Ceramide Selectively Decreases Tau Levels in Differentiated PC12 Cells Through Modulation of Calpain I

Abstract

Ceramide has been recently proposed to be a signal mediator in several important physiological processes including apoptosis, cellular growth, and differentiation. Because the microtubule-associated protein tau plays an important role in the establishment and maintenance of neuronal morphology, the effects of ceramide on tau were examined. Treatment of differentiated P012 cells with the cell-permeable ceramide derivative N-acetylsphingosine (C2) resulted in a significant reduction in tau levels. Significant decreases in tau levels were also observed when the cells were treated with another ceramide derivative, N-hexanoylsphingosine (06). In addition, C2 treatment increased the levels of a calpain-derived spectrin breakdown product but did not alter the levels of two cytoskeletal proteins, a-actin and a-tubulin. Because both tau and spectrin are proteolyzed in vitro by the calcium-activated cysteine protease calpain, the effects of ceramide analogues on the activity of this protease were examined. Treatment of P012 cells with C2 enhanced calcium-stimulated proteolytic activity significantly, as revealed by monitoring the hydrolysis of the membrane-permeable calpain-selective fluorescence probe N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin. This activity increase was not due to a direct effect of 02 on calpains, because 02 did not alter the activities of purified calpain I or II. In addition, 02 treatment of PC12 cells resulted in a significant increase in the levels of calpain I and, to a lesser extent, the levels of calpastatin (an endogenous calpain inhibitor protein), whereas the levels of calpain II were not changed. Moreover, treatment of the cells with the synthetic calpain-specific inhibitor N-carbobenzoxy-L-leucyl-L-leucyl-L-tyrosine diazomethyl ketone blocked the 02-induced decreases in tau levels. These results indicate that tau levels are regulated in response to a physiological factor and, thus, have implications for ceramidemediated changes in normal and pathological neuronal processes.

Molecular mechanisms of euplotin C-induced apoptosis: involvement of mitochondrial dysfunction, oxidative stress and proteases

“…In PC12 cells, we have found that the overall calpain activity increases after 1 h treatment with euplotin C. This can be expected since euplotin C induces a rapid Ca 2+ overload [21]. In particular, the euplotin C-induced [Ca 2+ ] i rise is within the submicromolar range, thus indicating that calpain-1 is the isoform that is likely to be coupled to euplotin C. This observation is consistent with the evidence that calpain-1 expression is up-regulated by euplotin C. Calpain-1, but not calpain-2, has been previously shown to mediate the signalling mechanisms of apoptotic agents in PC12 cells [65]. It is interesting to note that euplotin C-induced modulation of calpain-1 parallels the effects that this compound exerts on mitochondrial dysfunction, ROS generation and [Ca 2+ ] i increase.…”

Noncaspase proteases in apoptosis

“…In neuronally differentiated PC12 cells, induction of apoptosis by ceramide analogs stimulates calpain-mediated cleavage of Ns-LLVY-AMC. 80 Similar activation of N-s-LLVY-AMC cleavage activites are seen during apoptosis caused by reovirus infection of cultured fibroblasts (81) or in vivo ischemia-reperfusion injury in rat hepatocytes. 82 In addition to observations of calpain activation in experimental apoptosis, activation or overexpression of calpains has also been seen in human diseases marked by excessive cell death.…”

Ceramide triggers an NF-κB-dependent survival pathway through calpain

“…Independent studies have shown that C2 ceramide can activate calpain 5 and NF-kB, 31 or both. 24 In addition, calpain inhibitors can prevent NF-kB induction triggered by ceramide in dermal fibroblasts.…”

“…4 Moreover, C2 ceramide selectively modulates calpain I expression levels, thus decreasing tau levels in differentiated PC12 cells. 5 The final outcome of C2 ceramide treatment changes according to its concentration and the cellular context. C2 ceramide was shown to trigger growth arrest in a number of cell types, [6][7][8] and different types of cell death such as apoptosis, 9-11 necrosis 12,13 and autophagocytosis.…”