A. Marcilhac, UMR5539, EPHE-CNRS-UM2, cc107, Université de Montpellier II, place E. Bataillon, 34095 Montpellier cedex 5, France Fax: +33 0467144727 Tel: +33 0467144775 E-mail: email@example.com
Apoptotic neuronal cell death is the cardinal feature of aging and neurodegenerative 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, nuclear 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 disease in adult brain. Alzheimer's disease is characterized by extracellular plaques of amyloid–β 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 signaling pathways leading to chronic neurodegenerative disorders such as Alzheimer'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.
In recent years, enormous efforts have been made to clarify the apoptotic pathways involved in neuronal cell death. Indeed, programmed cell death, or apoptosis, is beneficial during embryonic development and adult life, but its dysregulation accompanies the pathogenesis 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 pathologies such as Alzheimer's disease (AD).
Calpain and the regulation of apoptosis
Proteolytic enzymes of the caspase family play a central role in initiating and sustaining the biochemical events that result in apoptotic cell death. In some 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 triggered by cytochrome c release from mitochondria . This process is critically regulated by Bcl-2 family proteins. 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 . Several members are processed by calpains . 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 . In this context, calpain cleaves Bax into a pro-apoptotic 18-kDa fragment which promotes cytochrome c release and apoptosis .
Moreover, cleavage of Bid (another pro-apoptotic Bcl-2 family member) by calpain has been implicated in mitochondrial permeabilization and cell death following ischemia/reperfusion in the heart . 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.  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. Calpain 1 then cleaves AIF, releasing truncated AIF, which could regulate apoptosis. So AIF is a novel calpain substrate that has been implicated in neuronal cell death (Fig. 1).
Cross-talk between calpains and caspases
The striking similarity between the substrates for caspases and calpains raises the possibility that both protease 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 . Moreover, calpains are able to be activated via caspase-mediated cleavage of calpastatin during initiation of apoptotic execution , and the disturbance of intracellular calcium storage associated with ischemic injury may induce apoptosis through calpain-mediated caspase 12 activation and Bcl-xL inactivation  (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. Yamashima  added to this dual cross-talk another effective candidate, cathepsins, which are implicated in neuronal cell death. He suggested a possible cascade of events involving three protease systems: calpain-induced cathepsin 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 implicated 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 proteasome in regulating the stability of p53 family members. Several recent reports have highlighted a possible role for calpains in the cleavage of p53. In particular, Kubbutat & Vousden  have shown that a preferential site for calpain cleavage exists within the N-terminus of p53. Calpain inhibition leads to p53 stabilization 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 . More recently, Munarriz et al.  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 degradation 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  have demonstrated in dissociated cultures of cerebellar granule cells that calpain-mediated Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV) proteolysis is an autoregulatory feedback response to sustained activation of a Ca2+/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-κB
The excitatory neurotransmitter glutamate is a key player in neuronal plasticity, development and neurodegeneration. Stimulation of glutamate receptors [N-methyl-d-aspartate receptors (NMDARs)] leads to Ca2+-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]. However, the literature is contradictory on this subject. Indeed, recent results show that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke . Scholzke et al.  have shown that glutamate activates NF-κB through calpain in neurons. Moreover, after glutamate exposure, the specific calpain inhibitor, calpeptin, prevents IκBα degradation and therefore NF-κB activation. However, the role of ΙκΒα degradation by calpain in glutamate-induced cell death is difficult to predict as both proapoptotic and antiapoptotic effects have been attributed to NF-κB [22,23].
Astrocytes contribute to the neuroprotection and survival of neurons: any astrocytic dysfunction seriously 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 neurodegenerative diseases. Calpain inhibitors, N-acetyl-Leu-Leu-norleucinal (calpain inhibitor 1) and N-acetyl-Leu-Leu-methioninal (calpain inhibitor 2), decrease Ca2+ reperfusion-induced H2O2 production and apoptotic cell death in cultured rat astrocytes. These findings suggest that calpain is involved in Ca2+-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-κB acts as a death-promoting factor in apoptosis of cultured astrocytes .
Calpain and Alzheimer's neurodegenerative disease
Neuronal cell death in acute and neurodegenerative disorders occurs by a variety of biochemical and morphological alterations .
AD leads to a progressive deterioration of cognitive function with loss of memory. Neuronal injury presents in the region of the brain containing the hippocampus and the cortex. AD is characterized by two pathological hallmarks consisting of extracellular plaques of amyloid–β peptide aggregates  and intracellular neurofibrillary tangles composed of hyperphosphorylated microtubular protein tau . The β-amyloid deposition that constitutes the plaques is composed of a 39–42 amino-acid peptide (Aβ), which is the proteolytic product of the amyloid precursor protein (APP) by β/γ secretases. Calpains modulate processes that govern the function and metabolism of key proteins in the pathogenesis of AD including tau and APP .
Calpains in the brains of patients with AD
Calpains are known to regulate the activities of various enzymes, including several protein kinases and phosphatases that modify the cytoskeleton in addition to direct cleavage of cytoskeletal proteins (Lebart & Benyamin, [28a]). Alterations in calcium homeostasis in AD pathogenesis  associated with calpain overactivation  have been proposed to play an important 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 . Moreover, calpain 1 is activated, which in turn cleaves and activates calcineurin in the AD brain and this phenomenon correlates with the number of neurofibrillary tangles . Calpastatin, a specific calpain inhibitor, is altered in AD. Indeed, it decreases as the number of plaques and tangles increase in AD brains . Moreover, calpain inhibitors were able to restore normal cognition and synaptic transmission in a transgenic model of AD .
Mutations in PS1 (presenilin) that cause early onset familial AD can sensitize cells to DNA-damage-induced death and increase the production of Aβ. In hippocampal neurons expressing mutant PS1, the hypersensitivity to DNA damage correlates with increased intracellular calcium concentrations, up-regulation of calpain 1, and induction of p53, leading to neuronal apoptosis . Moreover, calpains 1 and 2 have been shown to regulate PS1 activity by direct cleavage of this protein .
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 Aβ region by α-secretase to release a soluble N-terminal fragment (denoted sAPP). A number of studies have shown that enhancing α-secretase activity can reduce Aβ production. APP-α processing is sensitive to a variety of regulatory agents such as phorbol esters, glutamate, calcium ionophore. For example, some authors demonstrated that the stimulated sAPP release by phorbol esters involves calpain activation in the cells, suggesting that calpain, particularly calpain 1, is a potential candidate for an α-secretase substrate in the regulated APP α-processing [37,38]. These results support the hypothesis of a decrease in calpain activity during aging leading to selective accumulation of its substrates (e.g. APP) and an increase in Aβ42 production in cultured cells . Consistent with this, it has been reported that infusion of calpain inhibitors into the rat brain results in accumulation of Aβ or Aβ-containing fragments . 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 , this model remains controversial and awaits additional experimental tests.
Indeed, although there is evidence in favor of calpains acting as α-secretases, such as (a) the α-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 reagents that enhance α-secretase activity are well-known calpain activators, and (d) considering its vulnerability to oxidative stress, α-secretase may belong to the family 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 outside 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 proteins, including growth factor receptors, integrin and glutamate receptors, leading to the release of their extracellular domains .
During the extended time course of AD in humans, the calpain system plays multiple roles. Indeed, if reduced calpain activity were to promote AD pathology by increasing Aβ42 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. Consequently, the p25/cdk5 kinase hyperphosphorylates tau, disrupts the cytoskeleton, and promotes apoptosis of primary neurons. Application of the amyloid–β-peptide(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]. Moreover, a recent study showed that preaggregated Aβ induced the generation of a neurotoxic 17-kDa tau fragment, which is prevented by a calpain inhibitor in cultured hippocampal neurons . 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 microtubules could in turn reduce their stability and promote a more rapid depolymerization cycle and therefore the disruption of the microtubule network . Veeranna et al.  demonstrated that, under conditions of calcium injury in neurons, calpains are upstream activators 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 perturbation of the microtubule network in AD and ultimately lead to neuronal degeneration without accumulation of amyloid deposits. A recent study  showed that soluble Aβ oligomers induce time-dependent degradation of MAP1A, MAP1B and MAP2. Calpain activation is sufficient on its own to proteolyse MAP2a,b,c isoforms, whereas MAP1A and MAP1B sequential proteolysis 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.
In summary, the ubiquitous expression of calpains in distinct subcellular compartments at different maturational stages and the diversity of substrates indicate that they are multifunctional effectors of myriad intracellular processes.
Moreover, progressive cell loss in specific neuronal populations is a pathological hallmark of neurodegenerative diseases, and calpain is a Ca2+-activated proteolytic enzyme involved in neurodegeneration in a variety of injuries and diseases of the central nervous system.
Thus, identification of mechanisms that involve calpains and either promote or prevent cell apoptosis provides new approaches for treating diverse neurodegenerative disorders including AD.