Calpain and calpastatin expression in primary oligodendrocyte culture: Preferential localization of membrane calpain in cell processes

Abstract

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.

Distinct Regulatory Functions of Calpain 1 and 2 during Neural Stem Cell Self-Renewal and Differentiation

“…Our results showed that calpain 2 silencing elicited a significant decrease in GFAP expression during neural differentiation, while no significant differences were detected in β-III Tubulin expression. This finding, coupled with others showing that calpain 2 is mostly localized in glial cells, while calpain 1 is located primarily in neurons [52], [59], [60], suggests that calpain 2 activity is important for glial, but not neuronal differentiation. In fact, the inhibition of both calpains during neural differentiation by calpastatin resulted in a marked increase in β-III Tubulin expression and no differences in GFAP levels.…”

Sustained Calpain Inhibition Improves Locomotor Function and Tissue Sparing Following Contusive Spinal Cord Injury

“…Calpain overactivation is implicated in the death of motor Fisher's LSD post hoc test, *P < 0.05, **P < 0.01, and ***P < 0.001, for MDL28170 treatment as compared to Vehicle. Differences among the MDL28170 treatment groups were not significant neurons [17,24,25], axonal degeneration [12,26], oligodendrocyte loss, demyelination [27][28][29][30], and several aspects of the inflammatory response [31][32][33][34][35][36]. The sustained activation of calpain following SCI and its central role in secondary degeneration has led to investigations regarding the protective role of calpain inhibition following SCI [37].…”

Caspase-independent pathways of hair cell death induced by kanamycin in vivo

“…28,29 The best characterized calpains are two ubiquitously expressed isozymes, m-and m-calpain, heterodimeric proteins composed of a specific catalytic subunit (80 kDa) and a common regulatory subunit (30 kDa). Localization of activated calpains to membranes may induce membrane rupture, 30 especially in lysosomes with the resultant leakage of cathepsins which in turn can cleave the DNA repair enzyme PARP1, triggering necrotic-or apoptoticlike cell death. 31 In particular, m-calpain maybe an important 32 In agreement with the predominant cellular localization of aminoglycosides in lysosomes, we find the synthesis and activation of the lysosomal protease cathepsin D increased.…”