ReviewNeuroscience

Calpain in the CNS: From Synaptic Function to Neurotoxicity

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Sci. Signal.  08 Apr 2008:
Vol. 1, Issue 14, pp. re1
DOI: 10.1126/stke.114re1

Abstract

The calpains are a class of cellular cysteine proteases that require calcium and are functionally active at neutral pH. Calpain activation can take place in two modes: controlled activation under physiological conditions (in which only a few molecules of calpain are activated per cell), and hyperactivation under pathological conditions that involve sustained calcium overload (in which all available calpain molecules are activated). Regulated activation of calpain in the central nervous system (CNS) may be critical to synaptic function and memory formation, with possible substrates including various structural and scaffolding proteins, enzymes, and glutamate receptors. Hyperactivation of calpain in the central nervous system is generally associated with severe cellular challenge or damage. Calpain cleavage products may thus provide useful biomarkers for the presence of neurodegenerative processes or neuronal injury.

The Calpain Cysteine Proteases

The calpains constitute a family of cellular cysteine proteases that are activated by calcium at neutral pH (1, 2). Thus, unlike the cathepsins, which are not calcium dependent and are active in acidic compartments like lysosomes (3), calpains can be activated by calcium in the cytosol. An intriguing feature of calpains as regulators of cellular processes is that, being proteases, calpain-mediated protein modifications must be unidirectional.

The first two isoforms of calpain identified—calpain 1 [consisting of the calpain 1 large catalytic subunit (protein accession number P07384) and small subunit, CSS1 (protein accession number P04632), or its isoform CSS2 (protein accession number Q96L46)], and calpain 2 [consisting of the calpain 2 large subunit (protein accession number P17655) and a small subunit for which the structure has not yet been determined] (1, 4, 5)—are found in most organs, but are particularly abundant in the central nervous system (CNS). Calpain 1 (μ-calpain) and calpain 2 (m-calpain) differ in the calcium concentration required for their activation, being activated by micromolar (1 to 20 μM) or millimolar (0.250 to 0.750 mM) calcium concentrations (6), respectively, but they have similar physiological functions and pathological actions (7, 8). Several of the 15 calpain family members identified in humans have been implicated in pathological conditions, including traumatic brain and organ injury (calpain 1 and 2) (1); limb-girdle muscular dystrophy and Duchenne muscular dystrophy (calpain 1, 2, and 3) (9, 10); susceptibility to type 2 diabetes (calpain 10) (11); gastric cancer (calpain 9); and cataract formation (calpain 1 and 2) (1, 10). However, whereas calpain 1 and 2 have been extensively studied (4), with the exception of the skeletal muscle–localized calpain 3 (12), little is known about the physiological functions and substrates of the other human calpains.

All calpains can act in two modes: Under physiological conditions they undergo controlled activation (involving only a few molecules of calpain), whereas during sustained calcium overload under pathological conditions they undergo hyperactivation (involving all available calpain molecules). Calpain 1 and 2 are somewhat selective with regard to substrate selection; only about 5% of cellular proteins are degraded or fragmented by these calpains even under extreme conditions (13). Calpain substrates that have been linked to physiological functions outside of the CNS include focal adhesion kinase (FAK) and ezrin, which are involved in fibroblast cell attachment and mobility (14, 15), and phosphotyrosine phosphatase 1 B (PTP1B), which is involved in platelet aggregation (16). In muscle, troponin-I and -T, which regulate actin-myosin interactions, are calpain substrates that, when degraded, could contribute to destabilization and degradation of myofibers themselves (17, 18). In the CNS (1922), controlled activation of calpain, possibly in a sub-plasma membrane microenvironment, might be critical to synaptic function and memory formation. Possible postsynaptic substrates include αII- and βII-spectrin, the postsynaptic density–95 (PSD95) scaffolding protein, calcium- and calmodulin-dependent protein kinase II (CaMKII), and the N-methyl-𝒹-aspartate (NMDA)-type and other glutamate receptors (23, 24) (Fig. 1).

Fig. 1.

In glutaminergic neurons (A), a physiological increase in presynaptic calcium concentration results in controlled glutamate release, eliciting a transient increase in synaptic glutamate (small green spheres), which is cleared by a glutamate transporter (green oval in presynaptic membrane) and on glial cell membrane (not shown) (B). NMDA receptor activation leads to a transient increase in postsynaptic calcium concentration (red), allowing calcium (small red sphere) to bind to and activate calpain (C). The activated calcium-bound form of calpain (yellow) cleaves postsynaptic cytoskeletal and scaffolding proteins, thereby promoting reorganization of the PSD and synaptic remodeling (D). In addition, calpain-mediated cleavage of β-catenin (E) and SCOP (F) may stimulate gene transcription (G) and thereby the production of proteins involved in synaptic remodeling, and calpain returns to its inactive state as intracellular calcium concentration returns to normal, as a result of calcium removal by the Na+/Ca2+ exchanger-3 NCX3 and both the plasma membrane and endoplasmic reticulum calcium pumps (not shown) (H). [See also supplemental Animation 1 (http://stke.sciencemag.org/cgi/content/full/sigtrans;1/14/re1/DC1)]

Physiological Functions of Calpains in the CNS

Calpains in long-term potentiation of synaptic function.

Long-term potentiation (LTP) is a form of synaptic plasticity that is widely hypothesized to underlie memory; it resembles memory in that it is initially unstable and then, over about 30 min, becomes increasingly resistant to disruption (25). LTP is accompanied by anatomic changes in synapses and specifically in dendritic spines and postsynaptic densities (PSDs) (26). This synaptic remodeling, which is thought to account for the long duration of LTP, may involve the calpain-mediated proteolysis of spectrin (19), which is known to regulate the proteins expressed on the cell surface and morphology of cells. This is supported by the isolation of calpain 1 from synaptosomal plasma membranes (27) and the inhibition of LTP by administration of the nonspecific cysteine proteinase inhibitor leupeptin (28). Spectrin degradation would be expected to disrupt the spine cytoskeleton as well as organization of the PSD, a process that underlies the initial phase of LTP (29).

LTP also involves the rapid insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)–type glutamate receptors into the postsynaptic membrane (30). AMPA receptors are stabilized within the PSD through interaction with a family of anchoring proteins, and calpain 1 may participate in the process of receptor mobilization by modifying the structure of glutamate receptor anchoring molecules. Calpain activation results in truncation of the glutamate receptor–interacting protein (GRIP) anchoring protein, disrupting GRIP interactions with the AMPA glutamate receptor 2 (GluR2) subunit. This results in the rapid insertion of AMPA receptors into postsynaptic membranes (31). Furthermore, by cleaving PSD95, calpain 2 activation could produce changes in the organization of the PSD, thereby modifying the anchoring of NMDA receptors and enhancing the efficacy of synaptic functions (20) (Fig. 1).

LTP stabilization requires the synthesis of new proteins (25), and memory consolidation may depend on activation of the mitogen-activated protein kinase (MAPK) signaling pathway and the subsequent synthesis of new proteins by way of cAMP response element (CRE)–mediated transcription (32) and dendritic translation (33). Suprachiasmatic nucleus circadian oscillatory protein (SCOP), which is degraded by both calpain 1 and calpain 2, inhibits MAPK (34) and CRE-binding protein (CREB)–mediated transcription (35) by binding to K-Ras and blocking its activity. In the hippocampus, exposure to a calpain inhibitor elicits an increase in the abundance of SCOP protein and greatly reduces MAPK activity. SCOP degrades after training for novel object memory, and overexpression of SCOP in the hippocampus blocks memory for novel objects (35). Thus, calpain-catalyzed degradation of SCOP in response to activity-dependent increases in Ca2+ concentration appear to contribute to activation of MAPK during the formation of hippocampus-dependent memory (Fig. 1). Because of the broad role of MAPK and CRE-mediated transcription in neuroplasticity, other plastic processes may also be influenced by SCOP protein and its regulated degradation by calpain.

Calpain and β-catenin signaling.

Recently, β-catenin was added to the list of calpain substrates found in neurons (21) . Regulation of gene expression by β-catenin depends on Wnt–glycogen synthase kinase 3β (Gsk-3β) signaling (36). Without Wnt signals, GSK-3β constitutively phosphorylates the N-terminal region of cytosolic β-catenin, leading to its degradation. Wnt inhibites GSK-3β activity, thereby suppressing these proteolytic processes. NMDA receptor–dependent calpain activation results in N-terminal truncation of β-catenin, thereby removing the target for β-catenin phosphorylation by the constitutively active kinase GSK-3β (21). β-Catenin is thus made resistant to subsequent proteolysis and translocates to the nucleus, where it activates gene transcription by associating with Tcf/Lef (T cell factor/lymphoid enhancer factor) family transcription factors (36).

As a component of the cadherin-catenin complex, β-catenin is also essential for the stability of synaptic junctions (37). Calpain cleaves β-catenin in the cadherin-catenin complex (37); the resultant cleaved β-catenin is unable to stably associate with cadherin, and is no longer a substrate for GSK-3β. Thus, by cleaving β-catenin, calpain could stimulate both Tcf-dependent gene transcription and the structural modulation of synapses (Fig. 1). This would allow neurons to utilize β-catenin as a mediator of activity-dependent gene expression.

Calpain-induced burst firing.

In dopaminergic neurons, calpain proteases activated in response to Ca2+ influx through L-type voltage-gated calcium channels (VGCCs) cleave the protein kinase C (PKC) catalytic domain from the regulatory domain, releasing the short-lived, constitutively active protein kinase M (PKM) fragment (22), which regulates the development and maintenance of burst firing, a process that enhances synaptic dopamine release in response to signal novelty and salience (38) (Fig. 2). Exposure to a calpain inhibitor completely prevents burst firing induced by a Ca2+ channel opener. PKC has also been implicated in addiction and motivation (39, 40); calpain, which could proteolyze various PKC isoforms into their PKM counterparts, could thus play an important role in regulating these functions through the activation of PKM. PKM is also associated with learning and memory, suggesting that calpains could contribute to these processes through mechanisms independent of their contribution to LTP (41).

Fig. 2.

In dopaminergic neurons, Ca2+ influx through L-type voltage-gated calcium channels (VGCCs) activates calpain proteases, which cleave the protein kinase C (PKC) catalytic domain from the regulatory domain, releasing the short-lived, constitutively active protein kinase M (PKM) fragment (16), which regulates the development and maintenance of burst firing.

It is also important to point out that physiologic activation is calpain is short-lived. Through the combined activities of the plasma membrane and enodplasmic reticulum (ER) calcium pumps and sodium-calcium exchanger (e.g. NCX-3), transiently elevated intracullar free calcium levels are usually rapidly returned to normal. When this occurs, calpain molecules returns to its inactive resting state (Fig. 1).

Calpains in CNS Pathology

Activation of calpain 1 and 2 in the CNS in the hyperactivated mode in response to a sustained increase in intracellular calcium concentration, however, is generally secondary to severe cellular challenge or damage due to substantial physical trauma, or to chemical or ischemic insults

Furthermore, calpain activation has been implicated in several chronic neurodegenerative conditions, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD). There is widespread activation of calpains in the brain in AD (42), and calpains modulate processes that govern the function and metabolism of key proteins implicated in the pathogenesis of AD, including tau and amyloid precursor protein (APP), as well as various cytoskeletal proteins (18).

Activated calpain is also increased in HD, and its activity is enhanced by NMDA receptor activation (43). The Huntingtin protein (Htt) fragments generated by calpain cleavage are smaller than those generated from caspase cleavage; they can translocate to the nucleus and are therefore more toxic to cells (44). Calpain-resistant Htt mutants are less toxic to cells than is Htt in an in vitro cell culture model (45). Memantine, which leads to decreased concentrations of μ-calpain, reduces striatal cell death in HD (46).

There is increased calpain-related proteolytic activity in postmortem midbrain tissues from human PD cases that is not apparent in age-matched control subjects. Inhibition of calpains prevents neuronal and behavioral deficits in a N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD (47).

The following sections focus on calpain activation in response to calcium overload under conditions of acute neuroinjury. Under these circumstances, extensive proteolysis of functionally or structurally critical brain proteins leads to neuronal cell death and the loss of tissue and function (26).

Calcium overload in excitoxicity.

In the CNS, glutamate is the major excitatory neurotransmitter. If the concentration of synaptic glutamate ([Glu]s) is allowed to accumulate, however, it will pass a threshold and become neurotoxic (48). Excessive [Glu]s will overactivate various postsynaptic ionotrophic glutamate receptors (ligand-gated Na+ and Ca2+ channels), including the AMPA receptor and kainate receptor (KA-R) subtypes, as well as NMDA receptors (which are voltage dependent as well as ligand gated) (Fig. 3). Ca2+ influx through ionotrophic glutamate receptors contributes directly to an increase in the intracellular free calcium concentration ([Ca2+]i), whereas Na+ influx depolarizes the postsynaptic membrane, leading to opening of VGCCs and thereby further calcium influx (49). NMDA receptor activation can also follow AMPA receptor activation when Mg2+ block is relieved secondary to depolarization (50). [Glu]s also activates postsynaptic metabotropic Glu receptors (mGluRs), which are heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (51). The most notable example is mGluR1α, a class of metabotropic glutamate receptors that activate the phosphoinositide 3-kinase PI3K-Akt pathway; this is generally viewed as a neuroprotective mechanism or safeguard (see below).

Fig. 3.

In glutamatergic neurons (A) subjected to pathological conditions (B) (indicated by orange color), calcium (red) floods into the nerve terminal (C). This results in excessive synaptic glutamate release, which is exacerbated by decreased ATP synthesis and the ensuing decline in glutamate uptake. Glutamate then binds to various glutamate receptors. Sodium influx (blue spheres) through the AMPA receptor depolarizes the postsynaptic cell, enabling NMDA receptor activation. Calcium enters through the NMDA receptor and may also enter through voltage-gated calcium channels (VGCC, yellow) once the plasma membrane is sufficiently depolarized. Glutamate activation of the mGluR1α metabotropic glutamate receptor (D) stimulates activation of the neuroprotective PI3K-AKT pathway as well as stimulating the production of IP3 (small yellow sphere) (E) by phospholipase C (PLC, blue circle). IP3 stimulates the release of additional calcium from ER stores (F). Calpain cleavage of the C-terminal cytoplasmic tail of mGluR1α abrogates its activation of PI3K-AKT signaling (G).The calcium overload resulting from all of these processes triggers pathological calpain activation, leading to extensive neuroprotein degradation, inactivation of the Na+/Ca2+ exchanger (NCX3, pale blue oval) (76), and neuronal death (H). Protein breakdown products released into the extracellular compartment could be used as biomarkers for neuronal injury. [See also supplemental Animation 2 (http://stke.sciencemag.org/cgi/content/full/sigtrans;1/14/re1/DC1)]

"Excitotoxic" conditions are commonly found when the brain or spinal cord is challenged chemically, physically, or by ischemic or hypoxic conditions. For example, in stroke or other hypoxic conditions (such as coronary artery bypass graft, cardiac arrest, or hypoxia during birth), the lack of an adequate supply of oxygen to certain regions of the brain will result in impaired mitochondrial oxidative phosphorylation and thus decreased adenosine 5′-triphosphate (ATP) synthesis (52). Low cellular ATP concentrations will in turn impair active glutamate reuptake by glial cells and neurons (an ATP-dependent process) (Fig. 2), exacerbating buildup of [Glu]s. Physical trauma, as in traumatic brain injury (TBI) or spinal cord injury (SCI), can cause rapid local tissue necrosis, triggering massive nonspecific release of glutamate and physical disruption of local blood flow as TBI or SCI also results in additional ischemic insults.

Neurotoxins and calcium overload.

After treatment with neurotoxins, calcium- and calmodulin-dependent protein kinase II-α (CaMK-IIα) and neuronal nitric oxide synthase (nNOS) are proteolytically cleaved by calpain. This proteolytic degradation is reduced by calpain inhibitors (acetyl-Leu-Leu-Nle-CHO and PD151746). Several other calpain-sensitive calmodulin-binding proteins (the plasma membrane calcium pump [Ca2+-ATPase) (50), microtubule-associated protein 2, and calcineurin A] and protein kinase C-α are also degraded by calpain in neurotoxin-treated cultures (53).

Several neurotoxins are also known to exert detrimental effects through mitochondrial impairment. For example, the MPTP metabolite 1-methyl-4-phenylpyridinium (MPP+) (which mimics parkinsonism in animals), 3-nitropropionate, methamphetamine (METH), 3,4-methylenedioxymethamphetamine (MDMA, commonly called Ecstasy), and even dopamine (DA) target mitochondria and impair their function, triggering oxidative stress and calcium release either directly from the mitochondria or by means of triggered calcium-induced or free radical–enhanced calcium release from other calcium stores or through calcium influx through the plasma membrane (or both) (5456). METH and MDMA also appear to block dopamine reuptake, thus increasing [DA]s, which is also neurotoxic (57). Mitochondrial impairment can also lead to a decrease in cellular ATP concentrations, resulting in a vicious cycle of excessive glutamate (or DA) buildup in the synapse and the reduced ability to control [Ca2+]i by plasma membrane Ca2+ pumps, Na+-Ca2+ exchange and the endoplasmic reticulum (ER) calcium pump, and possible direct Ca2+ release from the mitochondria (58). The overall scenario is that neurotoxic conditions lead to a sustained increase in [Ca2+]i, especially in the postsynaptic regions of the cell body and dendrites (58). Over time, intra-axonal calcium will also be increased by propagation through calcium waves (59).

Calpain hyperactivation and structural proteins.

Sustained calcium overload leads to overactivation of calpains, which attack various structural proteins and enzymes throughout the cell, as well as other proteins important for neuronal functions (19). Among the key structural proteins known to act as calpain substrates is microtubule-associated protein 2 (MAP2). MAP2, which is one of the neuronal proteins most vulnerable to calpain, is usually extensively degraded in response to calcium, which likely results in destabilization of microtubules in the cell body and dendrites (60, 61) (Fig. 2). More recently, collapsin response mediator protein 2 (CRMP2), a structural protein that modulates neurite extension, has also been identified as a calpain substrate (62). When truncated by calpain, CRMP2 appears to redistribute to the cell body, a change that might be expected to have functional implications.

When the increase in [Ca2+]i extends to the axons of postsynaptic neurons, a subset of axonal structural proteins appears to be vulnerable to calpain attack. These include αII-spectrin, the microtubule-associated protein tau, and neurofilament protein triplets (H, M and L) (63). Like MAP2, tau is extensively degraded by calpain, likely resulting in destabilization of microtubule networks within the axon. αII-spectrin, together with βII-spectrin, although not present exclusively in the CNS (or in neurons), is highly enriched in central neurons. Degradation of αII- and βII-spectrin is followed by collapse of the cortical spectrin cytoskeleton (64). The αII- and βII-spectrin heterotetramer is part of the sub–plasma membrane actin-binding network that also interacts with the neurofilaments. Neurofilaments (NFs) are neuron-specific intermediate filaments responsible for the maintenance of axonal structure. They are composed of three NF proteins (H, M and L), with NF-L being perhaps the most vulnerable to calpain degradation (65) (Fig. 3). The combined αII- and βII-spectrin and NF degradation results in compromised axonal structure and axonal transport capability (66).

Calpain hyperactivation and glutamate receptors.

Another noteworthy group of postsynaptic calpain substrates are the glutamate receptors. Several isoforms of the ionotropic NMDA, AMPA, and KA receptors have been reported to undergo calpain-mediated truncation under excitotoxic conditions (23). It has been suggested that these truncations further enhance calcium entry.

The mGluR1α metabotropic glutamate receptor has the dual capability of being neuroprotective and contributing to neurotoxicity (67). Through G protein–mediated phospholipase C activation, leading to the production of inositol 1,4,5-trisphosphate (IP3) and the subsequent release of ER-stored calcium, mGluR1α can promote neurotoxicity. However, it can mediate neuroprotective effects through a C-terminal cytoplasmic tail interaction with Homer scaffolding proteins and the PI3K-AKT pathway. Thus, under physiological conditions, the potential neurotoxic effects of mGluR1α receptor activation are likely offset by its neuroprotective functions. However, in an excitotoxic paradigm, calpain cleavage of the C-terminal tail of mGluR1α uncouples its ability to exert neuroprotective effects through Akt activation and produces a positive-feedback loop that contributes to calcium overload (68) (Fig. 3).

Calpain and proteolytic brain injury biomarkers.

Emerging evidence now suggests that, upon neuronal cell injury, calpain and to a lesser extent capase-3 mediated neural protein breakdown products (BDPs), are released from injured cells into the extracellular component and ultimately into the cerebrospinal fluid and blood (Fig. 3). BDPs of αII-spectrin (e.g., SBDP150 and SBDP145 generated by calpain and SBDP120 generated by caspase-3) and cleaved tau (c-tau) are now viewed as potential biomarkers for various forms of neural injury, including cerebral ischemia, TBI, and METH-induced neurotoxicity (24, 49, 6872). An N-terminal fragment (about 2000 to 3000 daltons) of the NMDA receptor that circulates in the blood after acute brain injury (73) may also be viewed as a potential biomarker, although it is not clear if this protein fragment is directly generated by calpain action.

Perspective and Prospective

Thus, calpains are involved in both physiological and pathological processes in the CNS. New roles of calpains in these processes remain to be discovered. Building on our knowledge and with advances in technology, clinically relevant diagnostics could be developed based on calpain-generated proteolytic biomarkers. Lastly, selective and potent calpain inhibitors might have therapeutic potential in the treatment of calpain-related neural injury (74, 75).

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