The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC) (Pegg and McCann, 1988). Difluoromethylornithine (DFMO) is a suicide inhibitor of ODC. Administered to rats as a 2% solution in drinking water, DFMO lowers total polyamine levels significantly in all tissues examined (Danzin et al., 1979). Rats treated with DFMO for 2–21 days were used to determine the effects of reduced neuronal polyamine on CST. After 18 hr, 7 days, and 21 days of treatment, axonal MTs were labeled by injecting 35S-methionine into the vitreous of the eye and waiting 21 days for axonal transport to deliver labeled

tubulin to the optic nerve. Cold/Ca2+ fractionation of labeled optic nerve (Figure 1A) showed a significant decrease in CST after DFMO treatment (Figures

LY294002 manufacturer 1B and 1C). Fluorographs of S1, S2, and P2 fractions from control and DFMO-treated rats show a significant fraction of tubulin shifted from P2 to S1 fractions with DFMO treatment (Figure 1B). In control optic nerves, 52% of the total radiolabeled axonal tubulin was cold-insoluble tubulin, but in 7 day or 21 day DFMO-treated nerves, this fraction was <40% (Figure 1C; VX-809 manufacturer see also Figure S1 available online; Table S1) (p < 0.001), suggesting that polyamines are required for generation of cold-insoluble tubulin in axons. To determine whether a decrease in polyamines generally reduced cytoskeleton stability in axons or was specific for MTs, neurofilament (NFM) fractionation was analyzed in parallel. There was no change in NFM fractionation after DFMO treatment (Table S1). Next, we sought to determine whether polyamines were covalently added to tubulin in vivo and whether modified tubulin cofractionated with cold-insoluble tubulin. 14C-PUT axonal transport labeling experiments were until performed in rat optic nerves. Due

to high levels of endogenous polyamines and low specific activity of 14C-PUT, endogenous polyamine levels were lowered by 18 hr DFMO pretreatment. When axonal proteins were fractionated 21 days after 14C-PUT labeling, 70%–80% of the label was in P2 (not shown). Subsequent fractionation studies with higher-specific-activity 3H-PUT confirmed these results (Figure 2A). In optic nerves labeled by axonal transport of 3H-PUT, the only proteins with significant incorporation of labeled polyamines had the molecular weight (MW) of tubulin, although 35S-methionine labeled neurofilaments at the same time. Much lower levels of 3H-PUT were seen in S1, the cold-labile MT fraction. This suggested that polyamination of tubulin can occur before formation of stable MTs. Polyaminated tubulin may help nucleate and stimulate polymerization of tubulins and may also stabilize MTs after polymerization. 14C-PUT-labeled proteins in P2 were analyzed by gel filtration chromatography on a Toyopearl HW-55F (Supelco) column equilibrated in 6 M guanidine-HCl in MES to determine if 14C-PUT coeluted with tubulin.