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Abstract

The calpain family of proteases is causally linked to postischemic neurodegeneration. However, the precise mechanisms by which calpains contribute to postischemic neuronal death have not been fully elucidated. This review outlines the key features of the calpain system, and the evidence for its causal role in postischemic neuronal pathology. Furthermore, the consequences of specific calpain substrate cleavage at various subcellular locations are explored. Calpain substrates within synapses, plasma membrane, endoplasmic reticulum, lysosomes, mitochondria, and the nucleus, as well as the overall effect of postischemic calpain activity on calcium regulation and cell death signaling are considered. Finally, potential pathways for calpain-mediated neurodegeneration are outlined in an effort to guide future studies aimed at understanding the downstream pathology of postischemic calpain activity and identifying optimal therapeutic strategies.

Keywords

calpain ischemia neurodegeneration calcium brain neuron

Introduction

Since their discovery over 40 years ago, members of the calpain family of calcium-regulated cysteine proteases have been linked to a wide range of pathologic conditions including postischemic neuronal cell death. However, much remains unknown about the precise mechanism of calpain-mediated neuronal injury and the specific calpain substrates that play essential roles in postischemic neuronal death. This review presents an organized approach to explaining the potential roles of different calpain isoforms and substrates at key subcellular locations during postischemic neurodegeneration.

Overview of the Calpain—Calpastatin System

The calpain family of proteases was first established by the discovery of what is now known as μ-calpain in 1964 (Guroff, 1964). The cDNA for this protein was subsequently isolated in the mid-1980s (Ohno et al, 1984), and based on sequence homology, there are now 15 identified calpain family members within the human genome. The majority of these have been identified only as mRNA, and several are thought to be tissue specific (Goll et al, 2003). Only calpain catalytic subunit isoforms 1, 2, 3, 5, 10, and two small regulatory subunit isoforms have been identified in the brain. In addition to the various protease isoforms, the calpain system includes a single endogenous inhibitor, calpastatin. The various calpain isoforms are thought to have a common substrate profile. Despite multiple attempts at substrate sequence analysis (Tompa et al, 2004; Cuerrier et al, 2005), no definitive method exists to predict whether a given compound is a calpain substrate, or, if it is a substrate, to identify the cleavage site. Furthermore, there is evidence that cleavage can be regulated by modulation of secondary recognition sequences adjacent to the actual cleavage site (Wang and Yuen, 1999).

The most abundant and best-characterized brain calpains are the two major isoforms μ- and m-calpain. Both these proteases exist as heterodimers, each with a distinct 80-kDa catalytic subunit encoded by the CAPN1 gene on chromosome 11 or the CAPN2 gene on chromosome 1, respectively. Henceforth, the individual catalytic subunits will be referred to as calpains 1 and 2, whereas the heterodimeric peptides they form with their common regulatory subunit will be termed μ- and m-calpain, respectively. The 80-kDa calpain 1 and 2 subunits share approximately 50% sequence homology, and amino-acid sequence analysis of both predicts four functional domains (Ohno et al, 1984). The domain maps of calpains 1 and 2 are shown in Figures 1A and 1B, respectively. Calpain 2, the first to be crystallized (Hosfield et al, 1999), has an anchor domain at the N-terminus of the protein that undergoes autolytic cleavage, likely through an intermolecular mechanism. The catalytic site is present in domain II. The precise function of domain III is unknown, but it is likely important for phosopholipid and calcium binding (Tompa et al, 2001) as well as other electrostatic interactions (Strobl et al, 2000). Domain IV is the calcium-binding domain, which contains five EF-hand motifs. In the absence of calcium, the catalytic triad consisting of Cys105 in domain I and His262 and Asn286 in domain II is separated, indicating that a conformational change is required for activation (Hosfield et al, 1999). The crystal structure of a chimeric peptide containing 85% of the sequence of calpain 1 essentially mimics the structure of calpain 2 (Pal et al, 2003). The important distinctions between the two structures are that in calpain 1 the separation of the catalytic residues is less dramatic, and the calcium-binding region of domain IV is more flexible. These findings could explain the primary biochemical distinction between μ- and m-calpain, namely the concentration of calcium required for in vitro activation. μ-Calpain requires 3 to 50 μmol/L Ca²⁺ for half-maximal activity, whereas m-calpain requires higher levels, 400 to 800 μmol/L (Goll et al, 2003). Both major calpains share a common regulatory subunit, known as calpain small subunit (CSS)1 (also referred to as calpain 4; gene name, CAPNS1). This subunit consists of two domains (Figures 1A and 1B): the hydrophobic N-terminal domain V is glycine-rich and the C-terminal domain VI, like domain IV of the catalytic subunits, is a calcium-binding region containing five EF-hand motifs (Hosfield et al, 1999). More recently, one group has described a second small subunit, CSS2 (gene name, CAPNS2), which has 73% sequence homology with CSS1, but which binds calpains 1 and 2 with less affinity (Schád et al, 2002). They further described differential distribution of CSS1 and CSS2 within rat brain, with CSS1 present in dendrites and CSS2 in axons (Friedrich et al, 2004). However, given that knockout of CSS1 is lethal despite the presumed continued expression of CSS2, it does not appear that the more recently identified subunit is able to functionally replace CSS1.

Figure 1

Domain structure of brain calpain isoforms. Schematic representations based on crystallographic data for μ-calpain (A) and m-calpain (B), or sequence analysis of calpains 3, 5, and 10 (C to E; Swiss-Prot accession numbers P20807, O15484, and Q9HC96, respectively). μ- and m-Calpain (A and B) consist of heterodimers made up of catalytic subunits containing four major domains and a shared, two-domain regulatory subunit. Five EF-hand calcium-binding regions are present in domain IV of calpains 1 and 2, with an additional five EF-hands in domain VI of the regulatory subunit. Catalytic residues are present in domain II. Calpain 3 (C) consists of a single polypeptide with a structure similar to that of the catalytic subunits of μ- and m-calpain. Calpains 5 (C) and 10 (D) differ from the other brain calpains in that they lack the EF-hand-containing domain IV, and instead substitute a unique T domain.

Three other calpain family members have been identified in neural tissue. mRNA for calpain 3, previously believed to be skeletal muscle specific, has been identified in brain homogenates (Herasse et al, 1999). Antibodies raised from synthetic calpain 3 peptide sequences have been used to show calpain 3 protein in astrocytes (König et al, 2003) and a neuronal cell line (Marcilhac et al, 2006). Unlike the heterodimeric μ- and m-calpain, calpain 3 is a 94-kDa monomeric protease similar to the 80-kDa calpain 1 and 2 catalytic subunits (Figure 1C). mRNA for calpain 5 has been identified in human and rat brain. Like calpain 3, calpain 5 is a monomeric protein; however, it lacks domain IV, which is replaced by a unique T domain, the function of which is unknown (Figure 1D). The substitution of the T domain for domain IV is also seen in calpain 10 (Figure 1E), which has been identified in homogenates of rat retina, heart, skeletal muscle, and brain using an antibody generated from a synthetic peptide (Ma et al, 2001). The Caenorhabditis elegans homolog of calpain 5 is necessary for neurodegeneration in that species (Syntichaki et al, 2002), whereas the role of calpain 5 in other models of brain injury is not known. Similarly, calpains 3 and 10 may have roles in apoptotic cell death (Marcilhac et al, 2006) and mitochondrial dysfunction (Arrington et al, 2006), respectively, whereas their roles in brain physiology and pathology have yet to be determined.

Calpastatin is the only known protease inhibitor that appears to be completely specific to calpains. Both initial activation and subsequent calpain activity are inhibited by calpastatin, and because of its repeated domain structure, each calpastatin molecule is able to inhibit four calpains (Maki et al, 1987). Calpastatin alone does not bind calcium, whereas it binds to calpain only in the presence of calcium (Takano et al, 1999). Calpain alone possesses the ability to cleave calpastatin, but the proteolytic fragments retain their inhibitory function (DeMartino et al, 1988). One study has also suggested that the relative inhibitory effect of calpastatin differs between the two major isoforms, depending on its phosphorylation state. Dephosphorylated calpastatin has a 3- to 4-fold lower K_i against μ-calpain, whereas phosphorylated calpastatin has a 3- to 4-fold lower K_i against m-calpain (Salamino et al, 1994a). The other calpain isoforms are also believed to be inhibited by calpastatin, but detailed studies of the inhibitory effects have not been reported.

Physiologic Activity

The role of calpains in physiologic processes has been extensively reviewed (Goll et al, 2003) and encompasses functions including cell cycle regulation, differentiation, cell migration, adhesion, and signal transduction. A similarly wide range of physiologic functions has been attributed to calpain within the brain and neuronal culture systems. Calpain has been implicated in the differentiation of neuronal cell lines (Grynspan et al, 1997), neurite outgrowth in hippocampal neuronal culture (Song et al, 1994), as well as synaptic remodeling and long-term potentiation (Tomimatsu et al, 2002).

Through the use of transgenic mice and RNA interference (RNAi), there is an increasing literature concerning the isoform specificity of some physiologic functions of calpains. Perhaps the most convincing evidence for a functional difference between μ- and m-calpain is the fact that mice with a knockout of the calpain 1 catalytic subunit have been reported to have defects in platelet function (Azam et al, 2001) and to have normal long-term potentiation (Grammer et al, 2005), whereas knockout of calpain 2 (Dutt et al, 2006) or the common regulatory subunit (Zimmerman et al, 2000; Arthur et al, 2000) is embryonic lethal. Both calpain-3 and calpain-10 knockouts are viable, but the effects of these gene deletions on brain physiology and pathology have not been reported.

Studies using antisense oligonucleotides or RNAi have similarly shown isoform-specific physiologic effects. μ-Calpain has a demonstrated role in muscle cell differentiation (Moyen et al, 2004) and cell motility and migration (Satish et al, 2005; Wu et al, 2006). m-Calpain is involved in myoblast fusion (Balcerzak et al, 1995), membrane protrusion (Franco et al, 2004), and chromosome alignment during mitosis (Honda et al, 2004). Antisense oligonucleotides targeting the calpain 1 catalytic subunit reduced the effects of n -methyl-d-aspartic acid (NMDA) excitotoxicity in hippocampal slice culture (Bednarski et al, 1995). Similarly, RNAi for calpain 1, but not calpain 2, has been shown to be protective in dissociated neuronal culture (Cao et al, 2007). Unfortunately, the effects of translational suppression of calpains 3 and 10 on brain injury have not been reported, nor the effects of isoform-specific knockdown on in vivo injury.

Localization

The localization of calpain within the central nervous system has been repeatedly investigated. Immunohistochemical results have varied widely, with most studies of μ-calpain agreeing that it is expressed in neurons, and some noting glial expression as well. Specific brain regions identified include cerebellar Purkinje cells (Siman et al, 1985; Hamakubo et al, 1986), spinal cord nuclei (Siman et al, 1985; Hamakubo et al, 1986; Fukuda et al, 1990), cortex (Perlmutter et al, 1990), regions of diencephalon (Siman et al, 1985; Perlmutter et al, 1990), caudate nucleus (Siman et al, 1985; Perlmutter et al, 1990), and both granular and pyramidal neurons within the hippocampus (Siman et al, 1985; Hamakubo et al, 1986; Perlmutter et al, 1988; Fukuda et al, 1990). One immunohistochemical study specifically examining m-calpain noted that its expression was limited largely to glia (Hamakubo et al, 1986), whereas another observed m-calpain in hippocampal interneurons (Fukuda et al, 1990). Variable results such as these are common among calpain localization studies: in another example, Siman et al (1985) identified μ-calpain only in the hippocampal dentate gyrus, whereas Hamakubo et al (1986) and Fukuda et al (1990) reported that it is present in pyramidal cells throughout the hippocampus. Furthermore, Hamakubo et al (1986) observed only m-calpain in glial cells, whereas Perlmutter et al (1990) observed μ-calpain in glia. The major drawback of these studies, and undoubtedly the source of their variable results, is the fact that each used a different antibody. Although the isoform specificity of these antibodies was established in western blots, it is not clear as to what level of cross-reactivity each might have for other calpain isoforms when used in immuno-histochemical studies.

To avoid this problem, more recent studies have examined mRNA, rather than protein, expression. The most definitive of these used a combination of reverse transcriptase-polymerase chain reaction and in situ hybridization to examine the relative abundance and localization of mRNAs for calpains 1 and 2 and calpastatin (Li et al, 1996). Expression of calpain 2 mRNA was found to be 15-fold higher than calpain 1 in whole-brain homogenates. Calpastatin mRNA expression was three-fold higher than calpain 1. Calpain 1 and calpastatin message were observed to be diffused throughout the entire brain in both neurons and glia, whereas message for calpain 2 localized to distinct neuronal populations, including all cornu ammonis regions of hippocampus, cortical pyramidal neurons, and cerebellar Purkinje cells.

Less well studied is the subcellular localization of various components of the calpain system. Early immunoelectron microscopy studies were able to identify μ-calpain throughout neuronal and glial cytoplasm, typically associated with cytoskeletal elements. In neurons, staining extended into axons and dendrites, and, more specifically, was associated with dendritic spines and postsynaptic densities (Perlmutter et al, 1988). As a result of this study and of rough localization claims in earlier immunohistochemistry studies, calpain has typically been considered a ‘cytoplasmic’ protein. More recent immunoelectron microscopic studies have also placed calpain in the cytoplasm, but associated with the membranes of the endoplasmic reticulum (ER) and Golgi apparatus (Hood et al, 2003). Calpains' involvement in nuclear processes such as mitosis (Honda et al, 2004), however, suggests that calpain at least possesses the ability to translocate to the nucleus. In fact, fluorescein-tagged μ-calpain has been shown to be selectively transported to the nucleus under conditions known to allow energy-dependent nuclear transport of proteins (Mellgren and Lu, 1994), both μ- and m-calpain have been identified in the nuclear fraction of rabbit hippocampal homogenates (Ostwald et al, 1994), and m-calpain has been observed in cerebellar granule cell nuclei by immunohistochemistry and subcellular fractionation (Tremper-Wells and Vallano, 2005). Additionally, a recent study showed the presence of μ-calpain within isolated mitochondria (Garcia et al, 2005). Further studies have localized μ-calpain specifically to the mitochondrial intermembrane space (Cao et al, 2007). Calpain 10 has also been localized to mitochondria (Arrington et al, 2006), whereas calpain 3 is present in the cytoplasm and nucleus (Marcilhac et al, 2006). The subcellular localization of calpain 5 within the central nervous system has not been reported.

In addition to the localization of calpain under baseline conditions, there have been numerous studies of calpain translocation throughout the cell. Activation of calpain is associated with translocation to the plasma membrane in neutrophils (Pontremoli et al, 1989) and platelets (Ariyoshi et al, 1993). The importance of calpain translocation in the brain is not clear, and there is some evidence that calpain may be present at the membrane before any excitotoxic insult, and therefore no translocation is necessary before calpain activation (Hewitt et al, 1998). Given the similar substrate profiles of the two major calpain isoforms, it is likely that localization and translocation within the cell play a key role in differentiating their physiologic roles, and may impact their function in pathology as well.

Role of Calpain in Postischemic Neurodegeneration

Several forms of evidence have emerged since the early 1990s to support a role for calpain activity in acute neural injury. Calpain activation and activity occur in a spatial and temporal pattern that suggests a possible causal role for calpain in postischemic neurodegeneration (Saido et al, 1993; Roberts-Lewis et al, 1994; Neumar et al, 1996, 2001; Bartus et al, 1998). This evidence is bolstered by a series of studies showing that neurodegeneration is prevented by treatment with calpain inhibitors before (Lee et al, 1991; Rami and Krieglstein, 1993; Hong et al, 1994; Yokota et al, 1999) or after an ischemic insult (Bartus et al, 1994a, 1994b; Li et al, 1998; Markgraf et al, 1998).

Four major classes of calpain inhibitors have been used in in vitro and in vivo models of ischemic injury. There are several factors that can limit the utility of these inhibitor studies, including specificity, cell permeability, and blood—brain barrier penetration (for review, see Wang and Yuen, 1999; Donkor, 2000). The first class, the peptide aldehydes, bind reversibly to the active site and have poor specificity—in addition to calpain, these compounds can inhibit plasmin, trypsin, papain, and especially cathepsins. Early peptide aldehydes also had poor cell permeability, but this has been improved in later compounds such as the commonly used MDL28170. The newer peptide aldehyde SJA6017 is an even more potent inhibitor of calpains than MDL28170, but it continues to inhibit cathepsins as well (Inoue et al, 2003). Although it has been effective in treating traumatic brain injury (Kupina et al, 2001) and hypoxia in cardiac myocytes (Aki et al, 2002), use of SJA6017 in models of brain ischemia has not been reported. Another group of inhibitors, the epoxysuccinyl peptides, bind irreversibly to the calpain active site and have even better cell permeability. While they have been neuroprotective in in vitro excitotoxic models (Brorson et al, 1995; Rami et al, 1997), these compounds also inhibit other cysteine proteases in addition to calpain. The third major class of calpain inhibitors, the ketoamides, has several advantages over previous compounds, including high cell permeability and water solubility. However, early compounds were effective only after direct application to the brain (Bartus et al, 1994a) or intraarterial administration (Bartus et al, 1994b). Newer ketoamides, including A705239, have high oral bioavailability (Lubisch et al, 2003). Although they still inhibit cathepsin B, these drugs have no crossinhibition of either papain or caspase-3. A705239 is neuroprotective after traumatic brain injury (Lubisch et al, 2003) and preserves myocardial function after cardiac ischemia (Neuhof et al, 2003; Trumbeckaite et al, 2003). The development of more specific calpain inhibitors has been further limited by the fact that formation of the calpain catalytic site only occurs once the protease has been activated (Hosfield et al, 1999). As a result, it is difficult to design active site inhibitors because the active form cannot be crystallized and therefore the exact structure of the catalytic site is unknown. An alternate approach has been to target regions outside the catalytic site. This approach is exemplified by the fourth class of inhibitors, α-mercaptoacrylic acids such as PD150606. This compound is highly calpain specific and binds outside the active site (Wang et al, 1996). The only reported in vivo use of PD150606 required intracerebroventricular infusion of the drug, limiting its clinical applicability (Farkas et al, 2004).

To address the problems inherent in synthetic calpain inhibitors, an attempt was made to create a fusion protein consisting of the endogenous calpain inhibitor calpastatin coupled to the HIV Tat transducing protein. Although the Tat—calpastatin fusion product was able to inhibit calpain in cell-free assays, and was readily taken up into neurons, it remained encapsulated in endosomes and displayed no inhibitory activity in the cell (Sengoku et al, 2004). Even if such an approach were to be successful, calpastatin, like all the other available inhibitors, is not isoform specific (Saito and Nixon, 1993). As a result, most studies implicating calpain in the pathology of postischemic neurodegeneration are unable to identify the specific isoform involved; therefore, henceforth, the generic term ‘calpain’ will be used in cases where a specific isoform has not been identified.

The limitations of these inhibitory strategies have more recently been overcome through the use of genetic techniques. Twenty-fold overexpression of calpastatin in human SH-SY5Y neuroblastoma cells was protective against low-dose staurosporine injury, but this effect was lost with more substantial injuries (Neumar et al, 2003). Subsequently, two papers have described the effects of acute brain-injury models in mice with neuron-specific overexpression of calpastatin. In both cases, the consequent reduction in calpain activity was associated with neuroprotection in a kainic acid-based excitotoxicity model (Takano et al, 2005; Higuchi et al, 2005). These last studies have definitively established the role of calpain in mediating acute brain injury. What remains unknown is the precise calpain isoform or isoforms responsible for postischemic pathologic calpain activity. One study using antisense oligonucleotides to reduce μ-calpain expression in organotypic hippocampal slice culture has shown protection against excitotoxicity (Bednarski et al, 1995). More recently, RNAi-mediated knockdown of μ-calpain, but not m-calpain, was protective in an in vitro model of apoptosis-inducing factor (AIF)-mediated cell death (Cao et al, 2007). The in vivo effects of isoform-specific knockdown on brain injury have not been investigated. The importance of identifying the isoform responsible for pathologic calpain activity is highlighted by the differing physiologic roles discussed above. This is particularly important because m-calpain appears to be essential for normal mitosis. Additionally, given the potentially different subcellular localization of the major brain calpain isoforms, identifying a single isoform responsible for pathologic activity may allow one to narrow down the number of calpain substrates that play a key role in neuronal cell death. This is perhaps the largest unanswered question in the study of postischemic calpain proteolysis, and the subject of the remainder of this review—by what mechanisms do calpains cause neuronal death?

Subcellular Consequences of Pathologic Calpain Activity

Given the large number and wide range of known calpain substrates, it has been difficult to develop a unified model of how calpain proteolysis leads to eventual cell death. In an effort to develop such a model, this paper will take two approaches for categorizing and discussing calpain substrate cleavage. The first of these is to consider calpain substrates by the subcellular location. The pathology of ischemic neuronal injury involves alterations in the function of multiple cellular components, including the synapse, plasma membrane, ER, mitochondria, lysosomes, and nucleus. All these neuronal regions or organelles contain calpain substrates, and, therefore, a systematic consideration of the subcellular localization of pathologic calpain activity is one method by which one can understand the mechanism of calpain-mediated cell death. Regional differences in calcium fluxes within neurons during ischemia and reperfusion further support this theoretical approach.

Calpain substrates also exist across a wide range of functional categories. Three important categories include postsynaptic structural proteins (Table 1), calcium regulatory proteins (Table 2), and signaling proteins (Table 3). Calpains require elevated calcium concentrations not only for initial activation but also for subsequent activity, although activity can be sustained with a lower degree of elevation (Goll et al, 2003). As a result, it makes sense that calpain itself may play some role in regulating calcium homeostasis. In fact, most neuronal calcium regulatory proteins are known calpain substrates. A second set of calpain substrates can be defined as those contributing to normal cell structure. Calpain cleavage of this set of targets leads directly to cellular breakdown that is a hallmark of postischemic neuronal death. However, it remains to be determined if cytoskeletal proteolysis is causally related to cell death or primarily a postmortem phenomenon. In addition to directly damaging the cell structure, calpains also engage in complex proteolytic signaling cascades. This final group of calpain substrates consists of other proteins that undergo changes in function, rather than simply loss of function, in response to calpain cleavage. As we consider each subcellular location, it will be important to evaluate the contribution each of these three categories of substrates makes toward eventual cell death.

Table 1

Postsynaptic structural proteins cleaved by calpain

Substrate	Location	Effect of calpain cleavage	References
PSD-95	Synapse	Alters anchoring of NMDA receptors	Lu et al (2000a) and Vinade et al (2001)
SAP-97, glutamate receptor-activating protein	Synapse	Destabilization of AMPA receptors	Jourdi et al (2005)
Spectrin	Plasma membrane	Spectrin cleavage is a hallmark of calpain activity; cleavage likely alters membrane structure and function	Saido et al (1993), Roberts-Lewis et al (1994), Neumar et al (2001), and Siman et al (2004)
MAP2	Dendrites	Likely alters microtubule transport, and dendritic and synaptic structure	Siman and Noszek (1988), Inuzuka et al (1990), Pettigrew et al (1996), Buddle et al (2003), and Ziemka-Nałecz et al (2003)

Abbreviations: AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; MAP, microtubule-associated protein; NMDA, N-methyl-d-aspartic acid; PSD, postsynaptic density protein; SAP-97, synapse associated protein-97.

Table 2

Calcium regulatory proteins cleaved by calpain

Substrate	Location	Effect of calpain cleavage	Potential effect on cytosolic calcium	References
NMDA receptor NR2B subunit	Synapse	May alter interaction with signaling molecules	Unknown	Guttmann et al (2002), Simpkins et al (2003), and Dong et al (2006)
PMCA	Plasma membrane	No longer regulated by calmodulin/internalizes	Increase	James et al (1989) and Pottorf et al (2006)
NCX	Plasma membrane	No longer able to expel calcium from the cell	Increase	Bano et al (2005)
l-type Ca²⁺ channel	Plasma membrane	Removes phosphorylation site	Increase	Wei et al (1994), De Jongh et al (1994), and Hell et al (1996)
SERCA	Endoplasmic reticulum	Degrades/ATPase activity uncouples from Ca²⁺ uptake	Increase	Parsons et al (1999) and French et al (2006)
IP₃R	Endoplasmic reticulum	Reduces binding of the IP₃ ligand	Unknown	Nagata et al (1994), and Dahl et al (2000)
Ryanodine receptor	Endoplasmic reticulum	Increase in percentage of open time	Increase	Rardon et al (1990)

Abbreviations: IP₃R, inositol triphosphate receptor; NMDA, N-methyl-d-aspartic acid; PMCA, plasma membrane calcium ATPase; SERCA, sarcoplasmic/endoplasmic reticulum calcium ATPase.

Table 3

Signaling cascades influenced by calpain

Substrate	Location	Effect of calpain cleavage	References
CaMKIIα	Cytosol (synapse)	Removes calmodulin regulation; retains kinase activity	Hajimohammadreza et al (1997)
Protein kinase C	Cytosol (synapse)	Produces a catalytically active fragment	Kishimoto et al (1989)
Calcineurin	Cytosol (synapse)	Removes calmodulin regulation producing a constitutively active phosphatase	Shioda et al (2006)
CaMKIV	Nucleus	Reduces phosphatase activity	Tremper-Wells and Vallano (2005)
β-catenin	Cytosol/nucleus	Translocates to the nucleus; induces gene transcription	Abe and Takeichi (2007)
GSK-3	Cytosol	Increases kinase activity	Goñi-Oliver et al (2007)
P35/p39	Cytosol/nucleus	Produces more stable p25/p29 fragments, which bind CDK5 and translocate to the nucleus	O'Hare et al (2005) and Saito et al (2007)
CRMP	Cytosol/nucleus	Translocates to the nucleus; overexpression of truncated form is neurotoxic	Hou et al (2006)
P53	Cytosol/nucleus	Enhances p53-dependent apoptosis	Atencio et al (2000) and Pariat et al (1997)
Bid	Cytosol/mitochondria	Promotes mitochondria permeability transition and release of proapoptotic factors from mitochondria	Chen et al (2001) and Takano et al (2005)
Bax	Cytosol/mitochondria	Promotes insertion into mitochondria and subsequent cytochrome c release	Choi et al (2001)
Bcl-xL	Cytosol	Function alters to become proapoptotic	Nakagawa and Yuan (2000)
AIF	Mitochondria/nucleus	Released from the mitochondria and subsequently translocates to the nucleus	Cao et al (2007) and Polster et al (2005)
APAF-1	Cytosol	No longer able to activate caspase cascade	Reimertz et al (2001)
Caspase-9	Cytosol	No longer able to activate caspase-3	Chua et al (2000) and Volbracht et al (2005)
Caspase-3	Cytosol	Unclear—calpain cleavage has been shown to be both inhibitory and activating	McGinnis et al (1999) and Blomgren et al (2001)
Caspase-7	Cytosol	Converts procaspase-7 to the active form	Ruiz-Vela et al (1999)

Abbreviations: AIF, apoptosis-inducing factor; APAF, apoptosis-activating factor 1; CaMKIIα, calmodulin-dependent protein kinase IIα; CaMKIV, calmodulin-dependent protein kinase IV; CDK, cyclin-dependent kinase; CRMP, collapsin response mediator protein; GSK, glycogen synthase kinase.

Plasma Membrane and Synapse

A major component of the postischemic injury cascade in the brain is the membrane depolarization that occurs after ATP depletion (for review, see Neumar, 2000). This membrane depolarization results in the opening of voltage-gated sodium and calcium channels and in the release of excitatory neurotransmitters, such as glutamate. Transmitter release further alters ion homeostasis as it results in activation of calcium-permeable NMDA receptors and sodium-permeable AMPA receptors. Resulting loss of the normal Na gradient then leads to reversal of the Na⁺/Ca²⁺ exchanger. Although these events are likely sufficient to activate calpain, the initial change in ion balance is reversible within minutes after a mild or moderate ischemic insult. However, there is a secondary disruption of ion homeostasis that occurs in many neurons between 4 and 48 h after reperfusion. This further disruption of calcium regulatory processes is necessary to produce the delayed calpain activation that potentially contributes to delayed postischemic neurodegeneration. Calpain itself may contribute to the sustained or secondary calcium overload by cleaving and altering the function of many of these proteins during the recovery period.

Plasma Membrane: The most obvious effects of calpain activity on calcium regulation come through its cleavage of l-type calcium channels, the Na⁺/Ca²⁺ exchanger, and the plasma membrane Ca²⁺ ATPase (Figure 2A). Calpain-mediated truncation of the l-type calcium channel occurs both in vitro (De Jongh et al, 1994) and in hippocampal neurons exposed to NMDA (Hell et al, 1996). This truncation removes the major site of channel phosphorylation and results in a persistent increase in Ca²⁺ influx (Wei et al, 1994). A second potential contributor to calpain-mediated calcium dysregulation at the plasma membrane is the Na⁺/Ca²⁺ exchanger, which is a major extruder of intracellular calcium. After in vitro excitotoxicity and in vivo brain ischemia, the Na⁺/Ca²⁺ exchanger undergoes a cleavage that is prevented by calpain inhibitors or calpastatin overexpression (Bano et al, 2005). The cleaved exchanger is unable to expel calcium.

Figure 2

Subcellular localization of calpain pathology in postischemic neurons. Postischemic calpain activity occurs in a wide range of subcellular regions including the synapse and plasma membrane (A), endoplasmic reticulum and lysosomes (B), and mitochondria and nucleus (C). The substrate profile within each location suggests multiple distinct mechanisms by which calpain activity can contribute to postischemic neurodegeneration. Calpain-mediated proteolysis of calcium regulatory proteins at the synapse, plasma membrane, and endoplasmic reticulum could work synergistically in a feed-forward pathway of disrupted calcium homeostasis. Calpain-mediated proteolysis of signaling proteins in the cytosol, mitochondria, and nucleus potentially modulates caspase-dependent and caspase-independent cell death. Finally, calpain-mediated proteolysis of cytoskeletal and membrane proteins could result in structural failure of the plasma membrane and lysosomal membranes. AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; NMDA, n -methyl-d-aspartic acid receptor; mGluR1α, metabotropic glutamate receptor 1α; CamKIIα, calmodulin-dependent protein kinase IIα; PKC, protein kinase C; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; MAP2, microtubule-associated protein 2; PMCA, plasma membrane calcium ATPase; NCX, sodium—calcium exchanger; RYR, ryanodine receptor; IP₃R, inositol 3-phosphate receptor; SERCA, sarcoplasmic/endoplasmic reticulum calcium ATPase; AIF, apoptosis-inducing factor; CytC, cytochrome c; APAF-1, apoptosis-activating factor 1; β-cat, β-catenin; GSK-3, glycogen synthase kinase 3; CDK5, cyclin-dependent kinase 5; CRMP, collapsin response mediator protein; PARP, poly(ADP-ribose) polymerase; CamKIV, calmodulin-dependent protein kinase IV.

The plasma membrane calcium ATPase (PMCA) has long been known to be a calpain substrate in erythrocytes, where truncation results in an active form of the protein that is no longer regulated by calmodulin (James et al, 1989). However, at least one study has shown that calpain proteolysis of erythrocyte PMCA in situ actually causes a decrease in function (Salamino et al, 1994b). More recently, glutamate excitotoxicity has been shown to result in internalization of PMCA in hippocampal neurons, an effect that was blocked by calpain inhibition (Pottorf et al, 2006). This result is consistent with studies showing loss of PMCA activity (Oguro et al, 1995) and expression (Lehotský et al, 1999) in postischemic gerbil hippocampus. The result of calpain-mediated PMCA truncation therefore appears to parallel the effects seen for calpain cleavage of l-type channels and the Na⁺/Ca²⁺ exchanger; cleavage of all three proteins reduces the ability of the cell to maintain a low internal calcium concentration and therefore likely contributes to a feed-forward pathway of cytosolic calcium overload.

Synapse: The manner in which calpain activity may alter calcium regulation through cleavage of neurotransmitter receptors is less straightforward (Figure 2A). Classic NMDA-type glutamate receptors are heteromeric proteins composed of two NR1 subunits and two NR2 subunits, selected from subtypes NR2A, NR2B, NR2C, and NR2D. Of these, NR2A, -2B, and -2C are known to undergo C-terminal proteolysis by calpain in vitro (Guttmann et al, 2002), a truncation that does not appear to prevent any aspects of receptor function. However, only the NR2B subunit has been (Xu et al, 2007) shown to be cleaved by calpain in mature neurons (Dong et al, 2006). This cleavage occurs both in vivo after transient bilateral carotid artery occlusion and in vitro after excitotoxic insults in hippocampal neurons (Simpkins et al, 2003). The cleaved receptor remains on the cell surface, and, although it has not been shown to be active in neurons, it is predicted to be so based on the cleavage sites. Separate published reports have shown either a reduction in NMDA-mediated transmission (Wu et al, 2005) or essentially no change in electrophysiologic properties (Guttmann et al, 2001) after calpain proteolysis. What should be altered is the ability of the truncated C-terminus to interact with intracellular signaling molecules. The downstream effects of this modification on signaling and calcium regulation are unknown.

Activation of NMDA receptors is also associated with calpain-mediated cleavage of a member of the metabotropic family of glutamate receptors. The C-terminus of the mGluR1α G-protein-coupled receptor is cleaved by calpain after NMDA exposure (Xu et al, 2007). The truncated receptor retains its ability to signal for the release of calcium from intracellular stores, but is unable to activate the neuroprotective PI₃ kinase—Akt signaling pathway. The calpain-cleaved mGluR1α is therefore able to contribute to cytosolic calcium overload without any balancing neuroprotective effect.

Calpain also cleaves the GluR1, GluR2, and GluR3 subunits of AMPA-type ionotropic glutamate receptors (Bi et al, 2000). Again, calpain proteolysis produces a C-terminal truncation of the receptor, an effect that is seen in vitro and after NMDA excitotoxicity in hippocampal slice cultures (Bi et al, 1998). The effects of calpain cleavage are clearer for AMPA receptors than for NMDA receptors, with studies suggesting that calpain activation leads to both a loss of total GluR1–3 protein (Jourdi et al, 2005) and their dissociation from postsynaptic densities (Lu et al, 2000b). The overall conclusion from these studies appears to be that calpain cleavage reduces AMPA-mediated currents and increases turnover of AMPA receptors. This is particularly interesting in light of studies showing a decrease in expression of mRNA for the calcium-impermeable GluR2 subunit after ischemia, suggesting that replacement of AMPA receptors after ischemia would be biased toward calcium-permeable isoforms (Pellegrini-Giampietro et al, 1997). This concept is supported by the fact that overexpression of a Ca²⁺-impermeable form of the receptor rescues neurons from ischemic damage (Liu et al, 2004). Conversely, it has recently been shown that the calpain-mediated downregulation of AMPA receptors acts primarily on the GluR1 subunit, leaving GluR2 at the cell surface both in cortical neuron culture and in rat brain (Yuen et al, 2007). It is not clear what effect this sparing of GluR2 subunits might have on potential changes in Ca²⁺ permeability associated with AMPA receptor turnover.

In addition to direct effects on neurotransmitter receptors, postischemic calpain activation may alter synapse structure and function through cleavage of postsynaptic cytoskeletal elements (summarized in Table 1 and Figure 2A). Postsynaptic density protein 95 ordinarily binds numerous receptors and protects NR2A from being cleaved by calpain (Dong et al, 2004). However, postsynaptic density protein 95 itself is cleaved by calpain in calcium-treated rat brain sections and NMDA-exposed hippocampal slice culture (Lu et al, 2000a), which may modify the anchoring of NMDA receptors (Vinade et al, 2001). Calpain also cleaves proteins associated with the stabilization of AMPA receptors, such as synapse associated protein-97 (SAP-97) and glutamate receptor-activating protein (Jourdi et al, 2005), which may have an additive effect with direct calpain cleavage of AMPA subunits on receptor turnover.

The structural changes that occur in synapses after an ischemic insult extend beyond tethering of receptors. Rapid changes in dendritic spine density have been observed within hours of in vitro excitotoxicity, where calpain appears to be involved in synaptic remodeling (Faddis et al, 1997). These changes in neuronal structure likely involve cleavage of cytoskeletal calpain substrates such as αII-spectrin and microtubule-associated protein II (MAP2).

Spectrin is the major cytoskeletal protein component of the cell membrane (Czogalla and Sikorski, 2005). Intact spectrin is a 280 kDa protein, calpain cleavage of which produces unique 150 and 145 kDa fragments. Antibodies specific to these fragments provide a means to identify calpain activity, both through Western blotting of tissue lysates and through immunohistochemistry. Studies in postischemic gerbil hippocampus have shown an initial rapid rise in calpain-cleaved spectrin that occurs within 15 mins of reperfusion and which is then followed by a secondary accumulation of spectrin breakdown product between 4 and 24 h after injury (Saido et al, 1993; Roberts-Lewis et al, 1994). Transient forebrain ischemia in rats produces a similar pattern, with a peak in spectrin breakdown products observed at 1 h after injury that subsides only to be followed by a second peak beginning between 24 and 48 h in those cornu ammonis 1 hippocampal neurons that subsequently degenerate (Neumar et al, 2001). Spectrin proteolysis is, in fact, a hallmark of postischemic brain injury to the degree that the appearance of spectrin breakdown products in cerebrospinal fluid can be used as a biomarker of ischemia (Siman et al, 2004). Given the interactions between spectrin and many other membrane proteins such as ankyrin, actin, calmodulin, and microtubules (Bennett, 1990), it is likely that postischemic spectrin cleavage by calpain has substantial effects on membrane structure and function.

Microtubule-associated protein II serves to stabilize microtubules and regulate the microtubule network and transport system in neuronal dendrites (Dehmelt and Halpain, 2005). Microtubule-associated protein II is susceptible to calpain-mediated cleavage, as in vivo excitotoxicity leads to calpain-mediated loss of MAP2 immunoreactivity (Siman and Noszek, 1988). Similar degradation of MAP2 occurs after oxygen—glucose deprivation in hippocampal slice culture (Buddle et al, 2003) and in in vivo focal (Pettigrew et al, 1996) and global ischemia (Ziemka-Nałecz et al, 2003). This degradation of MAP2 is preceded by calpain activation, and is prevented by calpain inhibition (Inuzuka et al, 1990). Taken together, evidence for calpain-mediated cleavage of spectrin and MAP2 strongly suggests an important role of calpain in postischemic degradation of the neuronal cytoskeleton and alterations in synaptic structure.

The final role of calpain at the plasma membrane and in the synapse is to proteolytically modify a number of signaling molecules, triggering downstream effects throughout the cell. Many of these molecules are regulated by calmodulin, and a common effect of calpain cleavage is to remove this regulation. Excitotoxicity in hippocampal neurons (Wu et al, 2004) and focal brain ischemia in mice (Shioda et al, 2006) induces calpain cleavage of the calmodulin-dependent phosphatase calcineurin, producing a constitutively active form. Calpain also produces an active form of type IIα calmodulin-dependent protein kinase and degrades neuronal nitric oxide synthase after both in vitro and in vivo administration of a variety of neurotoxins (Hajimohammadreza et al, 1997). The presynaptic growth-associated protein 43, which is involved in the regulation of neurotransmitter release, is cleaved by calpain, decreasing calmodulin binding (Zakharov et al, 2005). Although not regulated by calmodulin, protein kinase C is cleaved by calpain, again producing a catalytically active fragment (Kishimoto et al, 1989). The precise downstream effects of cleavage of these signaling molecules on postischemic neurodegeneration are not known.

Endoplasmic Reticulum

Although the influx of extracellular calcium is certainly an important component of postischemic injury, it is important to also consider ways in which intracellular calcium stores can contribute to alterations in ion homeostasis. As noted above, calpain has been shown to be associated with the membrane of the ER (Hood et al, 2003, 2004). It is therefore only natural to assume that it is able to cleave ER proteins, and, in fact, several calpain substrates have been identified in the membrane of the ER (Figure 2B).

The ryanodine receptor (RYR) and inositol triphosphate receptor (IP₃R) are the two Ca²⁺ channels found in the membrane of the ER. The primary physiologic mechanism of RYR channel activation is thought to be increased cytosolic Ca²⁺ (Ca²⁺-induced Ca²⁺ release). Calpain-mediated proteolysis of cardiac RYR2 has been studied in detail. Digestion of cardiac junctional sarcoplasmic reticulum vesicles by m-calpain causes cleavage of the 400 kDa RYR2 isoform into 350 and 315 kDa intermediates followed by generation of a 150 kDa limit protein (Rardon et al, 1990). Electron microscopic analysis of sarcoplasmic reticulum vesicles after m-calpain proteolysis revealed a ‘shaved-off’ appearance of the cytoplasmic extension of the proteins. The functional effect of calpain-mediated proteolysis of cardiac RYR2 has been studied with the protein reconstituted in lipid bilayers. In these studies, calpain-mediated cleavage of RYR2 renders the channel unable to close in the presence of elevated cytosolic Ca²⁺ levels. Specifically, addition of calpain to the cis (cytosolic) chamber containing 1 μmol/L Ca²⁺ causes the RYR2 percentage open time to increase from 36% to >90% with no change in unitary channel conductance (Rardon et al, 1990). Ryanodine receptor 2 channels in a ‘locked open’ state are a potential mechanism for permanent ER Ca²⁺ depletion and loss of the ER's ability to buffer elevations in cytosolic Ca²⁺.

Published work evaluating the effect of brain ischemia on RYR is limited to studies showing decreased ³H-ryanodine binding in situ after permanent unilateral carotid artery occlusion in gerbils (Nozaki et al, 1997, 1999). Interpretation of these studies is limited because ryanodine binding is not significantly altered by calpain cleavage (Rardon et al, 1990). However, postischemic administration of the RYR channel antagonist dantrolene has been reported to be neuroprotective both in vitro (Wei and Perry, 1996) and in vivo (Nakayama et al, 2002), suggesting that increased Ca²⁺ efflux via RYR could contribute to sustained or secondary postischemic calpain activation and eventual neuronal death.

The IP₃R is the second Ca²⁺ channel in the ER. Inositol triphosphate receptor channel opening is stimulated by binding of IP₃, a second messenger generated when phospholipase C converts phospha tidylinositol bisphosphate (PIP₂) to IP₃ and diacylglycerol. There is also evidence that IP₃R Ca²⁺ release is enhanced by increased intracellular Ca²⁺, similar to RYR. The Ca²⁺-dependent channel opening probability displays a bell-shaped curve with maximum probability of opening at 0.2 μmol/L free Ca²⁺, which is significantly lower than the RYR (Bezprozvanny et al, 1991). Fifty percent of brain ER Ca²⁺ can be released by IP₃ (Ferris and Snyder, 1992), making the IP₃R a major potential player in postischemic calcium overload.

Calpain-mediated proteolysis of IP₃R1 from the rat cerebellum or striatum results in loss of intact protein (260 kDa) and generation of 200, 130, and 95 kDa (predominant) fragments detected by antibody raised against a peptide corresponding to the C-terminal domain (Igwe and Filla, 1997). This proteolysis of the IP₃R resulted in decreased IP₃ binding, suggesting that site-specific cleavage decreases the affinity of the remaining protein species for the IP₃ ligand. A possible calpain-mediated reduction in ligand binding is consistent with studies of IP₃R in postischemic brain. Reduced ³H-IP₃ binding has been observed in cornu ammonis 1 (CA1) hippocampus after unilateral carotid occlusion in gerbils, an effect that was not associated with any decrease in IP₃R immunoreactivity (Nagata et al, 1994). A more recent study of global ischemia in the rat produced similar results—a reduction in ³H-IP₃ binding in CA1, but no change in receptor expression (Dahl et al, 2000). The loss of IP₃ binding has also been shown functionally in postischemic brain tissue. Flash-frozen hippocampal slices taken from gerbils after unilateral carotid artery occlusion showed normal uptake of ⁴⁵Ca²⁺, but failed to release that calcium when stimulated with IP₃. Although this would seem to indicate that calpain cleavage of the receptor prevents evoked calcium release, it is possible that the proteolysis simply removes the ligand regulation of the channel. It is easy to imagine a model in which calpain-mediated loss of the ligand-binding domain of the IP₃R leads to baseline calcium release from the ER, contributing to postischemic calcium overload.

In addition to its effects on the IP₃R, calpain may also alter ER calcium release by affecting processing of IP₃ itself. A major metabolic pathway that serves to limit the half-life of IP₃ is its phosphorylation to form IP₄ by a family of IP₃ kinases (Irvine et al, 1986). The B isoform of the IP₃ kinase (IP₃-KB) is cleaved by calpain (Pattni et al, 2003). This cleavage separates the catalytic and membrane-anchoring domains of the molecule, releasing IP₃-KB from the membrane of the ER. This could then prevent metabolism of IP₃, allowing it to act longer on the IP₃R, thereby potentiating calcium efflux from the ER.

The sarcoplasmic/ER Ca²⁺ ATPase (SERCA) is responsible for energy-dependent ER Ca²⁺ sequestration. Sarcoplasmic/ER Ca²⁺ ATPase has been shown to be susceptible to calpain proteolysis in vitro, but only when the ATPase is in its oxidized form. Although calpain-mediated degradation of SERCA has been repeatedly shown after ischemia—reperfusion injury in the heart (French et al, 2006), little data exist for the effects of ischemia on brain SERCA. One study has measured SERCA ATPase activity in brain microsomes after decapitation ischemia (Parsons et al, 1999). In this model, SERCA-mediated ⁴⁵Ca²⁺ uptake was decreased after as little as a 10-min ischemia, but SERCA-specific ATP hydrolysis was unaffected. This apparent uncoupling of SERCA ATPase activity and Ca²⁺ uptake could be explained by modification of the SERCA molecule itself or increased ER membrane Ca²⁺ conductance via RYR and/or IP₃R channels. One would not expect calpain-mediated proteolysis of SERCA in a model of decapitation ischemia because unoxidized SERCA is not susceptible to calpain-mediated proteolysis. Sarcoplasmic/ER Ca²⁺ ATPase function has not yet been examined in the reperfused brain where conditions favor SERCA oxidation. Still, loss of SERCA function because of calpain-mediated proteolysis, like calpain alteration of RYR and IP₃R systems, is a potential mechanism of Ca²⁺ overload in postischemic neurons.

Mitochondria

Mitochondrial dysfunction has a well-established role in ischemic brain injury. Like cytosolic calcium overload, mitochondrial calcium overload is known to occur both immediately after brain ischemia and again at later time points (Zaidan and Sims, 1994). The initial postischemic calcium overload is accompanied by a burst of free radical production (Hall et al, 1993), an effect that can be inhibited with mitochondrial complex I blockers (Piantadosi and Zhang, 1996). Mitochondrial permeability transition has been implicated in models of both focal (Shiga et al, 1992) and global (Uchino et al, 1995) brain ischemia by the neuroprotective effects of cyclosporine A, a mitochondrial permeability transition inhibitor. Although such mitochondrial dysfunction is classically associated with apoptotic cell death and activation of the caspase family of proteases, a number of studies have identified a role of calpain in cell death associated with mitochondrial dysfunction. Calpain inhibitors have been shown to preserve mitochondrial function in cardiac tissue after ischemia—reperfusion injury (Neuhof et al, 2003; Trumbeckaite et al, 2003). This effect is likely independent of any inhibition of caspases, as the specific inhibitors used have been shown not to inhibit caspase-3 (Lubisch et al, 2003).

Apoptosis Regulatory Proteins: One way in which calpain may contribute to mitochondrial dysfunction is through cleavage of a number of proteins that regulate apoptosis (Figure 2C). This includes the Bcl2 family of proteins, several of which are calpain substrates. Calpain cleavage of Bax produces an 18 kDa fragment that leads to cytochrome c release in dopaminergic neurons exposed to mitochondrial toxins (Choi et al, 2001). Similar results have been observed for Bid, another proapoptotic Bcl2 family protein. Calpain-mediated cleavage and activation of Bid have been observed in rabbit heart after ischemia reperfusion injury (Chen et al, 2001) and in mouse brain after kainate excitotoxicity (Takano et al, 2005). Bid activation in this model was enhanced in mice deficient for the endogenous calpain inhibitor calpastatin. There is also evidence that calpain cleaves the loop region of the antiapoptotic protein Bcl-xL, converting it to a proapoptotic molecule (Nakagawa and Yuan, 2000). Calpain cleavage of these molecules leads to mitochondrial permeability transition, and release of death trigger molecules such as AIF and cytochrome c. Calpain is also thought to regulate apoptosis through degradation of the transcription factor p53 (Pariat et al, 1997), a finding that is supported by the fact that inhibition of calpains increases activated p53 and enhances p53-dependent apoptosis in tumor cell lines (Atencio et al, 2000).

Caspase-Dependent Programmed Cell Death: Calpain may also play an important role in apoptotic cell death through direct and indirect interactions with members of the caspase family of proteases (Figure 2C). Apoptosis protease-activating factor-1, which combines with cytochrome c to bind and activate caspase-9, is cleaved by calpain. The resulting loss of apoptosis protease-activating factor-1 was correlated with reduced activity of caspase-3-like proteases (Reimertz et al, 2001). Direct calpain truncation of caspase-9 also results in a loss of ability to activate caspase-3 (Chua et al, 2000). Conversely, calpain cleavage converts procaspase-7 to the active form, independent of the activity of other caspases (Ruiz-Vela et al, 1999). Conflicting results have been reported for caspase-3, calpain cleavage of which has been shown in separate studies to be inhibitory (McGinnis et al, 1999) or activating (Blomgren et al, 2001). Crosstalk between the calpain and caspase systems also occurs via calpastatin. Caspases have been shown to cleave calpastatin and reduce its inhibitory ability (Wang et al, 1998), setting up a complex interaction between the calpain and calpastatin systems. Although overexpression of calpastatin initially enhances caspase-3 activity, likely by preventing calpain-mediated degradation of caspase-9 and caspase-3, calpastatin itself is degraded by caspase-3, resulting in a secondary increase in calpain activity (Neumar et al, 2003).

Caspase-Independent Programmed Cell Death: In addition to promoting formation of the mitochondrial permeability transition pore, calpain may play a more direct role in the release of AIF. Calpain has recently been shown to cleave AIF, and to induce its release from mitochondria (Polster et al, 2005; Cao et al, 2007). This cleavage can occur both with exposure to exogenous calpain and when the only calpain present is that already within the mitochondria. Release of AIF from the mitochondria has also been observed in concert with activation of caspase-12, another calpain substrate (Nakagawa and Yuan, 2000), in models of ER stress-induced apoptosis in retinal ganglion cells (Sanges et al, 2006). Release of AIF in response to excitotoxic stimuli is suppressed by adenovirus-mediated expression of calpastatin (Volbracht et al, 2005; Cao et al, 2007), in transgenic mice overexpressing calpastatin (Takano et al, 2005), and after RNAi-mediated knockdown of μ-calpain in neurons (Cao et al, 2007). Therefore, although calpain appears to downregulate forms of caspase-mediated cell death, it may be simultaneously working to promote caspase-independent programmed cell death via an AIF-mediated mechanism.

Lysosomes

One proposed mechanism by which calpain activation may bring about necrotic destruction of the cell is through release of the hydrolytic enzymes typically sequestered within lysosomes, primarily the cathepsins (Figure 2B). This process, described by Tetsumori Yamashima as the ‘calpain—cathepsin cascade’, consists of calpain-mediated disruption of lysosomal membranes followed by diffusion of cathepsin B throughout the cytoplasm and nucleus. The evidence supporting this hypothesis in models of brain ischemia has recently been reviewed (Yamashima, 2004). Independent support for the interaction between calpains and cathepsins comes from work in C. elegans where loss of function in the proteases CLP-1 and TRA-3 (equivalent to calpains) as well as ASP-3 and ASP-4 (equivalent to cathepsins) is neuroprotective (Syntichaki et al, 2002). Combined loss of both calpain and cathepsin equivalent proteases did not have an additive neuroprotective effect, suggesting that the two protease families may induce neurodegeneration via a common sequential pathway.

Nucleus

Calpain directly cleaves nuclear proteins as well as causes the translocation of cytosolic proteins to the nucleus after proteolysis (Figure 2C). Within the nucleus of primary cerebellar granule neurons, calpain cleaves Ca²⁺/calmodulin-dependent protein kinase type IV (CaMKIV), leading to a reduction in phosphorylation of CaMKIV targets (Tremper-Wells and Vallano, 2005). The role of calpain-mediated CaMKIV downregulation in neurodegeneration has not been investigated. Calpain also cleaves the nuclear protein poly(ADP-ribose) polymerase-1 (PARP). Poly(ADP-ribose) polymerase-1 is normally a DNA repair enzyme that becomes overactivated after numerous toxic insults, leading instead to NAD and ATP depletion and eventual cell death (Pellicciari et al, 2004). Evidence for a role in ischemic brain injury includes findings that PARP becomes activated after excitotoxicity in cerebellar granule neurons (Cosi et al, 1994) and that PARP gene expression increases after global brain ischemia or kainate injection in gerbil and rat brains (Liu et al, 2000). Knockout of PARP reduces infarct size in a mouse model of focal cerebral ischemia (Eliasson et al, 1997). There is also evidence that PARP plays a key role in AIF-mediated programmed cell death (Yu et al, 2006). Although PARP is a known calpain substrate (McGinnis et al, 1999), it is again not clear what role, if any, calpain-mediated PARP cleavage plays in postischemic neurodegeneration.

Calpain also cleaves a number of cytosolic molecules that subsequently translocate to the nucleus. One such molecule is β-catenin, which undergoes calpain-mediated truncation in neurons exposed to glutamate (Abe and Takeichi, 2007). The truncated molecule then translocates to the nucleus where it induces gene transcription. The precise role of this gene regulation in postischemic neuronal death has not been examined. A related molecule in the Wnt-signaling pathway, glycogen synthase kinase 3, is also cleaved by calpain. Like many other calpain substrates, cleaved glycogen synthase kinase remains functional, and, in fact, displays an increase in kinase activity (Goñi-Oliver et al, 2007). Interestingly, glycogen synthase kinase 3 phosphorylation of β-catenin should prevent its translocation to the nucleus, further complicating the role calpain cleavage of these molecules may have in postischemic pathology.

More substantial evidence for a causal role in calpain-mediated cell death exists for cyclin-dependent kinase 5 (CDK5). Cyclin-dependent kinase 5 is unique in the CDK family in that it is primarily active in the central nervous system, where it is involved in regulating neuronal migration and synaptic function (Dhavan and Tsai, 2001). Activity of CDK5 kinase is regulated by the binding of one of its activator proteins, p35 or p39, which are cleaved by calpain to form to p25 or p29, respectively (Kusakawa et al, 2000; Patzke and Tsai, 2002). Accumulation of p25 has been shown after excitotoxicity, hypoxia, calcium overload, and ER stress in primary neuronal culture (Lee et al, 2000; Saito et al, 2007), as well as after middle cerebral artery occlusion in vivo (Nath et al, 2000). Similar results have been reported for p29 (Patzke and Tsai, 2002). Calpain-mediated truncation of p35 to p25 produces a more stable form of the protein, which is still able to activate CDK5. The CDK5–p25 complex translocates from the cytoplasm to the nucleus, where it appears to play an essential role in neuronal cell death (O'Hare et al, 2005; Saito et al, 2007).

The collapsin response mediator proteins (CRMPs) are another family of proteins with important roles in normal neuronal development that undergo calpain cleavage and nuclear translocation in disease states. Both CRMP-2 (Zhang et al, 2007) and CRMP-3 (Hou et al, 2006) are cleaved by calpain in vitro after excitotoxic injury and in vivo after traumatic or ischemic brain injury, respectively. Calpain cleavage of both CRMP-2 and CRMP-3 leads to their translocation from the cytosol to the nucleus. Furthermore, overexpression of the calpain-cleaved form of CRMP-3 was sufficient to induce neuronal death, whereas knockdown of endogenous CRMP-3 was neuroprotective (Hou et al, 2006), suggesting a causal role of nuclear CRMP in neurodegeneration.

Summary

This review has attempted to outline the potential mechanisms by which pathologic calpain activity can contribute to postischemic neurodegeneration. Key considerations include the potential role of specific calpain isoforms, subcellular localization, and the dysregulatory effect that calpain-mediated proteolysis has on a wide variety of substrates involved in both essential cell functions and cell death signaling. The sensitivity of most calcium regulatory proteins to calpain-mediated proteolysis is the foundation for hypothesized feed-forward mechanisms by which pathologic calpain activity causes lethal neuronal calcium overload (Table 2). However, direct in vivo evidence is still lacking. Furthermore, disruption of neuronal calcium homeostasis is only one mechanism by which calpains have been proposed to cause cell death. Disruption of survival signals or activation of death signals (Table 3) through cytosolic, mitochondrial, lysosomal, and nuclear substrates remains as potential mechanisms that require further evaluation (Figure 2).

Moving forward, the field is likely to benefit from more effective and specific strategies to inhibit postischemic activity of various calpain isoforms in vivo. Furthermore, elucidation of the discrete pathways that contribute to neurodegeneration will require much more comprehensive and precise evaluation of the calpain substrates that are actually cleaved in postischemic neurons. Perhaps the greatest challenge is to elucidate the relative contribution of each cleaved substrate to subsequent neuronal death. Finally, the physiologic function of the calpain—calpastatin system cannot be ignored. Given the known role of calpains in key cell processes such as mitosis, nonspecific calpain inhibition is a potentially unacceptable therapeutic strategy. Approaches that might avoid toxicity include isoform-specific calpain inhibition, targeting nondividing cells, or targeting subcellular compartments where pathologic calpain activity contributes to neuronal death.

References

Abe

Takeichi

(2007) NMDA-receptor activation induces calpain-mediated beta-catenin cleavages for triggering gene expression. Neuron 53:387–97.

Aki

Yoshida

Fujimiya

(2002) Phosphoinositide 3-kinase accelerates calpain-dependent proteolysis of fodrin during hypoxic cell death. J Biochem (Tokyo) 132:921–6.

Ariyoshi

Shiba

Sakon

Kambayashi

Yoshida

Kawashima

Mori

(1993) Translocation of human platelet calpain-I. Biochem Mol Biol Int 30:63–72.

Arrington

Van Vleet

Schnellmann

(2006) Calpain 10: a mitochondrial calpain and its role in calcium-induced mitochondrial dysfunction. Am J Physiol Cell Physiol 291:C1159–71.

Arthur

Elce

Hegadorn

Williams

Greer

(2000) Disruption of the murine calpain small subunit gene, Capn4: calpain is essential for embryonic development but not for cell growth and division. Mol Cell Biol 20:4474–81.

Atencio

Ramachandra

Shabram

Demers

(2000) Calpain inhibitor 1 activates p53-dependent apoptosis in tumor cell lines. Cell Growth Differ 11: 247–53.

Azam

Andrabi

Sahr

Kamath

Kuliopulos

Chishti

(2001) Disruption of the mouse mu-calpain gene reveals an essential role in platelet function. Mol Cell Biol 21:2213–20.

Balcerzak

Poussard

Brustis

Elamrani

Soriano

Cottin

Ducastaing

(1995) An antisense oligodeoxyribonucleotide to m-calpain mRNA inhibits myoblast fusion. J Cell Sci 108(Pt 5):2077–82.

Bano

Young

Guerin

Lefeuvre

Rothwell

Naldini

Rizzuto

Carafoli

Nicotera

(2005) Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity. Cell 120:275–85.

10.

Bartus

Baker

Heiser

Sawyer

Dean

Elliott

Straub

(1994a) Postischemic administration of AK275, a calpain inhibitor, provides substantial protection against focal ischemic brain damage. J Cereb Blood Flow Metab 14:537–44.

11.

Bartus

Dean

Mennerick

Eveleth

Lynch

(1998) Temporal ordering of pathogenic events following transient global ischemia. Brain Bes 790:1–13.

12.

Bartus

Hayward

Elliott

Sawyer

Baker

Dean

Akiyama

Straub

Harbeson

(1994b) Calpain inhibitor AK295 protects neurons from focal brain ischemia. Effects of postocclusion intraarterial administration. Stroke 25:2265–70.

13.

Bednarski

Vanderklish

Gall

Saido

Bahr

Lynch

(1995) Translational suppression of calpain I reduces NMDA-induced spectrin proteolysis and pathophysiology in cultured hippocampal slices. Brain Bes 694:147–57.

14.

Bennett

(1990) Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm. Physiol Rev 70:1029–65.

15.

Bezprozvanny

Watras

Ehrlich

(1991) Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 351:751–4.

16.

Baudry

(2000) Calpain-mediated truncation of glutamate ionotropic receptors. Methods for studying the effects of calpain activation in brain tissue. Methods Mol Biol 144:203–17.

17.

Chen

Baudry

(1998) Calpain-mediated proteolysis of GluR1 subunits in organotypic hippocampal cultures following kainic acid treatment. Brain Bes 781:355–7.

18.

Blomgren

Zhu

Wang

Karlsson

Leverin

Bahr

Mallard

Hagberg

(2001) Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of ‘pathological apoptosis’? J Biol Chem 276:10191–8.

19.

Brorson

Marcuccilli

Miller

(1995) Delayed antagonism of calpain reduces excitotoxicity in cultured neurons. Stroke 26:1259–66; discussion 1267.

20.

Buddie

Eberhardt

Ciminello

Levin

Wing

DiPasquale

Raley-Susman

(2003) Microtubule-associated protein 2 (MAP2) the NMDA receptor and is spatially redistributed within rat hippocampal neurons after oxygen-glucose deprivation. Brain Res 978:38–50.

21.

Cao

Xing

Liou

AKF

Yin

X-M

Clark

RSB

Graham

Chen

(2007) Critical role of calpain I in mitochondrial release of apoptosis-inducing factor in ischemic neuronal injury. J Neurosci 27:9278–93.

22.

Chen

Zhan

Krajewski

Reed

Gottlieb

(2001) Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion. J Biol Chem 276:30724–8.

23.

Choi

Lee

Chung

Jung

Jin

Kim

Saido

(2001) Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2. J Neurochem 77:1531–41.

24.

Chua

Guo

(2000) Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases. J Biol Chem 275:5131–5.

25.

Cosi

Suzuki

Milani

Facci

Menegazzi

Vantini

Kanai

Skaper

(1994) Poly(ADP-ribose) polymerase: early involvement in glutamate-induced neurotoxicity in cultured cerebellar granule cells. J Neurosci Res 39:38–46.

26.

Cuerrier

Moldoveanu

Davies

(2005) Determination of peptide substrate specificity for mu-calpain by a peptide library-based approach: the importance of primed side interactions. J Biol Chem 280:40632–41.

27.

Czogalla

Sikorski

(2005) Spectrin and calpain: a ‘target’ and a ‘sniper’ in the pathology of neuronal cells. Cell Mol Life Sci 62:1913–24.

28.

Dahl

Haug

Spilsberg

Johansen

Ostvold

Diemer

(2000) Reduced [3H]IP3 binding but unchanged IP3 receptor levels in the rat hippocampus CA1 region following transient global ischemia and tolerance induction. Neurochem Int 36:379–88.

29.

De Jongh

Colvin

Wang

Catterall

(1994) Differential proteolysis of the full-length form of the L-type calcium channel alpha 1 subunit by calpain. J Neurochem 63:1558–64.

30.

Dehmelt

Halpain

(2005) The MAP2/Tau family of microtubule-associated proteins. Genome Biol 6:204.

31.

DeMartino

Wachendorfer

McGuire

Croall

(1988) Proteolysis of the protein inhibitor of calcium-dependent proteases produces lower molecular weight fragments that retain inhibitory activity. Arch Biochem Biophys 262:189–98.

32.

Dhavan

Tsai

(2001) A decade of CDK5. Nat Rev Mol Cell Biol 2:749–59.

33.

Dong

Waxman

Lynch

(2004) Interactions of postsynaptic density-95 and the NMDA receptor 2 subunit control calpain-mediated cleavage of the NMDA receptor. J Neurosci 24:11035–45.

34.

Dong

Hsu

Coulter

Lynch

(2006) Developmental and cell-selective variations in AT-methyl-D-aspartate receptor degradation by calpain. J Neurochem 99:206–17.

35.

Donkor

(2000) A survey of calpain inhibitors. Curr Med Chem 7:1171–88.

36.

Dutt

Croall

Arthur

De Veyra

Williams

Elce

Greer

(2006) m-Calpain is required for preimplantation embryonic development in mice. BMC Dev Biol 6:3.

37.

Eliasson

Sampei

Mandir

Hum

Traystman

Bao

Pieper

Wang

Dawson

Snyder

Dawson

(1997) Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia. Nat Med 3:1089–95.

38.

Faddis

Hasbani

Goldberg

(1997) Calpain activation contributes to dendritic remodeling after brief excitotoxic injury in vitro. J Neurosci 17:951–9.

39.

Farkas

Tantos

Schlett

Vilagi

Friedrich

(2004) Ischemia-induced increase in long-term potentiation is warded off by specific calpain inhibitor PD150606. Brain Res 1024:150–8.

40.

Ferris

Snyder

(1992) Inositol 1,4,5-trisphosphate-activated calcium channels. Annu Rev Physiol 54:469–88.

41.

Franco

Perrin

Huttenlocher

(2004) Isoform specific function of calpain 2 in regulating membrane protrusion. Exp Cell Res 299:179–87.

42.

French

Quindry

Falk

Staib

Lee

Wang

Powers

(2006) Ischemia-reperfusion-induced calpain activation and SERCA2a degradation are attenuated by exercise training and calpain inhibition. Am J Physiol Heart Circ Physiol 290:H128–36.

43.

Friedrich

Papp

Halasy

Farkas

Tompa

Kása

(2004) Differential distribution of calpain small subunit 1 and 2 in rat brain. Eur J Neurosci 19:1819–25.

44.

Fukuda

Adachi

Kawashima

Yoshiya

Hashimoto

(1990) Immunohistochemical distribution of calcium-activated neutral proteinases and endogenous CANP inhibitor in the rabbit hippocampus. J Comp Neurol 302:100–9.

45.

Garcia

Bondada

Geddes

(2005) Mitochondrial localization of mu-calpain. Biochem Biophys Res Commun 338:1241–7.

46.

Goll

Thompson

Wei

Cong

(2003) The calpain system. Physiol Rev 83:731–801.

47.

Goñi-Oliver

Lucas

Avila

Hernández

(2007) N-terminal cleavage of GSK-3 by calpain: a new form of GSK-3 regulation. J Biol Chem 282:22406–13.

48.

Grammer

Kuchay

Chishti

Baudry

(2005) Lack of phenotype for LTP and fear conditioning learning in calpain 1 knock-out mice. Neurobiol Learn Mem 84:222–7.

49.

Grynspan

Griffin

Mohan

Shea

Nixon

(1997) Calpains and calpastatin in SH-SY5Y neuroblastoma cells during retinoic acid-induced differentiation and neurite outgrowth: comparison with the human brain calpain system. J Neurosci Res 48:181–91.

50.

Guroff

(1964) A neutral, calcium-activated proteinase from the soluble fraction of rat brain. J Biol Chem 239:149–55.

51.

Guttmann

Baker

Seifert

Cohen

Coulter

Lynch

(2001) Specific proteolysis of the NR2 subunit at multiple sites by calpain. J Neurochem 78:1083–93.

52.

Guttmann

Sokol

Baker

Simpkins

Dong

Lynch

(2002) Proteolysis of the N-methyl-D-aspartate receptor by calpain in situ. J Pharmacol Exp Ther 302:1023–30.

53.

Hajimohammadreza

Raser

Nath

Nadimpalli

Scott

Wang

(1997) Neuronal nitric oxide synthase and calmodulin-dependent protein kinase IIalpha undergo neurotoxin-induced proteolysis. J Neurochem 69:1006–13.

54.

Hall

Andrus

Althaus

Von Voigtlander

(1993) Hydroxyl radical production and lipid peroxidation parallels selective post-ischemic vulnerability in gerbil brain. J Neurosci Res 34:107–12.

55.

Hamakubo

Kannagi

Murachi

Matus

(1986) Distribution of calpains I and II in rat brain. J Neurosci 6:3103–11.

56.

Hell

Westenbroek

Breeze

Wang

Chavkin

Catterall

(1996) AT-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons. Proc Natl Acad Sci USA 93:3362–7.

57.

Herasse

Ono

Fougerousse

Kimura

Stockholm

Beley

Montarras

Pinset

Sorimachi

Suzuki

Beckmann

Richard

(1999) Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events. Mol Cell Biol 19:4047–55.

58.

Hewitt

Lesiuk

Tauskela

Morley

Durkin

(1998) Selective coupling of mu-calpain activation with the NMDA receptor is independent of translocation and autolysis in primary cortical neurons. J Neurosci Res 54:223–32.

59.

Higuchi

Tomioka

Takano

Shirotani

Iwata

Masumoto

Maki

Itohara

Saido

(2005) Distinct mechanistic roles of calpain and caspase activation in neurodegeneration as revealed in mice overexpressing their specific inhibitors. J Biol Chem 280:15229–37.

60.

Honda

Marumoto

Hirota

Nitta

Arima

Ogawa

Saya

(2004) Activation of m-calpain is required for chromosome alignment on the metaphase plate during mitosis. J Biol Chem 279:10615–23.

61.

Hong

Lanzino

Goto

Kang

Schottler

Kassell

Lee

(1994) Calcium-activated proteolysis in rat neocortex induced by transient focal ischemia. Brain Res 661:43–50.

62.

Hood

Brooks

Roszman

(2004) Differential compartmentalization of the calpain/calpastatin network with the endoplasmic reticulum and Golgi apparatus. J Biol Chem 279:43126–35.

63.

Hood

Logan

Sinai

Brooks

Roszman

(2003) Association of the calpain/calpastatin network with subcellular organelles. Biochem Biophys Res Commun 310:1200–12.

64.

Hosfield

Elce

Davies

Jia

(1999) Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation. EMBO J 18:6880–9.

65.

Hou

Jiang

Desbois

Huang

Kelly

Tessier

Karchewski

Kappler

(2006) Calpain-cleaved collapsin response mediator protein-3 induces neuronal death after glutamate toxicity and cerebral ischemia. J Neurosci 26:2241–9.

66.

Igwe

Filla

(1997) Aging-related regulation of myoinositol 1,4,5-trisphosphate signal transduction pathway in the rat striatum. Brain Res Mol Brain Res 46:39–53.

67.

Inoue

Nakamura

Cui

Sakai

Hill

Wang

Yuen

(2003) Structure-activity relationship study and drug profile of N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (SJA6017) as a potent calpain inhibitor. J Med Chem 46:868–71.

68.

Inuzuka

Tamura

Sato

Kirino

Toyoshima

Miyatake

(1990) Suppressive effect of E-64c on ischemic degradation of cerebral proteins following occlusion of the middle cerebral artery in rats. Brain Res 526:177–9.

69.

Irvine

Letcher

Heslop

Berridge

(1986) The inositol tris/tetrakisphosphate pathway—demonstration of Ins(1,4,5)P3 3-kinase activity in animal tissues. Nature 320:631–4.

70.

James

Vorherr

Krebs

Morelli

Castello

McCormick

Penniston

De Flora

Carafoli

(1989) Modulation of erythrocyte Ca2+-ATPase by selective calpain cleavage of the calmodulin-binding domain. J Biol Chem 264:8289–96.

71.

Jourdi

Yanagihara

Lauterborn

Gall

Baudry

(2005) Prolonged positive modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors induces calpain-mediated PSD-95/Dlg/ZO-1 protein degradation and AMPA receptor down-regulation in cultured hippocampal slices. J Pharmacol Exp Ther 314:16–26.

72.

Kishimoto

Mikawa

Hashimoto

Yasuda

Tanaka

Tominaga

Kuroda

Nishizuka

(1989) Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain). J Biol Chem 264:4088–92.

73.

König

Raynaud

Feane

Durand

Mestre-Francès

Rossel

Ouali

Benyamin

(2003) Calpain 3 is expressed in astrocytes of rat and Microcebus brain. J Chem Neuroanat 25:129–36.

74.

Kupina

Nath

Bernath

Inoue

Mitsuyoshi

Yuen

Wang

Hall

(2001) The novel calpain inhibitor SJA6017 improves functional outcome after delayed administration in a mouse model of diffuse brain injury. J Neurotrauma 18:1229–40.

75.

Kusakawa

Saito

Onuki

Ishiguro

Kishimoto

Hisanaga

(2000) Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25. J Biol Chem 275:17166–72.

76.

Lee

Frank

Vanderklish

Arai

Lynch

(1991) Inhibition of proteolysis protects hippocampal neurons from ischemia. Proc Natl Acad Sci USA 88:7233–7.

77.

Lee

Kwon

Peng

Friedlander

Tsai

(2000) Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature 405:360–4.

78.

Lehotský

Kaplán

Racay

Mézesová

Raeymaekers

(1999) Distribution of plasma membrane Ca2+ pump (PMCA) isoforms in the gerbil brain: effect of ischemia-reperfusion injury. Neurochem Int 35:221–7.

79.

Grynspan

Berman

Nixon

Bursztajn

(1996) Regional differences in gene expression for calcium activated neutral proteases (calpains) and their endogenous inhibitor calpastatin in mouse brain and spinal cord. J Neurobiol 30:177–91.

80.

Howlett

Miyashita

Siddiqui

Shuaib

(1998) Postischemic treatment with calpain inhibitor MDL 28170 ameliorates brain damage in a gerbil model of global ischemia. Neurosci Lett 247:17–20.

81.

Liu

Ying

Massa

Duriez

Swanson

Poirier

Sharp

(2000) Effects of transient global ischemia and kainate on poly(ADP-ribose) polymerase (PARP) gene expression and proteolytic cleavage in gerbil and rat brains. Brain Res Mol Brain Res 80:7–16.

82.

Liu

Lau

Wei

Zhu

Zou

Sun

Liu

(2004) Expression of Ca(2+)-permeable AMPA receptor channels primes cell death in transient forebrain ischemia. Neuron 43:43–55.

83.

Rong

Baudry

(2000a) Calpain-mediated degradation of PSD-95 in developing and adult rat brain. Neurosci Lett 286:149–53.

84.

Rong

Baudry

(2000b) Calpain-mediated truncation of rat brain AMPA receptors increases their Triton X-100 solubility. Brain Res 863:143–50.

85.

Lubisch

Beckenbach

Bopp

Hofmann

Kartal

Kästel

Lindner

Metz-Garrecht

Reeb

Regner

Vierling

Möller

(2003) Benzoylalanine-derived ketoamides carrying vinylbenzyl amino residues: discovery of potent water-soluble calpain inhibitors with oral bioavailability. J Med Chem 46:2404–12.

86.

Fukiage

Kim

Duncan

Reed

Shih

Azuma

Shearer

(2001) Characterization and expression of calpain 10. A novel ubiquitous calpain with nuclear localization. J Biol Chem 276:28525–31.

87.

Maki

Takano

Mori

Sato

Murachi

Hatanaka

(1987) All four internally repetitive domains of pig calpastatin possess inhibitory activities against calpains I and II. FEES Lett 223:174–80.

88.

Marcilhac

Raynaud

Clerc

Benyamin

(2006) Detection and localization of calpain 3-like protease in a neuronal cell line: possible regulation of apoptotic cell death through degradation of nuclear IkappaBalpha. Int J Biochem Cell Biol 38:2128–40.

89.

Markgraf

Velayo

Johnson

McCarty

Medhi

Koehl

Chmielewski

Linnik

(1998) Six-hour window of opportunity for calpain inhibition in focal cerebral ischemia in rats. Stroke 29:152–8.

90.

McGinnis

Gnegy

Park

Mukerjee

Wang

(1999) Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates. Biochem Biophys Res Commun 263:94–9.

91.

Mellgren

(1994) Selective nuclear transport of mu-calpain. Biochem Biophys Res Commun 204: 544–550.

92.

Moyen

Goudenege

Poussard

Sassi

Brustis

Cottin

(2004) Involvement of micro-calpain (CAPN 1) in muscle cell differentiation. Int J Biochem Cell Biol 36:728–43.

93.

Nagata

Tanaka

Gomi

Mihara

Shirai

Nogawa

Nozaki

Mikoshiba

Fukuuchi

(1994) Alteration of inositol 1,4,5-trisphosphate receptor after six-hour hemispheric ischemia in the gerbil brain. Neuroscience 61:983–90.

94.

Nakagawa

Yuan

(2000) Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J Cell Biol 150:887–94.

95.

Nakayama

Yano

Ushijima

Abe

Terasaki

(2002) Effects of dantrolene on extracellular glutamate concentration and neuronal death in the rat hippocampal CA1 region subjected to transient ischemia. Anesthesiology 96:705–10.

96.

Nath

Davis

Probert

Kupina

Ren

Schielke

Wang

(2000) Processing of cdk5 activator p35 to its truncated form (p25) by calpain in acutely injured neuronal cells. Biochem Biophys Res Commun 274: 16–21.

97.

Neuhof

Götte

Trumbeckaite

Attenberger

Kuzkaya

Gellerich

Möller

Lubisch

Speth

Tillmanns

Neuhof

(2003) A novel water-soluble and cell-permeable calpain inhibitor protects myocardial and mitochondrial function in postischemic reperfusion. Biol Chem 384:1597–603.

98.

Neumar

(200) Molecular mechanisms of ischemic neuronal injury. Ann Emerg Med 36:483–506.

99.

Neumar

Hagle

DeGracia

Krause

White

(1996) Brain mu-calpain autolysis during global cerebral ischemia. J Neurochem 66:421–4.

100.

Neumar

Meng

Mills

Zhang

Welsh

Siman

(2001) Calpain activity in the rat brain after transient forebrain ischemia. Exp Neurol 170: 27–35.

101.

Neumar

Gada

Guttmann

Siman

(2003) Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis. J Biol Chem 278:14162–7.

102.

Nozaki

Tanaka

Gomi

Mihara

Nogawa

Nagata

Kondo

Fukuuchi

(1999) Role of the ryanodine receptor in ischemic brain damage—localized reduction of ryanodine receptor binding during ischemia in hippocampus CA1. Cell Mol Neurobiol 19:119–31.

103.

Nozaki

Tanaka

Nagata

Kondo

Koyama

Dembo

Fukuuchi

(1997) Rapid reduction in ryanodine binding of hippocampus CA1 in cerebral ischemia. Keio J Med 46:85–9.

104.

O'Hare

Kushwaha

Zhang

Aleyasin

Callaghan

Slack

Albert

Vincent

Park

(2005) Differential roles of nuclear and cytoplasmic cyclin-dependent kinase 5 in apoptotic and excitotoxic neuronal death. J Neurosci 25:8954–66.

105.

Oguro

Nakamura

Masuzawa

(1995) Histochemical study of Ca(2+)-ATPase activity in ischemic CA1 pyramidal neurons in the gerbil hippocampus. Acta Neuropathol 90:448–53.

106.

Ohno

Emori

Imajoh

Kawasaki

Kisaragi

Suzuki

(1984) Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein? Nature 312:566–70.

107.

Ostwald

Hayashi

Nakamura

Kawashima

(1994) Subcellular distribution of calpain and calpastatin immunoreactivity and fodrin proteolysis in rabbit hippocampus after hypoxia and glucocorticoid treatment. J Neurochem 63:1069–76.

108.

Pal

De Veyra

Elce

Jia

(2003) Crystal structure of a micro-like calpain reveals a partially activated conformation with low Ca2+ requirement. Structure 11:1521–6.

109.

Pariat

Carillo

Molinari

Salvat

Debüssche

Bracco

Milner

Piechaczyk

(1997) Proteolysis by calpains: a possible contribution to degradation of p53. Mol Cell Biol 17:2806–15.

110.

Parsons

Churn

DeLorenzo

(1999) Global ischemia-induced inhibition of the coupling ratio of calcium uptake and ATP hydrolysis by rat whole brain microsomal Mg(2+)/Ca(2+) ATPase. Brain Bes 834: 32–41.

111.

Pattni

Millard

Banting

(2003) Calpain cleavage of the B isoform of Ins(1,4,5)P3 3-kinase separates the catalytic domain from the membrane anchoring domain. Biochem J 375:643–51.

112.

Patzke

Tsai

(2002) Calpain-mediated cleavage of the cyclin-dependent kinase-5 activator p39 to p29. J Biol Chem 277:8054–60.

113.

Pellegrini-Giampietro

Gorter

Bennett

Zukin

(1997) The GluR2 (GluR-B) hypothesis: Ca(2+)-permeable AMPA receptors in neurological disorders. Trends Neurosci 20:464–70.

114.

Pellicciari

Camaioni

Costantino

(2004) 3. Life or death decisions: the cast of poly(ADP-ribose)polymerase (PARP) as a therapeutic target for brain ischaemia. Prog Med Chem 42:125–69.

115.

Perlmutter

Gall

Baudry

Lynch

(1990) Distribution of calcium-activated protease calpain in the rat brain. J Comp Neurol 296:269–76.

116.

Perlmutter

Siman

Gall

Seubert

Baudry

Lynch

(1988) The ultrastructural localization of calcium-activated protease ‘calpain’ in rat brain. Synapse 2:79–88.

117.

Pettigrew

Holtz

Craddock

Minger

Hall

Geddes

(1996) Microtubular proteolysis in focal cerebral ischemia. J Cereb Blood Flow Metab 16:1189–202.

118.

Piantadosi

Zhang

(1996) Mitochondrial generation of reactive oxygen species after brain ischemia in the rat. Stroke 27:327–31; discussion 332.

119.

Polster

Basañez

Etxebarria

Hardwick

Nicholls

(2005) Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria. J Biol Chem 280:6447–54.

120.

Pontremoli

Melloni

Salamino

Patrone

Michetti

Horecker

(1989) Activation of neutrophil calpain following its translocation to the plasma membrane induced by phorbol ester or fMet-Leu-Phe. Biochem Biophys Res Commun 160:737–43.

121.

Pottorf

Johanns

Derrington

Strehler

Enyedi

Thayer

(2006) Glutamate-induced protease-mediated loss of plasma membrane Ca2+ pump activity in rat hippocampal neurons. J Neurochem 98:1646–56.

122.

Rami

Ferger

Krieglstein

(1997) Blockade of calpain proteolytic activity rescues neurons from glutamate excitotoxicity. Neurosci Res 27:93–7.

123.

Rami

Krieglstein

(1993) Protective effects of calpain inhibitors against neuronal damage caused by cytotoxic hypoxia in vitro and ischemia in vivo. Brain Bes 609:67–70.

124.

Rardon

Cefali

Mitchell

Seiler

Hathaway

Jones

(1990) Digestion of cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles with calpain II. Effects on the Ca2+ release channel. Circ Res 67:84–96.

125.

Reimertz

Kögel

Lankiewicz

Poppe

Prehn

(2001) Ca(2+)-induced inhibition of apoptosis in human SH-SY5Y neuroblastoma cells: degradation of apoptotic protease-activating factor-1 (APAF-1). J Neurochem 78:1256–66.

126.

Roberts-Lewis

Savage

Marcy

Pinsker

Siman

(1994) Immunolocalization of calpain I-mediated spectrin degradation to vulnerable neurons in the ischemic gerbil brain. J Neurosci 14:3934–44.

127.

Ruiz-Vela

González de Buitrago

Martínez-A

(1999) Implication of calpain in caspase activation during B cell clonal deletion. EMBO J 18:4988–98.

128.

Saido

Yokota

Nagao

Yamaura

Tani

Tsuchiya

Suzuki

Kawashima

(1993) Spatial resolution of fodrin proteolysis in postischemic brain. J Biol Chem 268:25239–43.

129.

Saito

Nixon

(1993) Specificity of calcium-activated neutral proteinase (CANP) inhibitors for human mu CANP and mCANP. Neurochem Res 18:231–3.

130.

Saito

Konno

Hosokawa

Asada

Ishiguro

Hisanaga

(2007) p25/Cyclin-dependent kinase 5 promotes the progression of cell death in nucleus of endoplasmic reticulum-stressed neurons. J Neurochem 102:133–40.

131.

Salamino

De Tullio

Michetti

Mengotti

Melloni

Pontremoli

(1994a) Modulation of calpastatin specificity in rat tissues by reversible phosphorylation and dephosphorylation. Biochem Biophys Res Commun 199:1326–32.

132.

Salamino

Sparatore

Melloni

Michetti

Viotti

Pontremoli

Carafoli

(1994b) The plasma membrane calcium pump is the preferred calpain substrate within the erythrocyte. Cell Calcium 15:28–35.

133.

Sanges

Comitato

Tammaro

Marigo

(2006) Apoptosis in retinal degeneration involves cross-talk between apoptosis-inducing factor (AIF) and caspase-12 and is blocked by calpain inhibitors. Proc Natl Acad Sci USA 103:17366–71.

134.

Satish

Blair

Glading

Wells

(2005) Interferon-inducible protein 9 (CXCL11)-induced cell motility in keratinocytes requires calcium flux-dependent activation of mu-calpain. Mol Cell Biol 25:1922–41.

135.

Schád

Farkas

Jékely

Tompa

Friedrich

(2002) A novel human small subunit of calpains. Biochem J 362:383–8.

136.

Sengoku

Bondada

Hassane

Dubal

Geddes

(2004) Tat-calpastatin fusion proteins transduce primary rat cortical neurons but do not inhibit cellular calpain activity. Exp Neurol 188:161–70.

137.

Shiga

Onodera

Matsuo

Kogure

(1992) Cyclosporin A protects against ischemia-reperfusion injury in the brain. Brain Res 595:145–8.

138.

Shioda

Moriguchi

Shirasaki

Fukunaga

(2006) Generation of constitutively active calcineurin by calpain contributes to delayed neuronal death following mouse brain ischemia. J Neurochem 98:310–20.

139.

Siman

Gall

Perlmutter

Christian

Baudry

Lynch

(1985) Distribution of calpain I, an enzyme associated with degenerative activity, in rat brain. Brain Res 347:399–403.

140.

Siman

McIntosh

Soltesz

Chen

Neumar

Roberts

(2004) Proteins released from degenerating neurons are surrogate markers for acute brain damage. Neurobiol Dis 16:311–20.

141.

Siman

Noszek

(1988) Excitatory amino acids activate calpain I and induce structural protein breakdown in vivo. Neuron 1:279–87.

142.

Simpkins

Guttmann

Dong

Chen

Sokol

Neumar

Lynch

(2003) Selective activation induced cleavage of the NR2B subunit by calpain. J Neurosci 23:11322–31.

143.

Song

Malmstrom

Kater

Mykles

(1994) Calpain inhibitors block Ca(2+)-induced suppression of neurite outgrowth in isolated hippocampal pyramidal neurons. J Neurosci Res 39:474–81.

144.

Strobl

Fernandez-Catalan

Braun

Huber

Masumoto

Nakagawa

Irie

Sorimachi

Bourenkow

Bartunik

Suzuki

Bode

(2000) The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proc Natl Acad Sci USA 97: 588–92.

145.

Syntichaki

Driscoll

Tavernarakis

(2002) Specific aspartyl and calpain proteases are required for neurodegeneration in C elegans. Nature 419:939–44.

146.

Takano

Masatoshi

Yuen

(1999) Structure of calpastatin and its inhibitory control of calpain. In: Calpain: pharmacology and toxicology of calcium-dependent protease ( Wang

KKW

, ed), Philadelphia, PA: Taylor & Francis, 25–50.

147.

Takano

Tomioka

Tsubuki

Higuchi

Iwata

Itohara

Maki

Saido

(2005) Calpain mediates excitotoxic DNA fragmentation via mitochondrial pathways in adult brains: evidence from calpastatin mutant mice. J Biol Chem 280:16175–84.

148.

Tomimatsu

Idemoto

Moriguchi

Watanabe

Nakanishi

(2002) Proteases involved in long-term potentiation. Life Sci 72:355–61.

149.

Tompa

Buzder-Lantos

Tantos

Farkas

Szilágyi

Bánóczi

Hudecz

Friedrich

(2004) On the sequential determinants of calpain cleavage. J Biol Chem 279:20775–85.

150.

Tompa

Emori

Sorimachi

Suzuki

Friedrich

(2001) Domain III of calpain is a Ca2+-regulated phospholipid-binding domain. Biochem Biophys Res Commun 280:1333–9.

151.

Tremper-Wells

Vallano

(2005) Nuclear calpain regulates Ca2+-dependent signaling via proteolysis of nuclear Ca2+/calmodulin-dependent protein kinase type IV in cultured neurons. J Biol Chem 280:2165–75.

152.

Trumbeckaite

Neuhof

Zierz

Gellerich

(2003) Calpain inhibitor (BSF 409425) diminishes ischemia/reperfusion-induced damage of rabbit heart mitochondria. Biochem Pharmacol 65:911–6.

153.

Uchino

Elmér

Uchino

Lindvall

Siesjö

(1995) Cyclosporin A dramatically ameliorates CA1 hippocampal damage following transient forebrain ischaemia in the rat. Acta Physiol Scand 155:469–71.

154.

Vinade

Petersen

Dosemeci

Reese

(2001) Activation of calpain may alter the postsynaptic density structure and modulate anchoring of NMDA receptors. Synapse 40:302–9.

155.

Volbracht

Chua

Bahr

Hong

(2005) The critical role of calpain versus caspase activation in excitotoxic injury induced by nitric oxide. J Neurochem 93:1280–92.

156.

Wang

Yuen

(1999) Calpain substrates, assay methods, regulation and its inhibitory agents. In: Calpain: pharmacology and toxicology of calcium-dependent protease ( Wang

KKW

Yuen

, eds), Philadelphia, PA: Taylor & Francis, 77–102.

157.

Wang

Posmantur

Nadimpalli

Nath

Mohan

Nixon

Talanian

Keegan

Herzog

Allen

(1998) Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. Arch Biochem Biophys 356:187–96.

158.

Wang

Posner

Raser

Buroker-Kilgore

Nath

Hajimohammadreza

Probert

Marcoux

Lunney

Hays

Yuen

P-W

(1996) Alpha-mercaptoacrylic acid derivatives as novel selective calpain inhibitors. Adv Exp Med Biol 389:95–101.

159.

Wei

Perry

(1996) Dantrolene is cytoprotective in two models of neuronal cell death. J Neurochem 67: 2390–8.

160.

Wei

Neely

Lacerda

Olcese

Stefani

Perez-Reyes

Birnbaumer

(1994) Modification of Ca2+ channel activity by deletions at the carboxyl terminus of the cardiac alpha 1 subunit. J Biol Chem 269:1635–40.

161.

Tomizawa

Oda

Wei

Matsushita

Moriwaki

Matsui

(2004) Critical role of calpain-mediated cleavage of calcineurin in excitotoxic neurodegeneration. J Biol Chem 279:4929–40.

162.

Yuen

Matsushita

Matsui

Yan

Tomizawa

(2005) Regulation of N-methyl-D-aspartate receptors by calpain in cortical neurons. J Biol Chem 280:21588–93.

163.

Fan

Caron

Whiteway

Shen

(2006) Functional dissection of human protease mu-calpain in cell migration using RNAi. FEBS Lett 580:3246–56.

164.

Wong

Chery

Gaertner

Wang

Baudry

(2007) Calpain-mediated mGluR1alpha truncation: a key step in excitotoxicity. Neuron 53:399–412.

165.

Yamashima

(2004) Ca2+-dependent proteases in ischemic neuronal death: a conserved ‘calpain-cathepsin cascade’ from nematodes to primates. Cell Calcium 36: 285–93.

166.

Yokota

Tani

Tsubuki

Yamaura

Nakagaki

Hori

Saido

(1999) Calpain inhibitor entrapped in liposome rescues ischemic neuronal damage. Brain Res 819:8–14.

167.

Andrabi

Wang

Kim

Poirier

Dawson

(2006) Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death. Proc Natl Acad Sci USA 103:18314–9.

168.

Yuen

Yan

(2007) Calpain regulation of AMPA receptor channels in cortical pyramidal neurons. J Physiol 580:241–54.

169.

Zaidan

Sims

(1994) The calcium content of mitochondria from brain subregions following short-term forebrain ischemia and recirculation in the rat. J Neurochem 63:1812–9.

170.

Zakharov

Bogdanova

Mosevitsky

(2005) Specific proteolysis of neuronal protein GAP-43 by calpain: characterization, regulation, and physiological role. Biochemistry (Mosc) 70:897–907.

171.

Zhang

Ottens

Sadasivan

Kobeissy

Fang

Hayes

Wang

(2007) Calpain-mediated collapsin response mediator protein-1, -2, and -4 proteolysis after neurotoxic and traumatic brain injury. J Neurotrauma 24:460–72.

172.

Ziemka-Nalecz

Zalewska

Zajac

Domañska-Janik

(2003) Decrease of PKC precedes other cellular signs of calpain activation in area CA1 of the hippocampus after transient cerebral ischemia. Neurochem Int 42:205–14.

173.

Zimmerman

Boring

Pak

Mukerjee

Wang

(2000) The calpain small subunit gene is essential: its inactivation results in embryonic lethality. IUBMB Life 50:63–8.

Mechanistic Role of Calpains in Postischemic Neurodegeneration

Abstract

Keywords

Introduction

Overview of the Calpain—Calpastatin System

Physiologic Activity

Localization

Role of Calpain in Postischemic Neurodegeneration

Subcellular Consequences of Pathologic Calpain Activity

Plasma Membrane and Synapse

Endoplasmic Reticulum

Mitochondria

Lysosomes

Nucleus

Summary

References