Calpain and calpastatin expression in primary oligodendrocyte culture: preferential localization of membrane calpain in cell processes.
The cellular localization of calpain is important in understanding the roles that calpain may play in physiological function. We, therefore, examined calpain expression, activity, and immunofluorescent localization in primary cultures of rat oligodendrocytes. The mRNA expression of m-calpain was 64.8% (P = 0.0033) and 50.5% (P = 0.0254) higher than that of mu-calpain and calpastatin, respectively, in primary culture oligodendrocytes. The levels of mRNA expression of mu-calpain and calpastatin were not significantly different. As revealed by Western blotting, cultured oligodendrocytes contained a 70 kD major band identified by membrane m-calpain antibody, a 80 kD band recognized by cytosolic m-calpain antibody, and calpastatin bands ranging from 45 to 100 kD detected by a calpastatin antibody. Calpain activity in oligodendrocytes was determined by Ca(2+)-dependent 71.2% degradation of endogenous myelin basic protein compared with control; this activity was inhibited significantly (P = 0.0111) by EGTA and also substantially by calpeptin. Localization of calpain in cultured oligodendrocytes revealed strong membrane m-calpain immunofluorescence in the oligodendrocyte cell body and its processes. In contrast, the cytosolic antibody stained primarily the oligodendrocyte cell body, whereas the processes were stained very weakly or not at all. These results indicate that the major form of calpain in glial cells is myelin (membrane) m-calpain. The dissimilar localization of cytosolic and membrane m-calpain may indicate that each isoform has a unique role in oligodendrocyte function.
Ray
Swapan K
SK
Department of Neurology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Neuberger
Timothy J
TJ
Deadwyler
Gail
G
Wilford
Gloria
G
DeVries
George H
GH
Banik
Naren L
NL
D000818:Animals; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D002154:Calpain / Q000096:biosynthesis* / Q000235:genetics; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D022081:Cell Surface Extensions / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D008856:Microscopy, Fluorescence; D004676:Myelin Basic Protein / Q000378:metabolism; D009836:Oligodendroglia / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats
D002135:Calcium-Binding Proteins; D004676:Myelin Basic Protein; D012333:RNA, Messenger; C033668:calpastatin; D002154:Calpain; C449000:m-calpain; C449002:mu-calpain