Calpain activation in apoptosis of ventral spinal cord 4.1 (VSC4.1) motoneurons exposed to glutamate: Calpain inhibition provides functional neuroprotection

Glutamate toxicity has been implicated in cell death in neurodegenerative diseases and injuries. Glutamate‐induced Ca2+ influx may mediate activation of calpain, a Ca2+‐dependent cysteine protease, which in turn may degrade key cytoskeletal proteins. We investigated glutamate‐mediated apoptosis of V...

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Bibliographic Details
Published in:	Journal of neuroscience research Vol. 81; no. 4; pp. 551 - 562
Main Authors:	Das, Arabinda, Sribnick, Eric A., Wingrave, James M., Del Re, Angelo M., Woodward, John J., Appel, Stanley H., Banik, Naren L., Ray, Swapan K.
Format:	Journal Article
Language:	English
Published:	Hoboken Wiley Subscription Services, Inc., A Wiley Company 15-08-2005
Subjects:	Animals apoptosis Apoptosis - drug effects bcl-2-Associated X Protein Ca2+ influx Calcium - metabolism calpain Calpain - antagonists & inhibitors Calpain - metabolism Caspase 3 Caspases - metabolism Cell Fusion Cysteine Proteinase Inhibitors - pharmacology Cytochromes c - metabolism Dipeptides - pharmacology glutamate Glutamic Acid - pharmacology Humans Ionomycin - pharmacology Ionophores - pharmacology membrane potential Mice Mitochondria - metabolism motoneurons Motor Neurons - cytology Motor Neurons - drug effects Motor Neurons - enzymology Neuroblastoma Neuroprotective Agents - pharmacology Patch-Clamp Techniques Proto-Oncogene Proteins c-bcl-2 - metabolism Rats Spectrin - metabolism Spinal Cord - cytology
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Summary:	Glutamate toxicity has been implicated in cell death in neurodegenerative diseases and injuries. Glutamate‐induced Ca2+ influx may mediate activation of calpain, a Ca2+‐dependent cysteine protease, which in turn may degrade key cytoskeletal proteins. We investigated glutamate‐mediated apoptosis of VSC4.1 motoneurons and functional neuroprotection by calpain inhibition. Exposure of VSC4.1 cells to 10 μM glutamate for 24 hr caused significant increases in intracellular free [Ca2+], as determined by fura‐2 assay. Pretreatment of cells with 10 or 25 μM calpeptin (a cell‐permeable calpain‐specific inhibitor) for 1 hr prevented glutamate‐induced Ca2+ influx. Western blot analyses showed an increase in Bax:Bcl‐2 ratio, release of cytochrome c from mitochondria, and calpain and caspase‐3 activities during apoptosis. Cell morphology, as evaluated by Wright staining, indicated predominantly apoptotic features following glutamate exposure. ApopTag assay further substantiated apoptotic features morphologically as well as biochemically. Our data showed that calpeptin mainly prevented calpain‐mediated proteolysis and apoptosis and maintained whole‐cell membrane potential, indicating functional neuroprotection. The results imply that calpeptin may serve as a therapeutic agent for preventing motoneuron degeneration, which occurs in amyotrophic lateral sclerosis and spinal cord injury. In this investigation, we also examined glutamate receptor subtypes involved in the initiation of apoptosis in VSC4.1 cells following exposure to glutamate. Our results indicated that the N‐methyl‐D‐aspartate (NMDA) receptors contributed more than α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionic acid (AMPA) receptors to glutamate‐mediated Ca2+ influx and cell death mechanism. Inhibition of the activities of both NMDA and AMPA receptors protected VSC4.1 cells from glutamate toxicity and preserved whole‐cell membrane potential. © 2005 Wiley‐Liss, Inc.
Bibliography:	National Institutes of Health - No. R01 CA-91460; No. R01 NS-31622; No. R01 NS-38146; No. R01 NS-41088; No. R01 NS-45967; No. MSTP training grant GM-08716 ark:/67375/WNG-VCVNNT0B-6 Spinal Cord Injury Research Fund, State of South Carolina - No. SCIRF-0803 Muscular Dystrophy Association istex:70F0E68DC9C78B2394710E8AE4902FC8F71B21ED ArticleID:JNR20581
ISSN:	0360-4012 1097-4547
DOI:	10.1002/jnr.20581