FORMATION OF LIPOFUSCIN-LIKE AUTOFLUORESCENT MATERIALS IN NG108-15 CELLS - INVOLVEMENT OF LYSOSOMAL PROTEIN-DEGRADATION

We found that neuroblastoma x glioma hybrid NG108-15 cells accumulated lipofuscin-like autofluorescent materials during neuronal differentia tion in culture in a medium containing 1% fetal calf serum, I mM dibut yryl cyclic AMP and 1 mM theophylline. The emission maximum of the lip ofuscin-like autofluorescent materials was between 500 and 550 nm. Gra nules positive to acid phosphatase and periodic-acid Schiff were incre ased, as were the autofluorescent granules in NG108-15 cells. Thiolpro tease inhibitors, boxyoxirane-2-carbonyl)-L-leucin-4-aminobutylamide ( E-64) and acetyl-Leu-Leu-Arg (leupeptin), markedly accelerated the acc umulation of the lipofuscin-like autofluorescent materials in NG108-15 cells. On the other hand, activities of lysosomal thiolproteases, cat hepsin B, C and L, were increased during neuronal differentiation. Pro tein content in the cells was gradually increased with the neuronal di fferentiation, and the rise was significantly accelerated when proteol ysis was inhibited by E-64. These results suggest that the lipofuscin- like autofluorescent materials contain peptidic substances as a compon ent, and indicate that the increase in hydrolytic activities of thiolp roteases during neuronal differentiation is not enough for the hydroly sis of peptidic substrates, resulting in the accumulation of autofluor escent materials in NG108-15 cells.