Figure 4

Dietary restriction treatment partially ameliorates some TBI-induced phenotypes. DR-fed (1% YE), TBI-treated flies are shown in pink, and 1% YE control flies are shown in light grey. Standard-fed (6% YE), TBI-treated flies are shown in maroon, and 6% YE control flies are shown in grey. (A) 1% YE TBI-treated flies have significantly increased lifespan relative to both 6% YE TBI and control flies. TBI still leads to decreased lifespan in both 1% YE and 6% YE flies. All comparisons p < 0.0001, according to both Log-rank and Gehan-Breslow-Wilcoxon Survival comparison tests. One of three independent lifespan experiments displayed (sample size of 205–319 flies per group). (B) 1% YE TBI-treated flies exhibit significantly increased climbing ability at 2 weeks post-TBI, relative to 6% YE TBI flies (p < 0.01), according to two-way ANOVA, with Sidak’s multiple comparison correction (at least 16 groups of 20–25 flies each). TBI still induces a significant decrease in climbing ability at all timepoints, in both 1% YE and 6% YE groups (significance not indicated on graph). (C) The percent change in climbing ability of 1% YE flies (TBI in pink and control in light grey), relative to the average climbing ability of 6% YE flies of the same injury condition was calculated. At 2 weeks, the percent change in climbing ability of 1% YE relative to 6% YE flies is significantly increased (~ 40%) in TBI-treated flies but not control flies. DR-related change in climbing ability is also significantly increased in TBI-treated, but not control flies at 4 weeks post-TBI (data not shown). Both are determined by a two-way ANOVA, with Tukey’s multiple comparison test (p < 0.05). (D) Both 1% YE (p < 0.05) and 6% YE (p < 0.0001) TBI-treated fly heads show higher Drosomycin gene expression at 4 weeks post-TBI, when compared to age-matched, uninjured controls in each diet condition (six replicates of 20 fly heads each per condition). 1% YE TBI-treated flies display significantly decreased Drs gene expression relative to 6% YE TBI-treated flies (p < 0.01), but 1% YE controls do not exhibit significantly different Drs gene expression relative to 6% YE controls. Gene expression levels were calculated via standard curve, normalized to RpL1 gene expression levels, and then log-transformed. p values determined from one-way ANOVA pairwise comparisons between selected injury and diet conditions, with Sidak’s multiple comparisons test. (E) Mono- and poly-ubiquitinated protein between ~ 40 and 150 kDa, measured by P4D1 antibody, levels in head samples (from insoluble protein extracts of 6 replicates of 50 heads each) are significantly elevated (p < 0.05) in 6% YE TBI-treated flies, and trending (p = 0.081) elevated in 1% YE TBI-treated flies. TBI and control samples within each diet condition were run on the same western blot, normalized to total protein between 37 and ~ 200 kDa via Ponceau stain, and then normalized to a standard reference protein sample of mixed young and old flies, which was included on each western blot. Significance determined by two-way ANOVA, with Sidak’s multiple comparison test. Representative blot images shown. (F) 1% YE flies (TBI or control) do not exhibit a change in starvation sensitivity, relative to their 6% YE counterparts, at 2 weeks post-TBI. TBI-treated flies (both 1% YE and 6% YE) still exhibit a significant decrease in survival under starvation (sample size of 43–48 flies per condition).