CERAMIDE SELECTIVELY DECREASES TAU-LEVELS IN DIFFERENTIATED PC12 CELLS THROUGH MODULATION OF CALPAIN-I

Ceramide has been recently proposed to be a signal mediator in several important physiological processes including apoptosis, cellular growt h, and differentiation. Because the microtubule-associated protein tau plays an important role in the establishment and maintenance of neuro nal morphology, the effects of ceramide on tau were examined, Treatmen t of differentiated PC12 cells with the cell-permeable ceramide deriva tive N-acetylsphingosine (C2) resulted in a significant reduction in t au levels. Significant decreases in tau levels were also observed when the cells were treated with another ceramide derivative, N-hexanoylsp hingosine (C6). In addition, C2 treatment increased the levels of a ca lpain-derived spectrin breakdown product but did not alter the levels of two cytoskeletal proteins, alpha-actin and alpha-tubulin. Because b oth tau and spectrin are proteolyzed in vitro by the calcium-activated cysteine protease calpain, the effects of ceramide analogues on the a ctivity of this protease were examined. Treatment of PC12 cells with C 2 enhanced calcium-stimulated proteolytic activity significantly, as r evealed by monitoring the hydrolysis of the membrane-permeable calpain -selective fluorescence probe N-succinyl-L-leucyl-L-leucyl-L-valyl-L-t yrosine-7- amido-4-methylcoumarin. This activity increase was not due to a direct effect of C2 an calpains, because C2 did not alter the act ivities of purified calpain I or II, In addition, C2 treatment of PC12 cells resulted in a significant increase in the levels of calpain I a nd, to a lesser extent, the levels of calpastatin fan endogenous calpa in inhibitor protein), whereas the levels of calpain II were not chang ed. Moreover, treatment of the cells with the synthetic calpain-specif ic inhibitor N-carbobenzoxy-L-leucyl-L-leucyl-L-tyrosine diazomethyl k etone blocked the C2-induced decreases in tau levels. These results in dicate that tau levels are regulated in response to a physiological fa ctor and, thus, have implications for ceramide-mediated changes in nor mal and pathological neuronal processes.