDA receptor subunits.
Traditional PSD preparation involves first the purification of synaptosomes
followed by extraction of membranes with detergents. For our purpose, we
simplified the procedure in order to rapidly extract PSDs from crude synaptic
membrane fractions prepared from frozen-thawed brain sections. Thus, we used
Triton X-100 extraction of a crude mitochondrial/synaptic membrane fraction or
from a modified synaptic membrane fraction to prepare PSD-enriched fractions. The
pattern of proteins observed with Coomassie Blue staining of the Triton-insoluble
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| 4 -S n td fc
4 - Tabula
4 - a-C «M K n
4 - A c tte
4 - PSD -95
Fig. 14 Triton X-100 insolubility of GluRl. GluR2/4. GluR2/3. NR2A. NR2B and
aCaMKII. and effect of calcium on Coomassie blue staining pattern of PSP-enriched
fractions,
A. Triton-insolubility was calculated as the staining intensity measured in
Triton-insoluble fraction expressed as percentage of that measured in the membrane
fraction before Triton X-100 extraction (corrected for the amounts of proteins loaded
on the gels). Results are means ± S.E.M. of 4 separate experiments.
B. Coomassie blue staining pattern of proteins (about 20 pg protein) from
PDS-enriched fractions. Frozen-thawed brain sections were incubated in the
presence and absence of 2 mM calcium. After incubation, sections were collected
and subjected to subcellular fractionation and Triton X-100 extraction. Left:
molecular weight markers. Right: known proteins.
fraction was very similar to that reported by authors using traditional PSD
preparation procedures (Carlin et al., 1980; Kelly et al., 1984; Kennedy et al., 1983),
with clear identification of spectrin, PSD-95, tubulin, CaMKII, and actin (Fig. 14).
Western blots of Triton-insoluble and Triton-soluble fractions were stained with anti-
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GluRl, anti-GIuR2/4, anti-GluR2/3, anti-NR2A, anti-NR2B and anli-aCaMKII
antibodies to determine an index of Triton X-100 insolubility (Fig. 14). This index
was calculated as the staining intensity measured in the Triton-insoluble fraction
expressed as percent of that measured in the membrane fraction before Triton
extraction (adjusted for the amount of proteins loaded on the gels). This index ranged
from about 20 % for GluRl to 88 % for NR2B.
2. Effects of calpain activation on AMPA receptor properties and Triton X-
100 solubility.
Two methods were used to study the effects of calpain activation on the
properties of glutamate receptors. In the first method, we incubated membrane
fractions with purified calpain I and calcium. In the second one, frozen-thawed brain
sections (20 pm thick) were incubated at 37 °C in the presence and absence of 2 mM
calcium for 30 min to activate endogenous calpain. After incubation, sections were
collected, homogenized and subcellular fractionation was performed. In both cases,
adding leupeptin, a broad trypsin-like protease inhibitor, or calpain inhibitor III. a
more selective calpain inhibitor (Mehdi, 1991) assessed selectivity for calpain
activation. As previously reported (Bi et al., 1997; Bi et al., 1998), both treatment
resulted in the degradation of GluRl, GluR2 or NR2, and the effects were
completely blocked with either leupeptin or calpain inhibitor III (not shown).
Following calcium treatment of brain sections, samples from crude synaptic
membranes were subjected to Triton X-100 solubilization and aliquots from
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membranes, PSD-enriched, and Triton-soluble fractions were immunoblotted with
antibodies against the N-terminal domains of GluRl. Calcium treatment of tissue
sections resulted in the partial disappearance of the 105 kDa band and the formation
of a smaller molecular weight species, thus forming a doublet band in the membrane
fraction (Fig. 15), in agreement with our previous reports (Bi et al., 1996; Bi et al..
1997). The 98 kDa band was absent in all fractions under control conditions.
Following calcium treatment, it represented about 42 % of the 105 kDa band in the
crude synaptic membrane fraction. In contrast, it was almost completely absent in the
PSD-enriched fraction but was very prominent in the Triton-soluble fraction.
Quantification of the blots indicated that it was 10 times more abundant in the
Triton-soluble fraction as compared to the Triton-insoluble fraction. We also
compared total N-GluRl staining between control and calcium treated groups. In
PSD-enriched fractions, total staining after calcium treatment represented 66 % of
control, whereas no difference in total N-GluRl immunostaining between both
groups was observed in synaptic membranes, thus clearly indicating a loss of
immunoreactivity in the PSD-enriched fraction. In addition, the protein pattern
obtained with Coomassie Blue staining of PSD-enriched fractions was not changed
significantly after calcium treatment except for an evident decrease in the amount of
spectrin and a few other bands (Fig. 14). More importantly, 2 major PSD proteins, a-
CaMKII and the NR2B subunits of NMDA receptors, were not decreased in PSD-
enriched fractions under these conditions (see Fig. 17). Calpain treatment of synaptic
membranes resulted in an identical pattern of results, with a decrease in the 105 kDa
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A CSM PSD PSD-Sop CSM PSD PSD-Sap
CW C m * * CM C a r CM C*H C U C a* CM C rip CM C #
CSM PSD PSD-Sup CSM PSD PSD-Sap
Fig. 15 Effects of calpain activation on the properties and Triton X-100 solubility
of GluRl subunits.
Frozen-thawed brain sections were incubated with 2 mM calcium and were
collected and subjected to subcellular fractionation and Triton X-100 extraction.
Alternatively, synaptic membranes were treated with 5 units/ml calpain (Calp) plus 2
mM calcium and then subjected to Triton extraction. Western blots were performed
using antibodies against the N-terminal domain of GluRl subunits. CSM, crude
synaptic membranes; PSD, postsynaptic density-enriched (Triton-insoluble) fraction;
PSD-sup, Triton-soluble fraction.
A. Representative Western blots; left: effect of calcium treatment of
sections; right: effect of calpain treatment of synaptic membranes.
B. Quantification of the effects of calcium treatment of rat brain slices. The
amounts of the 105 kDa and 98 kDa bands were calculated as percent of the 105 kDa
present in control CSM, PSD and PSD-Sup, respectively. Results represent means ±
S.E.M. of 4 experiments. * p< 0.05 as compared to control, (Student’s t-test).
C. Quantification of the effects of calpain treatment of synaptic membranes.
The amounts of the 105 kDa and 98 kDa bands were calculated as percent of the 105
kDa present in control CSM, PSD and PSD-Sup, respectively. Results represent
means ± S.E.M. of 4 experiments. * p< 0.05 as compared to control, (Student’s t-
test).
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A
CSM PSD
PSD-Sap
C trl C«** LAC*** C trl C*** LAC*** C trl C«** LAC***
c u t C»h L& CrH- C trl C«++ LAC*m- C trl C*++ LAC*++
Fig. 16 Effects of calpain activation on the properties and Triton X-100 solubility
of GluR2 subunits.
Frozen-thawed brain sections were incubated in the presence and absence of
2 mM calcium (Ca**) in the absence or presence of leupeptin (100 pM, Leu). After
incubation, sections were collected and subjected to subcellular fractionation and
Triton X-100 extraction. Western blots were performed using antibodies against the
N-terminal domain of GluR2 subunits. Thirty pg proteins were loaded for CSM;
proteins prepared from 50 pg or from 150 pg of CSM proteins were loaded for PSD
and PSD-Sup, respectively. CSM, crude synaptic membranes; PSD, postsynaptic
density-enriched (Triton-insoluble) fraction; PSD-Sup, Triton soluble fraction.
(A) Representative Western blots. Arrows indicate the location of the 105
and 100 kDa species of GluR2/4.
(B) Quantification of the effects of calcium treatment of rat brain slices. The
amounts of the 105 kDa and 100 kDa bands were calculated as percent of the 105
kDa present in control CSM, PSD, and PSD-Sup, respectively. Results represent
means ± S.E.M. of 4 experiments. * p< 0.05 as compared to control, (Student’s t-
test).
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species of GluRl and the formation of a 98 kDa species of GluRl in crude synaptic
membrane fractions (Fig. 15). While the amount of the 105 kDa species was
markedly decreased in PSD-enriched fractions, the 98 kDa was hardly detected in
this fraction but was very prominent in the Triton-soluble fraction.
Western blots labeled with antibodies against the N-terminal domain of
GluR2 (anti-GluR2/4) exhibited a doublet band with apparent molecular weights of
105 and 100 kDa (Fig. 16). In the membrane fraction, the amount of the 100 kDa
species increased following calcium treatment of tissue sections, and the increase
was completely blocked by the calpain inhibitor, leupeptin. This lower molecular
weight species was absent in PSD-enriched fraction, while it was abundant and
increased by about 50 % in Triton-soluble fraction. In addition, the amount of the
105 kDa species in PSD-enriched fraction decreased by about 25 % following
calpain activation, an effect also blocked by leupeptin.
When Western blots were labeled with anti-GluR2/3 antibodies, a doublet
band was also observed under control conditions, and the amount of the upper band
was also decreased by about 25 % in PSD-enriched fractions (not shown).
3. Effects of calpain activation on NMDA receptor properties and Triton
X-100 solubility.
Similar experiments were performed using anti-NR2B antibodies in Western
blots. Incubation of frozen-thawed slices at 37 °C in the presence of 2 mM Ca^
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CSM PSP PSD-Sap
C trl C «H Ctrl C «t+ C trl C*++
CSM PSD
Fig. 17 Effects of calpain activation on the properties and Triton X-100 solubility
of NR2B subunits.
Frozen-thawed brain sections were incubated in the presence and absence of
2 mM calcium. After incubation, sections were collected and subjected to subcellular
fractionation and Triton X-100 extraction. Western blots were performed using
antibodies against the C-terminal domain of NR2B subunits. CSM, crude synaptic
membranes; PSD, postsynaptic density-enriched (Triton-insoluble) fraction; PSD-
sup, Triton-soluble fraction.
A. Typical western blots. Top: anti-NR2B; bottom: anti-a-CaMKII. Arrows
indicate the location of the 180 kDa and 150 kDa species of NR2B.
B. The amounts of the 180 and 150 kDa bands were calculated as percent of
the 180 kDa in control conditions. Results represent means ± S.E.M. of 4
experiments. * p< 0.05 as compared to control, (Student’s t-test).
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resulted in a significant decrease in the intensity of NR2B staining to 83 % of control
values in crude synaptic membrane fractions. However, as the intensity of the band
migrating with an apparent Mr of 180 kDa decreased, new immunoreactive bands
with lower molecular weights appeared, with the most prominent exhibiting an
apparent Mr of 150 kDa (Fig. 17). This 150 kDa band was previously found to be
produced following calpain treatment of synaptic membranes (Bi and Baudry,
unpublished data). The 150 kDa represented about 17 % of the control 180 kDa in
synaptic membranes. A similar pattern was obtained in PSD-enriched preparation, as
the amount of the 180 kDa band was decreased by 18 % and that of the 150 kDa
represented about 16 % of the control 180 kDa band. No significant amount of the
150 kDa species was present in Triton-soluble fraction with control or calcium
treatment.
DISCUSSION
The present results clearly indicate that calpain-mediated truncation of the C-
terminal domains of GluRl and GluR2 subunits of AMPA receptors is associated
with the removal of AMPA receptors from PSD-enriched fractions. We first
replicated our previous results that calpain treatment of synaptic membranes or
calcium treatment of frozen-thawed brain sections produced the formation of a 98
kDa species of GluRl that is recognized by an antibody against the N-terminal of
GluRl but not by an antibody against the C-terminal domain. In both cases, about 45
% of the receptors were truncated. We previously discussed possible reasons for this
limited truncation and hypothesized that phosphorylation could account for the
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protection of some receptor subunits from calpain-mediated truncation (Bi et al.,
1998). O f the synaptic membrane receptors, about 20 % were found in a Triton-
insoluble fraction, which we presumed to represent receptors incorporated in PSDs.
Our results are in good agreement with previous studies (Allison et al., 1998; Blahos
and Wenthold, 1996; Moon et al., 1994; Wenthold et al., 1996)indicating that, while
NMDA receptors are a key component of PSDs and are largely found in Triton-
insoluble fractions, AMPA receptors are relatively easily extracted by Triton X-100,
suggesting that only a small pool of AMPA receptors is present in PSDs.
Interestingly, a larger fraction of GluRl subunits than GIuR2 or GluR3 subunits were
found in Triton-soluble fractions, confirming that there might be homomeric GluRl
receptors that are not incorporated in PSDs (Wenthold et al., 1996).
GluRl and GluR2 subunits of AMPA receptors from both PSD-enriched and
Triton-soluble fractions were clearly truncated following calpain treatment of
synaptic membranes or calcium treatment of frozen-thawed brain slices, as
evidenced by the 45 % decrease in GluRl staining of the 105 kDa band with either
the C-terminal or the N-terminal antibodies and the 25 % decrease in GIuR2 staining
of the 105 kDa band. The fact that similar results were obtained with calpain
treatment o f membranes and calcium treatment of tissue sections argues strongly that
the major effect of calcium treatment of tissue sections on glutamate receptors is due
to endogenous calpain activation. This was further confirmed by showing that 2
different calpain inhibitors, leupeptin and calpain inhibitor III, completely blocked
the effects of calpain treatment of membranes or calcium treatment of tissue sections.
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Moreover, the total immunoreactivity against the N-terminal domain of GluRl or
GluR2 in crude synaptic membranes was not modified following either treatment and
the decreases in native receptor subunits match the increase in lower molecular
weight species. This strongly implies that the lower molecular weight species
appearing as a result of either treatment are true degradation products of the antigens
and do not represent artifactual contaminants.
The truncated species of GluRl subunits migrating at 98 kDa and the 100
kDa species of GluR2 subunits were barely detectable in PSD-enriched fractions
while they were clearly present in Triton-soluble fractions. In fact, the amount of the
98 kDa species of GluRl subunits recovered in the Triton-soluble fraction
corresponded to approximately 41 % of the 105 kDa found in the membrane
fractions before Triton extraction, indicating that most of the truncated species of
GluRl subunits were Triton extractable. As the total amount of intact GluRl
subunits remaining Triton-insoluble after calcium treatment of sections or calpain
treatment of membranes was decreased by about 35 %, it is likely that calpain does
truncate GluRl subunits in PSDs and not solely in Triton-soluble fractions.
Alternatively, calpain activation could result first in the removal of the receptors
from PSDs and then in the truncation of the subunits. Relatively similar results were
obtained with GluR2 and GluR3 subunits, suggesting that both GluR2 and GluR3
subunits are also removed from PSDs following calpain activation.
Thus, a possible function of calpain-mediated truncation of the C-terminal
domains of GluRl-3 subunits is to remove AMPA receptors from the PSDs. This is
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not completely unexpected as AMPA receptors are anchored in PSDs through the
interactions of the C-terminal domains of various subunits with a number of
anchoring proteins. For instance, GluR2 subunits have been shown to be associated
with GRIP and with NSF-like proteins via interactions with their C-terminal domains
(Dong et al., 1997; Nishimune et al., 1998; Song et al., 1998).
Very different results were obtained with NMDA receptors. As previously
reported (Bi et al., 1998), calpain treatment of synaptic membranes or calcium
treatment of frozen-thawed brain sections produced the truncation of NR2B subunits
of NMDA receptors and the formation of a truncated species with an Mr of about
150 kDa still recognized by an antibody raised against amino acid residues 984-1104
of NR2B. However, the truncated species of NR2B remained Triton-insoluble.
Further work is needed to understand the difference in behavior of AMPA and
NMDA receptors as both receptors are anchored in the membranes by interactions of
the C-terminal domains of various subunits to PDZ-domain containing proteins. In
particular, both GluRl and GluR2 (and possibly GluR3) are truncated in their C-
terminal domains by calpain activation. In contrast, NR1 subunits are not truncated
following calcium treatment of frozen-thawed brain slices. This could account for
the maintenance of NR2 subunits in PSDs.
In any event, these results might have important implications for our
understanding of mechanisms of synaptic plasticity. Phosphorylation and
dephosphorylation of AMPA receptors has been proposed to play critical roles in
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LTP/LTD in hippocampus and phosphorylation of AMPA receptors reduced calpain-
mediated truncation of its subunits (Bi et al., 1998). In addition, NMDA receptor
stimulation has been found to produce calpain activation (Vanderklish et al., 1995).
Thus, complex interactions between phosphorylation-dephosphorylation processes
and calpain-mediated truncation of C-terminal domains of AMPA receptor subunits
could lead to the maintenance or the removal of AMPA receptors from postsynaptic
sites.
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CHAPTER 6
GENERAL DISCUSSION
Our results demonstrated that AMPA and NMDA receptor anchoring
proteins, GRIP and PSD-95, are novel calpain substrates, and in both cases similar
proteolytic patterns have been observed for both in vitro calpain digestion of rat
synaptic membranes and in situ activation of endogenous calpain in frozen-thawed
brain sections and in hippocampal slice cultures. Using co-immunoprecipitation
techniques, GRIP degradation by calpain was further shown to cause disruption of
GluR2 and GRIP interactions, a mechanism that is currently believed to participate
in anchoring the AMPA receptors at the synapses. In addition, calpain-mediated
truncation of a-CaMKII was characterized and we found that calpain preferably
degraded the non-phosphorylated form while sparing the phosphorylated form of the
enzyme. Since a-CaMKII phosphorylation of AMPA receptors strongly increased
the channel conductance while the degradation of the non-active dephosphorylated
form of a-CaMKII decreased kinase activity, the consequence of calpain-mediated
proteolysis of a-CaMKII is likely to decrease AMPA receptor function and/or
contribute to the depression of synaptic efficacy. Finally, calpain activation caused
detachment of AMPA receptors from PSDs, but the dissociation of NMDA receptors
was not evident under the same conditions.
One of the difficulty to study biochemical pathways in synaptic modification
is that electrically-induced LTP/LTD is limited to only a few synapses and the
corresponding biochemical changes are therefore undetectable by assays performed
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on the whole section. Therefore, many research groups have decided to chemically
induce synaptic modifications throughout the slice and study the resulting
biochemical changes. Consequently, we tested whether a brief bath application of
NMDA, a manipulation that has been previously used to trigger chemo-LTD and
chemo-LTP (Broutman and Baudry, 2001; Lee et al., 1998), could produce calpain
activation and changes in calpain substrates. We found that a 5 min NMDA
treatment of hippocampal slice cultures indeed activates calpain and produces
degradation of several calpain substrates, including spectrin, GluRl, NR2, GRIP,
PSD-95, and a-CaMKII each with different time courses, which is compatible with
the involvement of calpain in synaptic plasticity.
I. Receptor location
The expression of AMPA and NMDA receptors on cell membranes is a
pivotal determinant for postsynaptic responsiveness to presynaptically released
glutamate and thus regulation of receptor location constitutes a key theme in studies
of synaptic plasticity. The functional receptors do not freely diffuse along the plasma
membrane, instead they are thought to be trapped in the postsynaptic densities (PSD)
by interacting with anchoring molecules such as GRIP and PSD-95. The isolated
NMDA receptor complex, which is a key component of PSD, has a molecular weight
of 2,000 kDa and comprises 77 structural and signaling proteins that form a huge
interconnected network (Husi et al., 2000) and is a very efficient postsynaptic
signaling machinery. Not surprisingly, molecules important in synaptic plasticity
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such as NMDA and AMPA receptors, their anchoring molecules, protein kinases and
proteases, such as calpain, have all been found within the complex.
The half-life of cell surface AMPA receptors, calculated by monitoring
antibody-labeled surface receptors (Man et al., 2000) or derived from
electrophysiological data (Luscher et al., 2000; Luscher et al., 1999), is very short
(between 20 min and one hr) , which makes interfering with receptor trafficking a
feasible mechanism for regulation of synaptic efficacy. Indeed, AMPA receptors are
constantly internalized through clathrin-coated pits (Ehlers, 2000; Lin et al., 2000;
Man et al., 2000). Since AMPA receptors have a metabolic half-life of about 30 to
50 hr (Archibald et al., 1998; Horikawa and Nawa, 1998; Mammen et al., 1 9 9 7 ), it is
postulated that most of the internalized receptors are re-inserted in the membranes. In
fact, the direct visualization of AMPA re-insertion has recently been reported in
hippocampal neurons (Passafaro et al., 2001). Manipulations that halt endocytosis
cause a rapid rundown of AMPA transmission by 30% and block LTD while drugs
interfering with exocytosis block LTP (Luscher et al., 19 9 9 ).
Even though a variety of manipulations, including activity and ligand
binding, could trigger AMPA receptor internalization, down stream events are not
fully clarified (Beattie et al., 2000; Carroll et al., 1999; Ehlers, 2000; Lissin et al.,
1998; Man et al., 2000). Nevertheless, based on current understanding of AMPA
receptor anchoring mechanisms, dissociation of the receptor subunits from anchoring
proteins is believed to be a necessary initial step (Luscher et al., 2000; Passafaro et
al., 2001; Turrigiano, 2000). In cultured Purkinje cells, disruption of GluR2 and
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GRIP interactions leads to un-clustering and internalization of AMPA receptors and
is correlated with cerebellar LTD (Matsuda et al., 2000). We showed that calpain
activation results in disruption of the AMPA receptor anchoring merhankm, and
thus could contribute to synaptic depression. GRIP is more sensitive to calpain than
GluR2, and thus, by digesting rat synaptic membranes with a calpain concentration
that produced no apparent proteolysis of GluR2, we demonstrated that degradation of
GRIP dramatically decreased the GRIP and GluR2 interactions. The dissociation of
GRIP/GIuR2 interaction was also observed following a brief NMDA treatment of
acute hippocampal slices.
Since GluRl and GluR2 subunits of AMPA receptors are substrates of
calpain and the hypothesized anchoring binding sites located in the last 7 amino
acids of the C-terminal tails (Dong et al., 1997), it is likely that degradation of either
GluRl or GluR2 would produce the exact same disruption. To further determine
whether calpain activation might contribute to internalization of AMPA receptors,
we used Triton X-100 solubilization of synaptic membranes to isolate PSDs that
have been shown to be Triton X-100 insoluble. We showed that, in frozen-thawed rat
forebrain sections, calpain activation truncated several glutamate receptor subunits
and the fragmented AMPA receptor subunits that were originally located in PSDs
translocated to Triton-soluble fraction, demonstrating that calpain activation indeed
releases the receptor from the “trapped” synaptic sites. However, those AMPA
receptors internalized as a result of degradation of receptor subunits are not likely to
end up in the re-cycling pool but are more likely to be rapidly and completely
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degraded, since partially degraded AMPA receptors were not observed in the brain
under normal conditions. The degraded Triton X-100 insoluble NMDA receptor
subunits, however, were not released into the Triton X-100 soluble fraction,
suggesting a more tightly tethered anchoring mechanism for NMDA receptors than
for AMPA receptors. The differential effect of calpain activation observed between
AMPA and NMDA receptors is in good agreement with the notion that AMPA
receptors are relatively mobile while NMDA receptors are more static (Allison et al.,
1998; Ehlers, 2000; Lin et al., 2000; Luscher et al., 1999; Roche et al., 2001).
Overall our data strongly argues for a role of calpain in the internalization process of
synaptic AMPA receptors by disrupting their anchoring mechanisms.
Inserting cytosolic receptors into the synapses has long been suggested to
constitute a critical mechanism for LTP expression (Lynch and Baudry, 1984) and
has been recently directly demonstrated (Passafaro et al., 2001; Shi et al., 1999). The
fact that a-CaMKII can drive GluRl subunits into the synapses was visualized by
monitoring GFP-tagged GluRl that were co-transfected with a-CaMKII in
hippocampal slices (Passafaro et al., 2001). This was also demonstrated by
electrophysiological experiments using insertion of recombinant GluRl subunits
detected by the appearance of in-ward rectifying AMPA current (Hayashi et al.,
2000). Interestingly, the “driving force” for receptor insertion appeared to be
unrelated to the phosphorylation of GluRl, since point mutation at Ser 831, an a-
CaMKII phosphorylation site, had no effect, while mutation at a potential PDZ
binding motif site blocked the synaptic delivery, suggesting that exocytosis is
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probably mediated by protein-protein interaction mechanisms (Hayashi et al., 2000;
Passafaro et al., 2001). Even though NSF, a GluR2 binding protein, might also be
involved in the synaptic delivery of AMPA receptors, current data cannot rule out
the possibility that NSF-mediated synaptic AMPA receptor accumulation could be
due to the effects on endocytosis or receptor synaptic anchoring procedure (Luscher
et al., 1999; Nishimune et al., 1998; Song et al., 1998).
Lynch and Baudry proposed in 1984 that calpain might promote synaptic
AMPA receptor delivery as the expression mechanism for LTP. Consistent with such
a hypothesis, LTP induction produced an increase in AMPA receptor binding in rat
hippocampus, which seems to be a result of increased maximal number of binding
sites rather than an increased affinity and this increase was blocked by the calpain
inhibitor leupeptin (Maren et al., 1993). An increase in synaptic AMPA receptors has
also been observed in hippocampal slices following chemically induced LTP in
hippocampal slices, in which the surface receptors are assessed by subcellular
fractionation and chemical cross-linking methods (Broutman and Baudry, 2001).
Since inhibition of either CaMKII or calpain is sufficient to block AMPA receptor
insertion, it seems that the two enzymes are located at distinct points along the same
“insertion cascade”. Nevertheless, despite the established role of calpain in receptor
insertion under certain circumstances, the underlying molecular events remain
unknown. Lynch and Baudry proposed earlier that fragmentation of cytoskeleton
proteins could facilitate receptor insertion by destabilization of the densely packed
postsynaptic structure (Lynch and Baudry, 1984). In our studies, we showed that a
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major postsynaptic density protein, PSD-95, is degraded by calpain and we observed
a more intensive calpain-mediated PSD-95 degradation during postnatal
development, a time period when a drastic synaptic remodeling occurs and PSD-95
rapidly turns over in the synapses as visualized by time-lapse fluorescence imaging
of GFP-PSD95 (Okabe et al., 1999). Therefore we propose that PSD-95
fragmentation could contribute significantly to the structural remodeling of the
PSDs, which may somehow facilitate the receptor insertion process.
II. Protein phosphorylation and long-term synaptic plasticity
Overwhelming evidence accumulated over the past decade has demonstrated
that CaMKII is a key component of the molecular machinery for LTP. Induction of
LTP in hippocampal slices results in NMDA receptor dependent activation of
CaMKII within 1 min, and the constitutive activity is stable for at least one hour
(Fukunaga et al., 1993). Postsynaptic injection of KN-62, a potent CaMKII inhibitor,
or genetic deletion of a-CaMKII blocks LTP (Barria et al., 1997; Malinow et al.,
1989; Silva et al., 1992). On the other hand, postsynaptic infusion of activated
CaMKII potentiates synaptic currents and occludes subsequent induction of LTP
(Lledo et al., 1995). Further study showed that the relevant substrate for such
potentiation is likely GluRl subunit of AMPA receptor, since LTP induction
protocol induced the phosphorylation of GluRl on Ser831 residue and
phosphorylated AMPA receptors exhibit enhanced AMPA current (Barria et al.,
1997). The activation of CaMKII is through autophosphorylation at Thr286 and
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dephosphoryation by PP1 and PP2A inactivates the enzyme (Lisman and
Zhabotinsky, 2001). Calpain-mediated proteolysis decreases the activity of non-
phosphorylated CaMKII (Kwiatkowski and King, 1989). Using in vitro cal pain
digestion of rat synaptic membranes and NMDA stimulation of organotypical
hippocampal culture, we have confirmed that CaMKII is a substrate of calpain. Even
though calpain digestion of CaMKII yields three distinct species, only the 33 kDa
fragment was also oberserved in hippocampal cultures treated with NMDA and
blocked by cell permeable calpain inhibitor III, indicating that this site is the
preferable cutting site in vivo. Furthermore, we also demonstrated by using
immunoprecipitation techniques, that calpain preferably degrades inactive CaMKII
while sparing the active autophosphorylated kinase. Thus the functional relevance of
calpain-mediated proteolysis of CaMKII would be that to bias synaptic strength
toward depression when the majority of the kinases exist in inactive form following
LTD induction protocol.
The contribution of calcium sensitive PKC to LTP has also been indicated by
a variety of experiments. PKC can phosphorylate Ser831 in GluRl (Roche et al..
1996) and PKC activators greatly increase mEPSC frequency as well as mEPSC
amplitude in neuronal cultures (Carroll et al., 1998). Furthermore, induction of LTP
generates prolonged activation of PKC (Klann et al., 1993; Sacktor et al., 1993) and
blockade of PKC activity blocks LTP (Abeliovich et al., 1993; Malinow et al., 1989).
Calpain increases PKC activity by limited proteolysis (Kishimoto et al., 1989;
Touyarot et al., 2000), a mechanism that could contribute synaptic potentiation.
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Though PKA phosphorylation of GluRl at Ser845 also increases AMPA receptor
currents (Malinow et al., 1989; Roche et al., 1996), it has been proposed that the
GluRl PKA phosphorylation site is saturated under basal condition, since a
membrane permeable analog of cAMP did not significantly increase the
phosphorylation of Ser84S in hippocampal slices, and PKA probably functions to
prevent dephosphorylation of Ser845, a key molecular event underlying LTD
(Kameyama et al., 1998; Lee et al., 1998).
Protein phosphatases, on the other hand, have been suggested to paly
important role in LTD induction. Low frequency stimulation might trigger
dephosphorylation via activation of PP1, PP2B and PP2A, and protein phosphatase
inhibitors block LTD (Bear and Malenka, 1994; Kirkwood and Bear, 1994; Lisman,
1989; Mulkey et al., 1994; O'Dell and Kandel, 1994). Kameyama et al. (1998) have
shown that dephosphorylation of GluRl at Ser845, a PKA site, is an important
mechanism that underlies homosynaptic LTD. Dephosphorylation of Ser845 of
GluRl is mediated by PPL While PP1 is not directly activated by calcium influx
through NMDA receptors, experimental evidence suggested that calcium might first
activate PP2B, which activates PP1 by dephosphoryiating PP1 inhibitor (Mulkey et
al., 1994).
III. Hypothetical mechanisms of synaptic plasticity
Our results obtained by studying calpain-mediated digestion of critical
postsynaptic molecules, provided biochemical evidence for calpain's involvement in
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synaptic modifications, including both LTP and LTD. As in the study of many
biomolecules, it is of pivotal importance to distinguish the conditions that lead to
bidirectional modifications. Strong evidence has shown that LTP induction produces
calcium spikes in dendritic spine heads, which may later spread through out the cell
by calcium-induced calcium release mechanisms (Regehr and Tank, 1990; Sabatini
et al., 2001). A recent study further suggested that an intracellular calcium
concentration of 180-500 nM leads to LTD while calcium concentrations above 600
nM result in LTP. There is evidence that calpain is likely to be activated by calcium
concentrations lower than 0.5 pM (Inomata et al., 1989), which is consistent with our
proposed role of calpain in both LTP and LTD.
In our model, inspired by previously published as well as newly obtained
data, LTP and LTD are opposite processes regulated by several common calcium-
dependent processes. At low calcium concentrations, following weak and prolonged
NMDA receptor stimulation, various protein phosphatases are activated and AMPA
receptors undergo dephosphorylation resulting in decreased channel activity. Calpain
activation preferably truncates the non-active dephosphorylated form of a-CaMKil
and decreases kinase activity, further shifting the system to a de-phosphorylated
state. In addition, activated calpain degrades the AMPA receptor anchoring protein
GRIP, thereby facilitating the internalization of the receptors. Furthermore, since
dephosphorylation increases AMPA receptor’s susceptibility to calpain-mediated
proteolysis, AMPA receptors themselves are degraded and targeted for further
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destruction within the cell. Thus, synaptic transmission is reduced as a combined
result of decreased single channel activity and decreased surface receptor expression.
At higher calcium concentrations following strong NMDA receptor
stimulation, calpain as well as various kinases including a-CaMKII and PKC are
activated resulting in phosphorylation of AMPA receptors and increased channel
activity. Since a large fraction of a-CaMKII is activated, cal pain-mediated
degradation of the non-active dephosphorylated form of a-CaMKII has only a minor
effect on overall kinase activity while degradation of PKC further increases the
catalytic activity of the kinase. PKC and a-CaMKII phosphorylate AMPA receptors
at distinct serine sites and cause enhancement of AMPA receptor currents. In
addition, AMPA receptors are translocated into the spine heads and PSDs due to a-
CaMKII regulated GluRl targeting mechanisms, calpain-mediated proteolysis of
spectrin and PSD-95 and probably other unidentified calcium-dependent processes.
The degradation of AMPA receptor subunits and GRIP, which happens during LTD,
are blocked probably by protein phosphorylation (Bi et al., 1998). The trafficking
events lead to an increase in the size of the PSDs and along with calpain-mediated
remodeling of the spine structure, eventually, result in the production of perforated
synapses and new spine formation.
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Asset Metadata

Creator Lu, Xiaoying (author)

Core Title Biochemical evidence for calpain's involvement in long term synaptic plasticity

Contributor Digitized by ProQuest (provenance)

School Graduate School

Degree Doctor of Philosophy

Degree Program Biology

Degree Conferral Date 2001-12

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, neuroscience,OAI-PMH Harvest

Language English

Advisor Baudry, Michel (committee chair), Maarek, Jean-Michel (committee member), Thompson, Richard (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-198116

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Rights Lu, Xiaoying

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