COLCHICINE INDUCES APOPTOSIS IN CEREBELLAR GRANULE CELLS

Exposure to 1 mu M colchicine, a microtubule disrupting agent, trigger ed apoptosis in rat cerebellar granule cells (CGC), Apoptotic nuclei b egan to appear after 12 h followed by oligonucleosomal DNA laddering, whereas inhibition of the mitochondrial methylthiazol-2-yl)-2,5-diphen yltetrazoliumbromide metabolism became significant between 18 and 24 h , when most cells already had apoptotic nuclei. These events were prec eded by loss of tau protein and fragmentation of alpha and beta tubuli ns. Colchicine treatment also caused alterations in Ca2+ responses to chemical depolarization and a moderate, but progressive, increase in t he resting intracellular Ca2+ concentration. Nearly all neurons expres sed c-Fos after the treatment with colchicine. However, while in part of the cell population c-Fos levels subsequently declined, in the neur ons undergoing apoptosis the protein was still expressed, but had an a bnormal intracellular localization. An increased expression of the con stitutive nitric oxide synthase (NOS-I) was also detected at 12 h and was followed by increased nitrite production. Treatment with 100 nM ta xol to stabilize the microtubuli prevented DNA laddering and apoptotic body formation induced by colchicine. In contrast, pretreatment with the N-methyl-D-aspartate receptor-antagonist, MK-801, or L-type Ca2+ c hannel blockers did not prevent colchicine-induced CGC apoptosis, Inhi bitors of NOS were also ineffective in preventing apoptotic body forma tion and DNA laddering, whereas they delayed the secondary cell lysis. These results support the idea that colchicine-induced cytoskeletal a lterations directly initiate the genetic and structural modifications that result in CGC apoptosis. (C) 1995 Academic Press, Inc.