PROTEASOME INHIBITORS WHICH INDUCE NEURITE OUTGROWTH FROM PC12H CELLSCAUSE DIFFERENT SUBCELLULAR ACCUMULATIONS OF MULTI-UBIQUITIN CHAINS

The effects of two proteasome inhibitors on neurite outgrowth from PC1 2h cells were investigated in terms of the mean length of the neurites and the frequency of occurrence of cells with long neurites. Benzylox ycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and zyloxycarbonyl-isoleucyl -t-butyl-glutamyl-leucinal (PSI) caused a significant elongation of PC 12h cell neurites. Since ZLLLal is known to inhibit both calpain and p roteasome activity, we examined the effects of benzyloxycarbonyl-leucy l-leucinal (ZLLal) which inhibits calpain activity to the same degree as ZLLLal, but which inhibits proteasome activity only weakly. ZLLal d id not induce the significant elongation of neurites at any of the con centrations we studied. These results show that the inhibition of prot easome activity causes neurite elongation. We also quantified subcellu lar levels of multi-ubiquitin chains and free ubiquitin after treatmen ts with PSI, ZLLLal and ZLLal. Treatment with ZLLal had no effects on levels of water- and urea-soluble multi-ubiquitin chains or of free ub iquitin either in the nucleus or in the cytoplasm. PSI and ZLLLal indu ced a large accumulation of water- and urea-soluble multiubiquitin cha ins and free ubiquitin in the nucleus. Similarly, PSI and ZLLLal incre ased cytoplasmic levels of urea-soluble multi-ubiquitin chains. On the contrary, PSI and ZLLLal had no effect on levels of water-soluble mul ti-ubiquitin chains or free ubiquitin in the cytoplasm. This is the fi rst study to demonstrate subcellular differences in the accumulation o f multi-ubiquitin chains and free ubiquitin during the neurite elongat ion induced by proteasome inhibitors.