calpastatin and Spinal-Cord-Injuries

Spinal cord injury (SCI) evokes an increase in intracellular free Ca(2+) level resulting in activation of calpain, a Ca(2+)-dependent cysteine protease, which cleaves many cytoskeletal and myelin proteins. Calpain is widely expressed in the central nervous system (CNS) and regulated by calpastatin, an endogenous calpain-specific inhibitor. Calpastatin degraded by overactivation of calpain after SCI may lose its regulatory efficiency. Evidence accumulated over the years indicates that uncontrolled calpain activity mediates the degradation of many cytoskeletal and membrane proteins in the course of neuronal death and contributes to the pathophysiology of SCI. Cleavage of the key cytoskeletal and membrane proteins by calpain is an irreversible process that perturbs the integrity and stability of CNS cells leading to cell death. Calpain in conjunction with caspases, most notably caspase-3, can cause apoptosis of the CNS cells following trauma. Aberrant Ca(2+) homeostasis following SCI inevitably activates calpain, which has been shown to play a crucial role in the pathophysiology of SCI. Therefore, calpain appears to be a potential therapeutic target in SCI. Substantial research effort has been focused upon the development of highly specific inhibitors of calpain and caspase-3 for therapeutic applications. Administration of cell permeable and specific inhibitors of calpain and caspase-3 in experimental animal models of SCI has provided significant neuroprotection, raising the hope that humans suffering from SCI may be treated with these inhibitors in the near future.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Glycoproteins; Humans; Spinal Cord Injuries

Calpain is a Ca(2+)-activated proteolytic enzyme involved in neurodegeneration in a variety of injuries and diseases of the central nervous system (CNS). Many calpain homologs have been discovered. Depending on the tissue distribution, calpains are broadly classified as ubiquitous and tissue-specific. Ubiquitous calpain isoforms, -calpain and m-calpain, are abundantly expressed in the CNS. Calpastatin, an endogenous protein inhibitor, regulates the activity of ubiquitous calpain. Overactivation of calpain may degrade calpastatin, limiting its regulatory efficiency. Molecular structures of calpain and calpastatin have been deduced from cDNA cloning. The precise physiological function of calpain remains elusive. However, experimental evidence strongly suggests an important role for calpain in causing neurodegeneration in various injuries and diseases of the CNS. The increase in intracellular free Ca(2+) levels in the course of injuries and diseases in the CNS causes overactivation of calpain, promoting degradation of key cytoskeletal and membrane proteins. Cleavage of these key proteins by calpain is an irreversible process that perturbs the integrity and stability of CNS cells, leading to programmed cell death or apoptosis. Calpain in conjunction with caspases can cause apoptosis of the CNS cells. An aberrant Ca(2+) homeostasis inevitably activates calpain, which plays a crucial role in the pathophysiology of the CNS injuries and diseases. Therefore, calpain is a potential therapeutic target to prevent neurodegeneration. To this end, various cell-permeable calpain inhibitors have been synthesized for pharmacological inhibition of calpain activity. Some calpain inhibitors have shown significant neuroprotection in animal models of the CNS injuries and diseases, indicating their therapeutic potential.

Topics: Brain Injuries; Calcium-Binding Proteins; Calpain; Drug Evaluation, Preclinical; Enzyme Inhibitors; Forecasting; Humans; Isoenzymes; Neurodegenerative Diseases; Spinal Cord Injuries

While the average age for patients admitted with spinal cord injury is 32 years, patients under the age of 16 account for 5% of spinal cord injured persons. For these younger patients, an increased mortality up to 24 h post-injury has been reported, however, survivors may regain more function than their adult counterparts, suggesting that age may play a role in injury tolerance. While the use of growth factors as a therapy for spinal cord injury is well researched, the response of the developing cord to secondary injury has not been thoroughly investigated. Following spinal cord injury, Ca(2+) influx can activate enzymes such as calpain, a Ca(2+)-dependent protease, which plays a role in the pathogenesis of spinal cord injury in rats. The present investigation revealed that following spinal cord injury, calpain upregulation was significantly less (15.3%) in the 21-day-old rats than in either 45-day-old (70%) or 90-day-old (99.6%) rats, as shown by Western blot and in situ immunofluorescent studies. Expression of the endogenous calpain inhibitor, calpastatin, was significantly higher in juvenile rats than adult rats. Juvenile rats with spinal cord injury also showed a reduced Bax:Bcl-2 ratio (4:1 vs. 6:1), reduced caspase-3 staining, reduced myelin loss (3% vs. 18%), and less neuronal DNA damage, as compared to older rats. These results suggest that increased calpastatin levels found in juvenile rats muted calpain activity and neuronal apoptosis, following spinal cord injury.

Topics: Age Factors; Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Female; Hydrolysis; Neurons; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries

Calpain, a Ca(2+)-dependent cysteine protease, has been implicated in cytoskeletal protein degradation and neurodegeneration in the lesion and adjacent areas following spinal cord injury (SCI). To attenuate apoptosis or programmed cell death (PCD) in SCI, we treated injured rats with E-64-d, a cell permeable and selective inhibitor of calpain. SCI was induced on T12 by the weight-drop (40 g-cm force) method. Within 15 min, E-64-d (1 mg/kg) in 1.5% DMSO was administered i.v. to the SCI rats. Following 24 h treatment, a 5-cm long spinal cord section with the lesion in the center was collected. The spinal cord section was divided equally into five 1-cm segments (S1: distant rostral, S2: near rostral, S3: lesion or injury, S4: near caudal and S5: distant caudal) for analysis. Determination of mRNA levels by reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that ratios of bax/bcl-2 and calpain/calpastatin were increased in spinal cord segments from injured rats compared to controls. Degradation of the 68-kD neurofilament protein and internucleosomal DNA fragmentation were also increased. All of these changes were maximally increased in the lesion and gradually decreased in the adjacent areas of SCI rats, while largely undetectable in E-64-d treated rats and absent in sham controls. The results indicate that apoptosis in rat SCI appears to be associated with calpain activity which can be attenuated by the calpain inhibitor E-64-d.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; DNA Fragmentation; DNA Primers; Female; Gene Expression; Leucine; Neurofilament Proteins; Nucleosomes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord Injuries