Báo cáo khoa học: Implication of calpain in neuronal apoptosis A possible regulation of Alzheimer’s disease doc

MINIREVIEW
Implication of calpain in neuronal apoptosis
A possible regulation of Alzheimer’s disease
F. Raynaud and A. Marcilhac
UMR5539, EPHE-CNRS-UM2, cc107, Universite
´
de Montpellier II, France
Introduction
In recent years, enormous efforts have been made to
clarify the apoptotic pathways involved in neuronal
cell death. Indeed, programmed cell death, or apopto-
sis, is beneficial during embryonic development and
adult life, but its dysregulation accompanies the patho-
genesis of many diseases. Calpain appears to play an
important role in apoptosis in many cases, as discerned
by its up-regulation and the blockade of apoptosis by
calpain inhibitors.
In this review, we summarize the most recent work
on calpain-dependent apoptotic neuronal cell death
and the regulation of intracellular pathways involving
calpain which may lead to neurodegenerative patholo-
gies such as Alzheimer’s disease (AD).
Calpain and the regulation of apoptosis
Proteolytic enzymes of the caspase family play a cen-
tral role in initiating and sustaining the biochemical
events that result in apoptotic cell death. In some
Keywords
Alzheimer’s disease; apoptosis; calpain;
neurodegenerative disease; neuron
Correspondence
A. Marcilhac, UMR5539, EPHE-CNRS-UM2,

cc107, Universite
´
de Montpellier II, place E.
Bataillon, 34095 Montpellier cedex 5, France
Fax: +33 0467144727
Tel: +33 0467144775
E-mail:
(Received 23 March 2006, accepted 31 May
2006)
doi:10.1111/j.1742-4658.2006.05352.x
Apoptotic neuronal cell death is the cardinal feature of aging and neurode-
generative diseases, but its mechanisms remain obscure. Caspases, members
of the cysteine protease family, are known to be critical effectors in central
nervous system cellular apoptosis. More recently, the calcium-dependent
proteases, calpains, have been implicated in cellular apoptotic processes.
Indeed, several members of the Bcl-2 family of cell death regulators, nuc-
lear transcription factors (p53) and caspases themselves are processed by
calpains. Progressive regional loss of neurons underlies the irreversible
pathogenesis of various neurodegenerative diseases such as Alzheimer’s dis-
ease in adult brain. Alzheimer’s disease is characterized by extracellular
plaques of amyloid–b peptide aggregates and intracellular neurofibrillary
tangles composed of hyperphosphorylated tau leading to apoptotic cell
death. In this review, we summarize the arguments showing that calpains
modulate processes that govern the function and metabolism of these two
key proteins in the pathogenesis of Alzheimer’s disease. To conclude, this
article reviews our understanding of calpain-dependent apoptotic neuronal
cell death and the ability of these proteases to regulate intracellular signa-
ling pathways leading to chronic neurodegenerative disorders such as Alz-
heimer’s disease. Further research on these calpain-dependent mechanisms
which promote or prevent cell apoptosis should help us to develop new

approaches for preventing and treating neurodegenerative disorders.
Abbreviations
AD, Alzheimer’s disease; AIF, apoptosis-inducing factor; APP, amyloid precursor protein; CaMKIV, calmodulin-dependent protein kinase type
IV; cdk5, cyclin-dependent kinase 5; MAP, microtubule-associated protein; NMDAR, N-methyl-
D-aspartate receptor.
FEBS Journal 273 (2006) 3437–3443 ª 2006 The Authors Journal compilation ª 2006 FEBS 3437
forms of apoptosis, the extrinsic apoptotic pathway is
initiated by activation of caspase 8 after death receptor
ligation. In other forms, activation of the intrinsic
apoptotic pathway is initiated by caspase 9 and is trig-
gered by cytochrome c release from mitochondria [1].
This process is critically regulated by Bcl-2 family pro-
teins. These pathways converge to activation of the
executioner caspases (e.g. caspase 3).
Calpains and the Bcl-2 family
Members of the Bcl-2 family of proteins either promote
or repress programmed cell death [2]. Several members
are processed by calpains [3]. Using the model of
trophic factor deprivation in sympathetic neurons, it
has been shown that Bax translocation from the cytosol
to the mitochondria is a critical event in neuronal
apoptosis [4]. In this context, calpain cleaves Bax into a
pro-apoptotic 18-kDa fragment which promotes cyto-
chrome c release and apoptosis [5].
Moreover, cleavage of Bid (another pro-apoptotic
Bcl-2 family member) by calpain has been implicated
in mitochondrial permeabilization and cell death fol-
lowing ischemia ⁄ reperfusion in the heart [6]. Indeed,
truncated Bid induced cytochrome c release from brain
mitochondria and apoptosis-inducing factor (AIF)

release only in the presence of active calpain. AIF has
been shown to translocate from mitochondria to the
cytosol as well as the nucleus when apoptosis is
induced. In a recent study, Polster et al. [7] suggested a
novel mechanism of AIF release that is mediated by
direct proteolysis of the protein by calpain, removing
its association with the mitochondrial inner membrane.
They proposed an experimental scheme in which the
calpain 1 cleaves Bid into a more active form in order
to permeabilize mitochondrial outer membrane and
allow calpain access to the intermembrane space. Cal-
pain 1 then cleaves AIF, releasing truncated AIF,
which could regulate apoptosis. So AIF is a novel cal-
pain substrate that has been implicated in neuronal cell
death [7] (Fig. 1).
Cross-talk between calpains and caspases
The striking similarity between the substrates for casp-
ases and calpains raises the possibility that both prote-
ase families contribute to structural dysregulation and
functional loss of nerve cells under neurodegenerative
conditions. Cross-talk between calpains and caspases
has been reported during apoptosis of neuronal cells
induced by a prion protein fragment [8]. Moreover,
calpains are able to be activated via caspase-mediated
cleavage of calpastatin during initiation of apoptotic
execution [9], and the disturbance of intracellular cal-
cium storage associated with ischemic injury may
induce apoptosis through calpain-mediated caspase 12
activation and Bcl-xL inactivation [10] (Fig. 1).
Although calpains may enhance caspase activity, they

can also function to block the activation of caspases.
For example, calpains can cleave caspase 9 rendering it
incapable of activating caspase 3 and preventing the
subsequent release of cytochrome c [11]. Yamashima
[12] added to this dual cross-talk another effective can-
didate, cathepsins, which are implicated in neuronal
cell death. He suggested a possible cascade of events
involving three protease systems: calpain-induced cath-
epsin release, cathepsin-mediated caspase activation
and caspase-mediated calpastatin degradation leading
to enhancement of calpain activity.
Calpains and transcription factor regulation
DNA damage is an initiator of neuronal death impli-
cated in neuropathological conditions such as stroke.
Previous evidence has shown that apoptotic death of
embryonic cortical neurons treated with the DNA-
damaging agent camptothecin is dependent on the
tumor suppressor p53, an upstream death mediator,
and more distal death effectors such as caspases.
Calpains can act as an alternative system to the pro-
teasome in regulating the stability of p53 family mem-
bers. Several recent reports have highlighted a possible
role for calpains in the cleavage of p53. In particular,
Kubbutat & Vousden [13] have shown that a pre-
ferential site for calpain cleavage exists within the
N-terminus of p53. Calpain inhibition leads to p53 sta-
bilization and to altered cell cycle progression. Both
calpain 1 and 2 can cleave p53 with a different degree
of susceptibility to cleavage in various p53 mutants.
The cleavage of p53 by calpains can occur under

pathological conditions and contributes to the DNA
damage response [14]. More recently, Munarriz et al.
[15] have shown that p73, another component of the
DNA damage response, which belongs to the family of
transcription factors that includes p53 and p63, is also
a substrate for calpains. In this case, calpain regulation
can control the steady-state protein concentration of
different isoforms of p73, and calpain-mediated degra-
dation of p73 may have a regulatory, physiological
function, in addition to a potential role in cell death.
So, several nuclear transcription factors are calpain
substrates leading to the suggestion that calpains can
regulate transcriptional events.
Moreover, translocation of calpain to the nucleus may
play a role in apoptosis. For example, Tremper-Wells
& Vallano [16] have demonstrated in dissociated
Calpain and neuronal apoptosis F. Raynaud and A. Marcilhac
3438 FEBS Journal 273 (2006) 3437–3443 ª 2006 The Authors Journal compilation ª 2006 FEBS
cultures of cerebellar granule cells that calpain-
mediated Ca
2+
⁄ calmodulin-dependent protein kinase
type IV (CaMKIV) proteolysis is an autoregulatory
feedback response to sustained activation of a
Ca
2+
⁄ CaMKIV signaling pathway. (CaMKIV mediates
phosphorylation of different transcription factors such
as CREB and regulates expression of prosurvival ⁄
antiapoptosis genes.)

Calpains, glutamate receptor and NF-jB
The excitatory neurotransmitter glutamate is a key
player in neuronal plasticity, development and neuro-
degeneration. Stimulation of glutamate receptors
[N-methyl-d-aspartate receptors (NMDARs)] leads
to Ca
2+
-mediated apoptosis, a response that may
contribute to excitatory neuronal toxicity. Calpain
activation in neurons has been predominantly linked
to cell death during ischemia and stroke [17–19]. How-
ever, the literature is contradictory on this subject.
Indeed, recent results show that prolonged activation
of NMDARs in neurons activates calpain, and activa-
ted calpain in turn down-regulates the function of
NMDARs, which provides a neuroprotective mechan-
ism against NMDAR overstimulation accompanying
ischemia and stroke [20]. Scholzke et al. [21] have
shown that glutamate activates NF-jB through calpain
in neurons. Moreover, after glutamate exposure, the
specific calpain inhibitor, calpeptin, prevents IjBa
degradation and therefore NF-jB activation.
However, the role of IjBa degradation by calpain in
glutamate-induced cell death is difficult to predict as
both proapoptotic and antiapoptotic effects have been
attributed to NF-jB [22,23].
Fig. 1. Scheme illustrating the regulation of
Bcl-2 family proteins, cytochrome c release
and apoptosis by calpains. (1) Cleavage of
Bax by calpain, formation of truncated Bax

(tBax) and stimulation of cytochrome c
release; (2) cleavage of Bid by calpain and
formation of truncated Bid (tBid) leading to
(2a) direct release of cytochrome c and ⁄ or
(2b) permeabilization of mitochondrial outer
membrane, translocation of calpain in the
intermembrane space and AIF cleavage
(tAIF) and release; (3) calpain-mediated
Bcl-xL inactivation and cytochrome c
release; (4) calpain-mediated p53 activation:
p53 induces Bcl-xL inactivation, Bax
stimulation and cytochrome c release.
F. Raynaud and A. Marcilhac Calpain and neuronal apoptosis
FEBS Journal 273 (2006) 3437–3443 ª 2006 The Authors Journal compilation ª 2006 FEBS 3439
Astrocytes contribute to the neuroprotection and
survival of neurons: any astrocytic dysfunction seri-
ously affects neuronal viability. The study of astrocytes
is particularly important, considering the coexistence
of the apoptotic death of neurons and astrocytes in
damaged brains suffering from ischemia and neurode-
generative diseases. Calpain inhibitors, N-acetyl-Leu-
Leu-norleucinal (calpain inhibitor 1) and N-acetyl-Leu-
Leu-methioninal (calpain inhibitor 2), decrease Ca
2+
reperfusion-induced H
2
O
2
production and apoptotic
cell death in cultured rat astrocytes. These findings

suggest that calpain is involved in Ca
2+
-mediated
apoptosis in astrocytes. In this model of astrocyte
apoptosis, translocation of the NF-kB p65 subunit to
the nucleus is observed. These findings indicate that, in
this case, NF-jB acts as a death-promoting factor in
apoptosis of cultured astrocytes [24].
Calpain and Alzheimer’s neuro-
degenerative disease
Neuronal cell death in acute and neurodegenerative
disorders occurs by a variety of biochemical and mor-
phological alterations [25].
AD leads to a progressive deterioration of cognitive
function with loss of memory. Neuronal injury pre-
sents in the region of the brain containing the hippo-
campus and the cortex. AD is characterized by two
pathological hallmarks consisting of extracellular
plaques of amyloid–b peptide aggregates [26] and
intracellular neurofibrillary tangles composed of
hyperphosphorylated microtubular protein tau [27].
The b-amyloid deposition that constitutes the plaques
is composed of a 39–42 amino-acid peptide (Ab),
which is the proteolytic product of the amyloid precur-
sor protein (APP) by b ⁄ c secretases. Calpains modu-
late processes that govern the function and metabolism
of key proteins in the pathogenesis of AD including
tau and APP [28].
Calpains in the brains of patients with AD
Calpains are known to regulate the activities of various

enzymes, including several protein kinases and phos-
phatases that modify the cytoskeleton in addition to
direct cleavage of cytoskeletal proteins (Lebart &
Benyamin, [28a]). Alterations in calcium homeostasis
in AD pathogenesis [29] associated with calpain over-
activation [30] have been proposed to play an import-
ant role in the development of cytoskeletal pathology
and neurodegeneration. Calpain activation has been
found in clinical brain specimens of AD. For example,
calpain 2 was demonstrated to be present in  75% of
neurofibrillary tangles [31]. Moreover, calpain 1 is acti-
vated, which in turn cleaves and activates calcineurin
in the AD brain and this phenomenon correlates with
the number of neurofibrillary tangles [32]. Calpastatin,
a specific calpain inhibitor, is altered in AD. Indeed, it
decreases as the number of plaques and tangles
increase in AD brains [33]. Moreover, calpain inhibi-
tors were able to restore normal cognition and synap-
tic transmission in a transgenic model of AD [34].
Mutations in PS1 (presenilin) that cause early onset
familial AD can sensitize cells to DNA-damage-
induced death and increase the production of Ab.In
hippocampal neurons expressing mutant PS1, the
hypersensitivity to DNA damage correlates with
increased intracellular calcium concentrations, up-regu-
lation of calpain 1, and induction of p53, leading to
neuronal apoptosis [35]. Moreover, calpains 1 and 2
have been shown to regulate PS1 activity by direct
cleavage of this protein [36].
Calpains and amyloid formations in AD

Different data support the hypothesis that calpains are
involved in the alpha cleavage of APP in vivo and thus
are able to stimulate the nonamyloidogenic pathway,
leading to a decrease in Ab42 release.
Indeed, APP is cleaved within its Ab region by
a-secretase to release a soluble N-terminal fragment
(denoted sAPP). A number of studies have shown that
enhancing a-secretase activity can reduce Ab produc-
tion. APP-a processing is sensitive to a variety of
regulatory agents such as phorbol esters, glutamate,
calcium ionophore. For example, some authors demon-
strated that the stimulated sAPP release by phorbol
esters involves calpain activation in the cells, suggesting
that calpain, particularly calpain 1, is a potential candi-
date for an a-secretase substrate in the regulated APP
a-processing [37,38]. These results support the hypothe-
sis of a decrease in calpain activity during aging leading
to selective accumulation of its substrates (e.g. APP)
and an increase in Ab42 production in cultured cells
[39]. Consistent with this, it has been reported that
infusion of calpain inhibitors into the rat brain results
in accumulation of Ab or Ab-containing fragments
[40]. This effect is not unexpected because calpain is
essential for life, and severe inhibition of its activity
could be harmful to cells. Although this novel model
for plaque formation is in agreement with both the
property of calpain that consists of specific and limited
cleavage of target proteins, and data that show that
other calpain susbtrates such as spectrin (fragment) are
also deposited during aging [41], this model remains

controversial and awaits additional experimental tests.
Calpain and neuronal apoptosis F. Raynaud and A. Marcilhac
3440 FEBS Journal 273 (2006) 3437–3443 ª 2006 The Authors Journal compilation ª 2006 FEBS
Indeed, although there is evidence in favor of cal-
pains acting as a-secretases, such as (a) the a-secretase
cleavage site is identical with a cleavage site of calpain
in protein kinase C, (b) calpains are colocalized with
APP in situ in different structures including neurons,
astroglia, senile plaques, and synapses, (c) some rea-
gents that enhance a-secretase activity are well-known
calpain activators, and (d) considering its vulnerability
to oxidative stress, a-secretase may belong to the fam-
ily of cysteine proteases, a serious problem with this is
that calpains are intracellular and may be not able to
reach the cleavage site of APP, which is located out-
side of the membrane. However, in support of calpains
acting as secretases is the fact that these proteases are
involved in cleavage of other membrane surface pro-
teins, including growth factor receptors, integrin and
glutamate receptors, leading to the release of their
extracellular domains [42].
During the extended time course of AD in humans,
the calpain system plays multiple roles. Indeed, if
reduced calpain activity were to promote AD pathol-
ogy by increasing Ab42 generation, this would contrast
with increased calpain activity promoting pathological
changes in tau.
Calpains and tau regulation in AD
Based on a growing literature, cyclin-dependent kinase
5 (cdk5), which promote phosphorylation of tau, has

been implicated in the pathological processes that
contribute to neurodegeneration in AD. p35 is a
neuron-specific activator of cdk5, and conversion of
p35 into p25 by calpain-dependent proteolysis causes
prolonged activation and mislocalization of cdk5. Con-
sequently, the p25 ⁄ cdk5 kinase hyperphosphorylates
tau, disrupts the cytoskeleton, and promotes apoptosis
of primary neurons. Application of the amyloid–b-pep-
tide(1–42) induces the conversion of p35 into p25 in
neurons, and inhibition of cdk5 or calpain activity
reduces cell death in these conditions [43,44]. More-
over, a recent study showed that preaggregated Ab
induced the generation of a neurotoxic 17-kDa tau
fragment, which is prevented by a calpain inhibitor in
cultured hippocampal neurons [45]. This proteolytic
cleavage may lead to neurite degeneration by reducing
the pool of full-length tau available for binding to
microtubules. The decrease in tau bound to microtu-
bules could in turn reduce their stability and promote
a more rapid depolymerization cycle and therefore the
disruption of the microtubule network [45]. Veeranna
et al. [46] demonstrated that, under conditions of cal-
cium injury in neurons, calpains are upstream activa-
tors of Erk1,2 signaling and probably mediate, in part,
the hyperphosphorylation of neurofilaments and tau
seen at early stages of AD.
Besides the alteration of the structure and properties
of tau [the most studied member of the microtubule-
associated protein (MAP) family], modifications of
other members of this family (such as MAP1A,

MAP1B and MAP2) may contribute to the perturba-
tion of the microtubule network in AD and ultimately
lead to neuronal degeneration without accumulation of
amyloid deposits. A recent study [47] showed that sol-
uble Ab oligomers induce time-dependent degradation
of MAP1A, MAP1B and MAP2. Calpain activation is
sufficient on its own to proteolyse MAP2a,b,c iso-
forms, whereas MAP1A and MAP1B sequential pro-
teolysis results from caspase 3 and calpain activation.
This work confirms the cross-talk between caspase and
calpain and identifies a novel mechanism associated
with the proteolysis of several MAPs and leading to
neuronal apoptosis in AD.
Conclusion
In summary, the ubiquitous expression of calpains in
distinct subcellular compartments at different matura-
tional stages and the diversity of substrates indicate
that they are multifunctional effectors of myriad intra-
cellular processes.
Moreover, progressive cell loss in specific neuronal
populations is a pathological hallmark of neurodegen-
erative diseases, and calpain is a Ca
2+
-activated pro-
teolytic enzyme involved in neurodegeneration in a
variety of injuries and diseases of the central nervous
system.
Thus, identification of mechanisms that involve cal-
pains and either promote or prevent cell apoptosis
provides new approaches for treating diverse neuro-

degenerative disorders including AD.
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