Calpain-mediated Proteolysis Research Articles

Initiation of tumor dormancy by the lymphovascular embolus.

Cancer dormancy followed by recurrence remains an enigma in cancer biology. Since both local and systemic recurrences are thought to emanate from dormant micrometastasis which take origin from lymphovascular tumor emboli we wondered whether the process of dormancy might initiate within lymphovascular emboli. This study combines experimental studies with a patient-derived xenograft (PDX) of inflammatory breast cancer (Mary-X) that spontaneously forms spheroids in vitro and budding lymphovascular tumor emboli in vivo with observational studies utilizing tissue microarrays (TMAs) of human breast cancers. In the experimental studies, Mary-X during both lymphovascular emboli formation in vivo and spheroidgenesis in vitro exhibited decreased proliferation, a G0/G1 cell cycle arrest and decreased mTOR signaling. This induction of dormancy required calpain-mediated E-cadherin proteolysis and was mediated by decreased P13K signaling, resulting in decreased mTOR activity. In observational human breast cancer studies, increased E-cadherin immunoreactivity due to increased E-cad/NTF-1 but both decreased Ki-67 and mTOR activity was observed selectively and differentially within the lymphovascular tumor emboli. Both our experimental as well as observational studies indicate that in vivo lymphovascular tumor emboli and their in vitro spheroid equivalent initiate dormancy through these pathways.

Targeting calpain-2-mediated junctophilin-2 cleavage delays heart failure progression following myocardial infarction

Coronary heart disease (CHD) is a prevalent cardiac disease that causes over 370,000 deaths annually in the USA. In CHD, occlusion of a coronary artery causes ischemia of the cardiac muscle, which results in myocardial infarction (MI). Junctophilin-2 (JPH2) is a membrane protein that ensures efficient calcium handling and proper excitation-contraction coupling. Studies have identified loss of JPH2 due to calpain-mediated proteolysis as a key pathogenic event in ischemia-induced heart failure (HF). Our findings show that calpain-2-mediated JPH2 cleavage yields increased levels of a C-terminal cleaved peptide (JPH2-CTP) in patients with ischemic cardiomyopathy and mice with experimental MI. We created a novel knock-in mouse model by removing residues 479-SPAGTPPQ-486 to prevent calpain-2-mediated cleavage at this site. Functional and molecular assessment of cardiac function post-MI in cleavage site deletion (CSD) mice showed preserved cardiac contractility and reduced dilation, reduced JPH2-CTP levels, attenuated adverse remodeling, improved T-tubular structure, and normalized SR Ca2+-handling. Adenovirus mediated calpain-2 knockdown in mice exhibited similar findings. Pulldown of CTP followed by proteomic analysis revealed valosin-containing protein (VCP) and BAG family molecular chaperone regulator 3 (BAG3) as novel binding partners of JPH2. Together, our findings suggest that blocking calpain-2-mediated JPH2 cleavage may be a promising new strategy for delaying the development of HF following MI.

Cardiac ischemic preconditioning promotes cMyBP-C phosphorylation by inhibiting the calpain-mediated proteolysis

Ischemic preconditioning (IPC) has been considered as the most important mean to protect against ischemia/reperfusion (I/R) induced heart injury. It has been reported that cardiac myosin binding protein-C (cMyBP-C) phosphorylation plays an essential role in cardiac protection against I/R-induced heart injury. However, it is still obscured whether IPC-mediated cardiac protection is causally related to cMyBP-C phosphorylation and proteolysis and, if so, what the underlying mechanism is. In this study, IPC was found to increase the phosphorylation level of cMyBP-C, companying with the decreased calpain activity in the collected perfusate samples. Mechanistically, we confirmed that IPC promoted cMyBP-C phosphorylation and inhibited calpain-mediated cMyBP-C proteolysis. Moreover, inhibition of calpain activity significantly increased the phosphorylated cMyBP-C level by using calpain inhibitor (MG-101), and subsequently promoted stabilization and secretion of cMyBP-C. Functionally, adeno-associated virus (AAV)-mediated overexpression of mutated phosphorylation motif site of cMyBP-C exhibited impaired IPC-mediated cardiac protection via proteolysis of the full-length cMyBP-C protein. We concluded that IPC promoted cMyBP-C phosphorylation via inhibition of calpain-mediated proteolysis and participated in IPC-mediated protection against I/R induced heart injury.

Effects of stepwise chilling with calcium incubation on proteolysis and tenderization in postmortem goose muscle

White Roman goose (12-wk-old male, N=30) carcasses were obtained from a local government-inspected slaughter plant at approximately ∼10-min postmortem. Each carcass was individually sealed in a zip-lock bag and chilled immediately in a water bath at 15°C for 1 h. Both sides of Pectoralis major muscles were excised from each carcass and incubated in 30 mM CaCl2 or 30 mM EDTA at 15°C for 5 h. After incubation, calcium-incubated and EDTA-incubated breast muscles were vacuum-packaged individually and stored at 5°C for 72 h. Control samples (without CaCl2 or EDTA incubation) were directly vacuum-packaged and chilled in a water bath at 15°C for 5 h and stored at 5°C for 72 h. Muscle specimens were taken from the left side of breast muscles at 1 h of chilling (∼1-h postmortem) and at 5 h of incubation at 15°C (∼6-h postmortem), as well as 24, 48, and 72 h of aging at 5°C for measuring the activities of calpain-1 and calpain-11 as well as the contents of 80 kDa calpain-1 subunit and desmin. The samples of shear force value and myofibril fragmentation index (MFI) were taken from the right side of breast muscle at 24 h and 72 h of 5°C storage. Our results showed that the decrease of the activities of calpain-1 and calpain-11 and the contents of 80 kDa calpain-1 subunit and desmin was more rapid (P < 0.05) in calcium-incubated samples than in control and EDTA-incubated samples. The shear force was lower, but the MFI was higher in calcium-incubated samples than in control and EDTA-incubated samples (P < 0.05). Therefore, our results suggest that the calpain-mediated proteolysis and tenderization in postmortem goose muscle could be greatly enhanced by combine effects of stepwise chilling with calcium incubation at 15°C and thereafter aging at 5°C. With applying this procedure, commercial slaughter plants may have an alternative way to improve the tenderness of goose meat.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Generation of myocyte agonal Ca2+ waves and contraction bands in perfused rat hearts following irreversible membrane permeabilisation

Although irreversible cardiomyocyte injury provokes intracellular Ca2+ ([Ca2+]i) overload, the underlying dynamics of this response and its effects on cellular morphology remain unknown. We therefore visualised rapid-scanning confocal fluo4-[Ca2+]i dynamics and morphology of cardiomyocytes in Langendorff-perfused rat hearts following saponin-membrane permeabilisation. Our data demonstrate that 0.4% saponin-treated myocytes immediately exhibited high-frequency Ca2+ waves (131.3 waves/min/cell) with asynchronous, oscillatory contractions having a mean propagation velocity of 117.8 μm/s. These waves slowly decreased in frequency, developed a prolonged decay phase, and disappeared in 10 min resulting in high-static, fluo4-fluorescence intensity. The myocytes showing these waves displayed contraction bands, i.e., band-like actin-fibre aggregates with disruption of sarcomeric α-actinin. The contraction bands were not attenuated by the abolition of Ca2+ waves under pretreatment with ryanodine plus thapsigargin, but were partially attenuated by the calpain inhibitor MDL28170, while mechanical arrest of the myocytes by 2,3-butanedione monoxime completely attenuated contraction-band formation. The depletion of adenosine 5′-triphosphate by the mitochondrial electron uncoupler carbonyl cyanide 4-trifluoromethoxy phenylhydrazone also attenuated Ca2+ waves and contraction bands. Overall, saponin-induced myocyte [Ca2+]i overload provokes agonal Ca2+ waves and contraction bands. Contraction bands are not the direct consequence of the waves but are caused by cross-bridge interactions of the myocytes under calpain-mediated proteolysis.

Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress.

Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by loss-of-function mutations in the E3 ubiquitin ligase adaptor gigaxonin, which is encoded by the KLHL16 gene. Gigaxonin regulates the degradation of multiple intermediate filament (IF) proteins, including neurofilaments, GFAP, and vimentin, which aggregate in GAN patient cells. Understanding how IFs and their aggregates are processed under stress can reveal new GAN disease mechanisms and potential targets for therapy. Here we tested the hypothesis that hypotonic stress-induced vimentin proteolysis is impaired in GAN. In both GAN and control fibroblasts exposed to hypotonic stress, we observed time-dependent vimentin cleavage that resulted in two prominent ∼40-45kDa fragments. However, vimentin proteolysis occurred more rapidly and extensively in GAN cells compared to unaffected controls as both fragments were generated earlier and at 4-6-fold higher levels. To test enzymatic involvement, we determined the expression levels and localization of the calcium-sensitive calpain proteases-1 and -2 and their endogenous inhibitor calpastatin. While the latter was not affected, the expression of both calpains was 2-fold higher in GAN cells compared to control cells. Moreover, pharmacologic inhibition of calpains with MDL-28170 or MG-132 attenuated vimentin cleavage. Imaging analysis revealed striking colocalization between large perinuclear vimentin aggregates and calpain-2 in GAN fibroblasts. This colocalization was dramatically altered by hypotonic stress, where selective breakdown of filaments over aggregates occurred rapidly in GAN cells and coincided with calpain-2 cytoplasmic redistribution. Finally, mass spectrometry-based proteomics revealed that phosphorylation at Ser-412, located at the junction between the central "rod" domain and C-terminal "tail" domain on vimentin, is involved in this stress response. Over-expression studies using phospho-deficient and phospho-mimic mutants revealed that Ser-412 is important for filament organization, solubility dynamics, and vimentin cleavage upon hypotonic stress exposure. Collectively, our work reveals that osmotic stress induces calpain- and proteasome-mediated vimentin degradation and IF network breakdown. These effects are significantly augmented in the presence of disease-causing KLHL16 mutations that alter intermediate filament organization. While the specific roles of calpain-generated vimentin IF fragments in GAN cells remain to be defined, this proteolytic pathway is translationally-relevant to GAN because maintaining osmotic homeostasis is critical for nervous system function.

Calpain-mediated proteolysis as driver and modulator of polyglutamine toxicity.

Among posttranslational modifications, directed proteolytic processes have the strongest impact on protein integrity. They are executed by a variety of cellular machineries and lead to a wide range of molecular consequences. Compared to other forms of proteolytic enzymes, the class of calcium-activated calpains is considered as modulator proteases due to their limited proteolytic activity, which changes the structure and function of their target substrates. In the context of neurodegeneration and - in particular - polyglutamine disorders, proteolytic events have been linked to modulatory effects on the molecular pathogenesis by generating harmful breakdown products of disease proteins. These findings led to the formulation of the toxic fragment hypothesis, and calpains appeared to be one of the key players and auspicious therapeutic targets in Huntington disease and Machado Joseph disease. This review provides a current survey of the role of calpains in proteolytic processes found in polyglutamine disorders. Together with insights into general concepts behind toxic fragments and findings in polyglutamine disorders, this work aims to inspire researchers to broaden and deepen the knowledge in this field, which will help to evaluate calpain-mediated proteolysis as a unifying and therapeutically targetable posttranslational mechanism in neurodegeneration.

Effects of intracellular calcium accumulation on proteins encoded by the major genes underlying amyotrophic lateral sclerosis

The aetiology of Amyotrophic Lateral Sclerosis (ALS) is still poorly understood. The discovery of genetic forms of ALS pointed out the mechanisms underlying this pathology, but also showed how complex these mechanisms are. Excitotoxicity is strongly suspected to play a role in ALS pathogenesis. Excitotoxicity is defined as neuron damage due to excessive intake of calcium ions (Ca2+) by the cell. This study aims to find a relationship between the proteins coded by the most relevant genes associated with ALS and intracellular Ca2+ accumulation. In detail, the profile of eight proteins (TDP-43, C9orf72, p62/sequestosome-1, matrin-3, VCP, FUS, SOD1 and profilin-1), was analysed in three different cell types induced to raise their cytoplasmic amount of Ca2+. Intracellular Ca2+ accumulation causes a decrease in the levels of TDP-43, C9orf72, matrin3, VCP, FUS, SOD1 and profilin-1 and an increase in those of p62/sequestosome-1. These events are associated with the proteolytic action of two proteases, calpains and caspases, as well as with the activation of autophagy. Interestingly, Ca2+ appears to both favour and hinder autophagy. Understanding how and why calpain-mediated proteolysis and autophagy, which are physiological processes, become pathological may elucidate the mechanisms responsible for ALS and help discover new therapeutic targets.

A novel methylated cation channel TRPM4 inhibited colorectal cancer metastasis through Ca2+/Calpain-mediated proteolysis of FAK and suppression of PI3K/Akt/mTOR signaling pathway.

Colorectal cancer (CRC) is an aggressive malignancy with poor prognosis. It is imperative to elucidate the potential molecular mechanisms that regulate CRC cell aggressiveness. In present study, the transient receptor potential melastatin 4 (TRPM4), a calcium-activated nonselective cation channel, is downregulated in CRC as a novel methylated tumor suppressor gene (TSG). The reduced mRNA level of TRPM4 is due to the epigenetic methylation of its promoter CpG island (CGI). Moreover, ectopic expression of TRPM4 inhibited tumor growth and metastasis both in vitro and in vivo. Our experiments also demonstrate that TRPM4 restructures the CRC cytoskeleton and activates the Ca2+-mediated calpain pathway through enhancing calcium influx. The western blot analysis shows that the expression of focal adhesion kinase (FAK), a calpain-mediated proteolytic substrate, is markedly suppressed after ectopic overexpression of TRPM4, besides, Akt (also known as protein kinase B, PKB), phosphatidylinositol 3-kinase (PI3K) as well as its central target mTOR have significantly decreased expression accompanied by elevated E-cadherin and restrained matrix metalloproteinases (MMP2/MMP9) expression. The inhibition of protease calpain effectively relieves the retard of FAK/Akt signals and reverses the migration suppression of TRPM4. Taken together, TRPM4, identified as a novel methylated TSG, employs intracellular Ca2+ signals to activate calpain-mediated cleavage of FAK and impede CRC migration and invasion through modulating the PI3K/Akt/mTOR signaling cascade, providing the first evidence that TRPM4 is likely to be a significant biomarker and potential target for CRC therapy.

Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress

Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress

Poststroke dendritic arbor regrowth requires the actin nucleator Cobl.

Ischemic stroke is a major cause of death and long-term disability. We demonstrate that middle cerebral artery occlusion (MCAO) in mice leads to a strong decline in dendritic arborization of penumbral neurons. These defects were subsequently repaired by an ipsilateral recovery process requiring the actin nucleator Cobl. Ischemic stroke and excitotoxicity, caused by calpain-mediated proteolysis, significantly reduced Cobl levels. In an apparently unique manner among excitotoxicity-affected proteins, this Cobl decline was rapidly restored by increased mRNA expression and Cobl then played a pivotal role in poststroke dendritic arbor repair in peri-infarct areas. In Cobl knockout (KO) mice, the dendritic repair window determined to span day 2 to 4 poststroke in wild-type (WT) strikingly passed without any dendritic regrowth. Instead, Cobl KO penumbral neurons of the primary motor cortex continued to show the dendritic impairments caused by stroke. Our results thereby highlight a powerful poststroke recovery process and identified causal molecular mechanisms critical during poststroke repair.

Proteasome- and Calpain-Mediated Proteolysis, but Not Autophagy, Is Required for Leucine-Induced Protein Synthesis in C2C12 Myotubes.

Muscle protein synthesis and proteolysis are tightly coupled processes. Given that muscle growth is promoted by increases in net protein balance, it stands to reason that bolstering protein synthesis through amino acids while reducing or inhibiting proteolysis could be a synergistic strategy in enhancing anabolism. However, there is contradictory evidence suggesting that the proper functioning of proteolytic systems in muscle is required for homeostasis. To add clarity to this issue, we sought to determine if inhibiting different proteolytic systems in C2C12 myotubes in conjunction with acute and chronic leucine treatments affected markers of anabolism. In Experiment 1, myotubes underwent 1-h, 6-h, and 24-h treatments with serum and leucine-free DMEM containing the following compounds (n = 6 wells per treatment): (i) DMSO vehicle (CTL), (ii) 2 mM leucine + vehicle (Leu-only), (iii) 2 mM leucine + 40 μM MG132 (20S proteasome inhibitor) (Leu + MG132), (iv) 2 mM leucine + 50 μM calpeptin (calpain inhibitor) (Leu + CALP), and (v) 2 mM leucine + 1 μM 3-methyladenine (autophagy inhibitor) (Leu + 3MA). Protein synthesis levels significantly increased (p < 0.05) in the Leu-only and Leu + 3MA 6-h treatments compared to CTL, and levels were significantly lower in Leu + MG132 and Leu + CALP versus Leu-only and CTL. With 24-h treatments, total protein yield was significantly lower in Leu + MG132 cells versus other treatments. Additionally, the intracellular essential amino acid (EAA) pool was significantly greater in 24-h Leu + MG132 treatments versus other treatments. In a follow-up experiment, myotubes were treated for 48 h with CTL, Leu-only, and Leu + MG132 for morphological assessments. Results indicated Leu + MG132 yielded significantly smaller myotubes compared to CTL and Leu-only. Our data are limited in scope due to the utilization of select proteolysis inhibitors. However, this is the first evidence to suggest proteasome and calpain inhibition with MG132 and CALP, respectively, abrogate leucine-induced protein synthesis in myotubes. Additionally, longer-term Leu + MG132 treatments translated to an atrophy phenotype. Whether or not proteasome inhibition in vivo reduces leucine- or EAA-induced anabolism remains to be determined.

Variants in Genes of Calpain System as Modifiers of Spinocerebellar Ataxia Type 3 Phenotype

Calpain-mediated proteolysis has been proposed to modulate the pathogenesis of spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), a disorder due to a CAG repeat expansion (CAGexp) at ATXN3. We aimedto investigate if single-nucleotide polymorphisms (SNPs) at calpain gene CAPN2 and at calpastatin gene CAST modulate the age at onset (AO) and disease progression in SCA3/MJD. A total of 287 SCA3/MJD symptomatic subjects (151 families) were included. AO was analyzed and controlled by the CAG repeat length of expanded allele and family. Candidate polymorphisms were chosen based on the literature and on a priori criteria. The CAG repeat length and SNPs were genotyped according to standard methods. AO of carriers of AA and AG + GGrs1559085 genotypes in CAST and with the median value of 75 repeats at the expanded allele were 34.23 (33.07-35.38) and 36.42years (34.50-38.34), respectively (p = 0.049, mixed model treating the expanded CAG repeat size as fixed effect and family as random effect). Carriers of haplotype Crs27852/Grs1559085 had mean AO of 37.23 (12.76) and 33.42years (12.20) (p = 0.047, Student's t test). Our data suggest an association between allele Grs1559085 and haplotype Crs27852/Grs1559085 at CAST and variations in the AO of SCA3/MJD subjects, independent from the effects of the CAGexp and family. The present results support the potential role of calpain cleavage pathway over modulation of SCA3/MJD phenotype.

Over-activation of NMDA receptors promotes ABCA1 degradation and foam cell formation

ATP-binding cassette transporter A1 (ABCA1) is an essential regulator of intracellular cholesterol efflux. Secreted cholesterol binds to lipid-free apolipoprotein A-I (apoA-I) in peripheral blood to constitute high-density lipoprotein cholesterol (HDL) complexes. ABCA1 protein on the surface of macrophages acts as a crucial controller in preventing cholesterol accumulation. Importantly, ABCA1 is unstable and easily degraded via a series of biochemical activities, including but not limited to calpain-mediated and ubiquitin-proteasome system-mediated processes. How accelerated ABCA1 degradation impacts disordered lipid metabolism in macrophages and foam cell formation is unclear. N-methyl d-aspartate receptors (NMDARs) are ionotropic glutamate receptors with high calcium permeability. Calcium influx via NMDARs activates downstream signaling pathways. Over-activation of NMDARs stimulated by NMDA contributes to dysfunctional lipid metabolism in macrophages and foam cell formation via promotion of calpain-mediated ABCA1 proteolysis. However, increased NMDAR activity does not affect liver X receptor expression or ABCA1 mRNA levels. Following NMDA receptor silencing or calpain inhibition, NMDA treatment did not reduce ABCA1 protein levels, nor caused lipid accumulation in macrophages. In addition, NMDAR over-activation activates NF-κB signaling to promote IL-1β and IL-6 macrophage marker expression. However, NMDAR silencing and calpain inhibition reduce inflammatory macrophage responses. In summary, our study suggests that NMDAR activation reduces surface ABCA1 protein, promotes lipid accumulation, and induces the production and secretion of many inflammatory mediators in macrophages, possibly through enhanced calpain-mediated ABCA1 protein degradation. Thus, the NMDAR receptor may be a novel pharmacologic target for atherosclerosis therapy.

A role for glia maturation factor dependent activation of mast cells and microglia in MPTP induced dopamine loss and behavioural deficits in mice

The molecular mechanism mediating degeneration of nigrostriatal dopaminergic neurons in Parkinson’s disease (PD) is not yet fully understood. Previously, we have shown the contribution of glia maturation factor (GMF), a proinflammatory protein in dopaminergic neurodegeneration mediated by activation of mast cells (MCs). In this study, methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal neurodegeneration and astro-glial activations were determined by western blot and immunofluorescence techniques in wild type (WT) mice, MC-deficient (MC-KO) mice and GMF-deficient (GMF-KO) mice, with or without MC reconstitution before MPTP administration. We show that GMF-KO in the MCs reduces the synergistic effects of MC and Calpain1 (calcium-activated cysteine protease enzyme)-dependent dopaminergic neuronal loss that reduces motor behavioral impairments in MPTP-treated mouse. Administration of MPTP increase in calpain-mediated proteolysis in nigral dopaminergic neurons further resulting in motor decline in mice. We found that MPTP administered WT mice exhibits oxidative stress due to significant increases in the levels of malondialdehyde, superoxide dismutase and reduction in the levels of reduced glutathione and glutathione peroxidase activity as compared with both MC-KO and GMF-KO mice. The number of TH-positive neurons in the ventral tegmental area, substantia nigra and the fibers in the striatum were significantly reduced while granulocyte macrophage colony-stimulating factor (GM-CSF), MC-Tryptase, GFAP, IBA1, Calpain1 and intracellular adhesion molecule 1 expression were significantly increased in WT mice. Similarly, tyrosine hydroxylase, dopamine transporters and vesicular monoamine transporters 2 proteins expression were significantly reduced in the SN of MPTP treated WT mice. The motor behavior as analyzed by rotarod and hang test was significantly reduced in WT mice as compared with both the MC-KO and GMF-KO mice. We conclude that GMF-dependent MC activation enhances the detrimental effect of astro-glial activation-mediated oxidative stress and neuroinflammation in the midbrain, and its inhibition may slowdown the progression of PD.

Calpain fosters the hyperexcitability of motoneurons after spinal cord injury and leads to spasticity.

Up-regulation of the persistent sodium current (INaP) and down-regulation of the potassium/chloride extruder KCC2 lead to spasticity after spinal cord injury (SCI). We here identified calpain as the driver of the up- and down-regulation of INaP and KCC2, respectively, in neonatal rat lumbar motoneurons. Few days after SCI, neonatal rats developed behavioral signs of spasticity with the emergence of both hyperreflexia and abnormal involuntary muscle contractions on hindlimbs. At the same time, in vitro isolated lumbar spinal cords became hyperreflexive and displayed numerous spontaneous motor outputs. Calpain-I expression paralleled with a proteolysis of voltage-gated sodium (Nav) channels and KCC2. Acute inhibition of calpains reduced this proteolysis, restored the motoneuronal expression of Nav and KCC2, normalized INaP and KCC2 function, and curtailed spasticity. In sum, by up- and down-regulating INaP and KCC2, the calpain-mediated proteolysis of Nav and KCC2 drives the hyperexcitability of motoneurons which leads to spasticity after SCI.

The calcium-activated protease calpain regulates netrin-1 receptor deleted in colorectal cancer-induced axon outgrowth in cortical neurons.

During development, neurons extend axons toward their appropriate synaptic targets to establish functional neuronal connections. The growth cone, a highly motile structure at the tip of the axon, is capable of recognizing extracellular guidance cues and translating them into directed axon outgrowth through modulation of the actin cytoskeleton. Netrin-1 mediates its attractive function through the receptor deleted in colorectal cancer (DCC) to promote axon outgrowth and guidance. The calcium-activated protease calpain is involved in the cleavage of cytoskeletal proteins, which plays an important role during adhesion turnover and cell migration. However, its function during neuronal development is less understood. Here we demonstrate that netrin-1 activated calpain in embryonic rat cortical neurons in an extracellular-regulated kinase 1/2-dependent manner. In addition, we found that netrin-1 stimulation led to an increase in calpain-1 localization in the axon, whereas its endogenous inhibitor calpastatin was decreased in the growth cones of cortical neurons by indirect immunofluorescence. Interestingly, calpain-1 was able to cleave DCC in vitro. Furthermore, netrin-1 induced the cleavage of the cytoskeletal proteins spectrin and focal adhesion kinase concomitantly with the intracellular domain of DCC in a calpain-dependent manner in embryonic rat cortical neurons. Cortical neurons over-expressing calpastatin or calpain-depleted neurons displayed increased basal axon length and were unresponsive to netrin-1 stimulation. Altogether, we propose a novel model whereby netrin-1/DCC-mediated axon outgrowth is modulated by calpain-mediated proteolysis of DCC and cytoskeletal targets in embryonic cortical neurons. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Differential regulation of Kidins220 isoforms in Huntington's disease.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by brain atrophy particularly in the striatum that produces motor impairment, and cognitive and psychiatric disturbances. Multiple pathogenic mechanisms have been proposed including dysfunctions in neurotrophic support and calpain-overactivation, among others. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is an essential mediator of neurotrophin signaling. In adult brain, Kidins220 presents two main isoforms that differ in their carboxy-terminal length and critical protein-protein interaction domains. These variants are generated through alternative terminal exon splicing of the conventional exon 32 (Kidins220-C32) and the recently identified exon 33 (Kidins220-C33). The lack of domains encoded by exon 32 involved in key neuronal functions, including those controlling neurotrophin pathways, pointed to Kidins220-C33 as a form detrimental for neurons. However, the functional role of Kidins220-C33 in neurodegeneration or other pathologies, including HD, has not been explored. In the present work, we discover an unexpected selective downregulation of Kidins220-C33, in the striatum of HD patients, as well as in the R6/1 HD mouse model starting at early symptomatic stages. These changes are C33-specific as Kidins220-C32 variant remains unchanged. We also find the early decrease in Kidins220-C33 levels takes place in neurons, suggesting an unanticipated neuroprotective role for this isoform. Finally, using ex vivo assays and primary neurons, we demonstrate that Kidins220-C33 is downregulated by mechanisms that depend on the activation of the protease calpain. Altogether, these results strongly suggest that calpain-mediated Kidins220-C33 proteolysis modulates onset and/or progression of HD.

Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection.