Figure 2

TBI induces long-term molecular hallmarks of neurodegeneration. TBI-treated flies = red; age-matched, uninjured control flies = dark grey. Insoluble protein extracted from heads of TBI-treated flies contains increased (A) mono- and poly-ubiquitinated protein between ~ 40 and 150 kDa, measured by P4D1 antibody, and (B) Ref2P, at 4 weeks post-TBI only, when compared to age-matched control flies. Insoluble protein extracted from bodies does not display a significant increase in (C) ubiquitinated protein. Six replicates of 50 heads each, or 20 bodies each, were extracted at each timepoint. TBI and control samples within each timepoint 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 old and young flies, which was included on each western blot. At 4 weeks post-TBI, TBI relative to control for Ubiquitin and Ref2P is p < 0.05, according to two-way ANOVA, with Sidak’s multiple comparison correction. Representative blot images for head samples are included below graph. (D–K) TBI-treated fly heads show higher AMP gene (Drosomycin (D,H), Diptericin (E,I), Cecropin (F,J), and Drosocin (G,K)) expression at multiple timepoints, as compared to age-matched, uninjured controls. Six cDNA replicates of 20 fly heads each were prepared for each condition, at each timepoint: 2 h, 4 h, 8 h, 48 h, 2 weeks, and 4 weeks post-TBI; gene expression levels were calculated via standard curve for each gene, normalized to RpL1 gene expression levels, and then log-transformed. Two-way ANOVA with Sidak’s multiple comparisons test was performed to determine statistical significance between TBI and control at each timepoint. For the acute timepoints: (D) Drosomycin (Drs), (E) Diptericin (Dipt), and (G) Drosocin (Dro) are all significantly elevated (p < 0.0001) at 2, 4, and 8 h following TBI. (F) Cecropin (Cec) is significantly elevated (p < 0.0001) at 2 and 4 h post-TBI, as well as (p < 0.01) at 8 h post-TBI. Following the acute injury phase, the highest induction of AMP gene expression was observed at 48 h post-TBI, with the pairwise comparison between TBI and control for each AMP gene being statistically significant. At 2 weeks post-TBI, Dipt, Cec, and Dro, but not Drs, were significantly elevated. At 4 weeks post-TBI, no pairwise comparisons were significant, although Drs was trending with a p value of 0.0763.