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,

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)

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

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

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

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

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,

degenerative disorders including AD.
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