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The calpain family of calcium-dependent proteases has been implicated in a variety of diseases and neurodegenerative pathologies. Prolonged activation of calpains results in proteolysis of numerous cellular substrates including cytoskeletal components and membrane receptors, contributing to cell demise despite coincident expression of calpastatin, the specific inhibitor of calpains. Pharmacological and gene-knockout strategies have targeted calpains to determine their contribution to neurodegenerative pathology; however, limitations associated with treatment paradigms, drug specificity, and genetic disruptions have produced inconsistent results and complicated interpretation. Specific, targeted calpain inhibition achieved by enhancing endogenous calpastatin levels offers unique advantages in studying pathological calpain activation. We have characterized a novel calpastatin-overexpressing transgenic mouse model, demonstrating a substantial increase in calpastatin expression within nervous system and peripheral tissues and associated reduction in protease activity. Experimental activation of calpains via traumatic brain injury resulted in cleavage of α-spectrin, collapsin response mediator protein-2, and voltage-gated sodium channel, critical proteins for the maintenance of neuronal structure and function. Calpastatin overexpression significantly attenuated calpain-mediated proteolysis of these selected substrates acutely following severe controlled cortical impact injury, but with no effect on acute hippocampal neurodegeneration. Augmenting calpastatin levels may be an effective method for calpain inhibition in traumatic brain injury and neurodegenerative disorders.
The calcium-dependent cysteine proteases, calpains, are multifaceted regulators of normal cell function. Although 15 isoforms of calpains are known to exist within cells (Sorimachi et al. 2011), the most commonly studied are the ubiquitously expressed isoforms, μ-calpain and m-calpain. While these two isoforms share a similar heterodimeric structure, they differ in their calcium requirements. Micromolar-range concentrations of ionic calcium are necessary to activate μ-calpains in vitro, while millimolar calcium concentrations activate m-calpains. Regulation of calpains' proteolytic activity occurs both by intracellular free-calcium concentrations and by a common endogenous inhibitor, calpastatin. Calpastatin is an intracellular 110-kDa protein consisting of an N-terminal leader domain followed by four identical inhibitory domains, each able to specifically inhibit one molecule of calpain (Maki et al. 1987). When free-calcium levels rise and activate calpains, a conformational change in the protease allows for inhibitor binding across the active site of calpain, blocking its access to substrates (Moldoveanu et al. 2008). Under physiologic conditions, calpains participate in cytoskeletal alterations, cell cycle and differentiation processes, apoptosis, and long-term potentiation (Goll et al. 2003), indicative of their importance to normal cell function.
Calpain activation contributes to the evolution of neurodegeneration in Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis as well as damage associated with stroke, traumatic brain injury (TBI), and spinal cord injury (Camins et al. 2006). Under pathological conditions, altered intracellular calcium homeostasis leads to calpain activation, resulting in the cleavage of cellular substrates including cytoskeletal elements, membrane receptors, cytosolic proteins, and cell death mediators (Saatman et al. 2010). As the most well-characterized calpain substrate following TBI, the cytoskeletal component α-spectrin is a valuable surrogate marker of calpain activation, and its early proteolysis may indicate the severity of cellular damage and subsequent neuronal death (Saatman et al. 1996). Through the use of calpain inhibitors and identification of calpain-specific breakdown products (BDPs), the number of calpain substrates verified in models of TBI is expanding. Collapsin response mediator protein-2 (CRMP-2) proteolysis was detected in response to excitotoxic insult and attenuated with in vitro calpain inhibitor application. Identical calpain-mediated CRMP-2 cleavage patterns were identified in brain homogenates after experimental TBI (Zhang et al. 2007). Similarly, voltage-gated sodium channel cleavage, triggered by exogenous calpain activation or using an in vitro model of TBI, was reversed with viral-mediated calpastatin overexpression or treatment with the calpain inhibitor MDL28170 (von Reyn et al. 2009). Limited cleavage characteristic of calpains may modulate ion flux and receptor function, contributing to exacerbated calcium dysfunction, further calpain activation, and neuronal damage associated with brain injury.
Genetic manipulation of calpastatin to enhance endogenous inhibitory mechanisms enables suppression of both μ- and m-calpain, providing a powerful research tool for understanding the role of pathological calpain proteolysis. Transgenic mice with calcium/calmodulin-dependent protein kinase II α (CaMKIIα)-driven calpastatin expression exhibited a three-fold reduction in in vitro m-calpain activity and significantly less hippocampal cell death in response to excitotoxic insult (Higuchi et al. 2005). Using these same mice, we recently showed that following severe contusion TBI, calpastatin overexpression reduced acute spectrin proteolysis and select behavioral deficits, but did not affect cortical tissue damage (Schoch et al. 2012).
Subsequently, we developed a novel transgenic mouse with human calpastatin (hCAST) under constitutive control of the ubiquitous prion promoter (Prp) to produce a more widespread cellular distribution of calpastatin overexpression. Here, we demonstrate that this hCAST transgenic mouse has cortical and hippocampal calpastatin levels approximately 80-fold greater than wildtype (WT) mice and use this new transgenic tool to verify the effectiveness of calpastatin in reducing calpain-mediated damage after TBI. To this end, we subjected WT and calpastatin-overexpressing (Prp-hCAST) transgenic mice to severe controlled cortical impact (CCI) injury and evaluated acute posttraumatic proteolysis of three proteins critical for neuronal structure and function: α-spectrin, CRMP-2, and voltage-gated sodium channel 1.2 (Nav1.2). In addition, we assessed acute regional hippocampal neurodegeneration in brain-injured WT and Prp-hCAST transgenic mice.
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Calpains are important mediators of neuronal damage and death under conditions of neurodegenerative disease and traumatic insults. Because of the risk calpain activation poses toward neuronal cell viability, we have investigated an avenue of calpain inhibition using overexpression of calpastatin in a new transgenic mouse line. Our results demonstrate that Prp-hCAST mice robustly express calpastatin throughout the brain, yielding potent inhibition of exogenous calpain. Following in vivo activation of calpains by experimental TBI, calpastatin overexpression reduced calpain-mediated proteolysis of the substrates α-spectrin, CRMP-2, and voltage-gated sodium channel. These findings confirm calpains as a pathological target and validate calpastatin as an agent for modulating calpain activity following TBI.
Prp-hCAST transgenic mice were found to express calpastatin at levels on the order of 80-fold higher than endogenous levels in WT mice in the cortex and hippocampus, with near complete inhibition of in vitro protease activity. Other transgenic models of constitutive calpastatin overexpression have also demonstrated significant elevations in calpastatin levels or inhibitory function. Neuronal expression of calpastatin via the CaMKIIα promoter produced a threefold greater calpastatin inhibitory activity, although hCAST and mCAST protein levels were not quantitatively compared (Higuchi et al. 2005). Use of the Prp promoter to initiate gene expression may yield a more widespread distribution throughout the brain, particularly concentrated in axons and terminals (Barmada et al. 2004). In addition to its central distribution, calpastatin expression was also noted in peripheral tissues, making this mouse an attractive tool for many models of both central and peripheral nerve injury and other pathologies involving non-neuronal tissues. An alternative calpastatin transgenic mouse, developed using the Thy1.1 promoter, achieved an approximately 15-fold greater calpastatin expression over WT, resulting in effective calpain-2 inhibition, but also in changes in basal levels of calpain-1 and calpain-2 and certain calpain substrates (Rao et al. 2008). Altered basal levels of proteases or calpain substrates was not evident in the Prp- or CaMKIIα-driven models, suggesting that constitutive overexpression of calpastatin did not produce compensatory up-regulation of proteases or widespread changes in substrate regulation. Both transgenic models (using CaMKIIα and Thy1.1 promoters) reduced pathological activation of calpain, and proteolysis of multiple calpain substrates after excitotoxic stimulus. Prp-driven hCAST overexpression was similarly successful in abating posttraumatic calpain-mediated proteolysis of structurally and functionally relevant proteins.
α-spectrin is an essential protein component of the neuronal cytoarchitecture that functions in both structural support and membrane protein anchoring. Its cleavage may contribute to neuronal pathology because of alterations in membrane stability or membrane-associated protein function. Severe TBI led to increased α-spectrin proteolysis into its characteristic 150- and 145-kDa fragments in WT mice, consistent with previous literature documenting potent and early spectrin cleavage in neurons of affected brain regions following experimental contusion injury (Saatman et al. 1996; Pike et al. 1998). Calpastatin overexpression in Prp-hCAST transgenic mice prevented appearance of the calpain-specific 145-kDa BDP in the cortex and hippocampus up to 24 h after severe CCI injury. Targeting calpain activity via administration of pharmacological calpain inhibitors has produced inconsistent results in the ability to decrease spectrin breakdown following TBI (Posmantur et al. 1997; Saatman et al. 2000; Kupina et al. 2001; Thompson et al. 2010), which may be a reflection of the differences in treatment parameters or limitations associated with the drugs themselves. Notably, some calpain inhibitors are not solely selective for calpains, have poor blood–brain barrier permeability, and are metabolically unstable (Carragher 2006). Newer inhibitors designed to mimic calpastatin show promise in preventing calpain-specific spectrin breakdown in response to elevated intracellular calcium (McCollum et al. 2006) or ischemic insult (Anagli et al. 2009).
Calpastatin overexpression in our transgenic model was unable to inhibit spectrin proteolysis into the 150-kDa fragment. Although a 150-kDa fragment can be generated through caspase activity, which is not inhibited by calpastatin, it is unlikely that caspase-3 contributed significantly to the accumulation of a 150-kDa BDP given the absence of the signature 120-kDa fragment (Pike et al. 1998). Rather, the 150-kDa product may represent the initial cleavage product of spectrin, later cleaved to a 145-kDa fragment (Zhang et al. 2009). Why calpain inhibition would be effective in inhibiting the second, but not the initial cleavage of α-spectrin is not clear. The 145-kDa BDP has been suggested to be a more sensitive marker for calpain activation (Zhang et al. 2009) and a biomarker for injury severity in experimental TBI (Pike et al. 2001; Ringger et al. 2004) and human TBI patients (Mondello et al. 2010). Thus, inhibition of spectrin breakdown in Prp-hCAST mice may be an indication of reduced injury severity.
The collapsin response mediator proteins are a family of intracellular proteins expressed within the CNS during development and periods of axonal growth (Quinn et al. 1999). CRMP-2, in particular, is important in protein trafficking (Rahajeng et al. 2010), microtubule assembly (Fukata et al. 2002), and neurite outgrowth (Quinn et al. 2003). Calpain digestion of CRMP-2 results in generation of a 55-kDa BDP, which is inhibited with calpain inhibitor, SJA6017, application in cortical lysates digested with calpain-2 (Zhang et al. 2007). Here, we demonstrate that CCI injury results in the appearance and subsequent accumulation of a 55-kDa CRMP-2 fragment as previously described (Zhang et al. 2007), which is attenuated by calpastatin overexpression. CRMP-2 cleavage by calpains following TBI may disrupt the protein's interactions with axonal transport proteins (Touma et al. 2007) and act to down-regulate surface expression of NMDA receptors (Bretin et al. 2006) and voltage-gated calcium channels (Brittain et al. 2009). Thus, CRMP-2 processing may not only affect axonal function, but also modulate receptors and channels, potentiating the characteristic ionic imbalance of TBI and neurodegenerative conditions. Inhibition of CRMP-2 cleavage in the presence of the calcium channel-binding domain prevents hippocampal cell death following TBI (Brittain et al. 2011), supporting a link between CRMP-2 cleavage and neuronal death.
Multiple voltage-gated sodium channel isoforms are present within the CNS, participating in action potential generation and propagation. The Nav1.2 protein is localized to axons and terminals of neurons (Westenbroek et al. 1989) and, with injury, is cleaved by calpains (Iwata et al. 2004; von Reyn et al. 2009). Because of the sodium channel's pivotal role in action potential generation and ion flux, injury-induced Nav1.2 damage or dysfunction may result in increased sodium influx and prolonged membrane depolarization, further exacerbating calcium dysregulation and calpain activation (Yuen et al. 2009). Severe CCI resulted in α-subunit breakdown, evidenced by the appearance of fragments of similar size to in vitro findings. Calpastatin overexpression inhibited the accumulation of two distinct channel fragments (85 kDa, 100 kDa) up to 24 h post-CCI, providing strong support that these products are calpain specific. We are the first to demonstrate attenuated sodium channel proteolysis into select fragments with calpain inhibition by calpastatin in vivo, confirming results with application of the calpain inhibitor, MDL28170, after in vitro stretch injury (von Reyn et al. 2009, 2012). Slight differences in the molecular weights of fragments between in vivo and neuronal stretch injury were evident, probably reflecting differences in injury pathology or gel migration patterns.
In cases where multiple breakdown products were evident, calpastatin overexpression in Prp-hCAST transgenic mice inhibited the appearance of some, but not all cleavage products. In particular, Nav1.2 proteolysis into various fragments was coincident with loss of full-length protein expression suggesting that calpastatin overexpression is most effective in inhibiting the progressive breakdown of the channel into smaller sized fragments. Alternatively, cleavages may indicate the activity of alternative proteolytic pathways that function independent of calpain activation and are therefore unaffected by calpastatin overexpression. It is unknown whether partial breakdown of spectrin, CRMP-2, or sodium channel imparts irreversible damage to the protein and cell or whether the fragments may exhibit independent functions in mediating cell death. Our results demonstrate an absence of acute hippocampal neuroprotection, consistent with previous data using CaMKIIα-driven calpastatin overexpression (Schoch et al. 2012). Nevertheless, reduced calpain-mediated proteolysis may delay neuronal damage, allowing the cell to repair itself or expanding the therapeutic window for additional survival interventions. While the accumulation of calpain-mediated fragments assessed in this study was definitively reduced in Prp-hCAST mice, subacute cell survival and functional improvements will need to be investigated.
Calpastatin overexpression within Prp-hCAST mice ensures inhibition of multiple calpain isoforms, thereby allowing for broad inhibition of calpains' proteolytic activity following TBI. However, we are unable to draw conclusions about the differential roles of calpain-1 versus calpain-2 in mediating posttraumatic damage. This study introduces a novel calpastatin-overexpressing transgenic mouse, and demonstrates reduced posttraumatic proteolysis of key cellular substrates, spectrin, CRMP-2, and sodium channel. Boosting endogenous calpastatin through genetic manipulation promotes inhibitory mechanisms at the initiation of damage and maintains this inhibition long past the primary insult, a strategy that may be critical for acute, overwhelming calpain activity. Continued studies investigating the ability of calpastatin overexpression to reduce TBI and neurodegenerative pathology may lead to the development of superior agents for calpain inhibition in vivo.