Traumatic injury induces stress granule formation and enhances motor dysfunctions in ALS/FTD models

Abstract

Traumatic brain injury (TBI) has been predicted to be a predisposing factor for amyotrophic lateral sclerosis (ALS) and other neurological disorders. Despite the importance of TBI in ALS progression, the underlying cellular and molecular mechanisms are still an enigma. Here, we examined the contribution of TBI as an extrinsic factor and investigated whether TBI influences the susceptibility of developing neurodegenerative symptoms. To evaluate the effects of TBI in vivo, we applied mild to severe trauma to Drosophila and found that TBI leads to the induction of stress granules (SGs) in the brain. The degree of SGs induction directly correlates with the level of trauma. Furthermore, we observed that the level of mortality is directly proportional to the number of traumatic hits. Interestingly, trauma-induced SGs are ubiquitin, p62 and TDP-43 positive, and persistently remain over time suggesting that SGs might be aggregates and exert toxicity in our fly models. Intriguingly, TBI on animals expressing ALS-linked genes increased mortality and locomotion dysfunction suggesting that mild trauma might aggravate neurodegenerative symptoms associated with ALS. Furthermore, we found elevated levels of high molecular weight ubiquitinated proteins and p62 in animals expressing ALS-causing genes with TBI, suggesting that TBI may lead to the defects in protein degradation pathways. Finally, we observed that genetic and pharmacological induction of autophagy enhanced the clearance of SGs and promoted survival of flies in vivo. Together, our study demonstrates that trauma can induce SG formation in vivo and might enhance neurodegenerative phenotypes in the fly models of ALS.

Figure 1.

Repetitive traumatic injury can lead to granule formation in Drosophila brain. Transgenomic Rasputin (SG assembly protein) protein tagged with green fluorescence protein (GFP), RIN-GFP, induction (arrows) in third instar Drosophila larval brain exposed to trauma. (A) Third instar larval brains that were subjected to traumatic hits (0, 1, 4 or 8 hits at 60°) and examined soon after trauma or 24 hours’ post-trauma. Anti-lamin (red) was used as a nuclear marker to highlight the nuclear envelope membrane. Quantification analysis using the box-and-whisker plot showed significant increase in the (B) number (#) and (C) area (µm²) of RIN-GFP puncta after injury (n = 7). RIN-GFP puncta remain 24 h after traumatic injury. (D) shows RIN-GFP puncta in larvae exposed to 42°C temperature for 30 min, which disassembles 24 h after the animal is removed from heat stress. (E) Poly-A binding protein (PABP), another SG marker, co-localized (merged, yellow) with RIN-GFP in larval brains post-injury. Control w1118 larvae brain stained with PABP (red) and the nuclear marker DAPI (blue) showed SG formation post-trauma. (F) Quantification of the % of RIN-GFP puncta that are PABP-positive. The insets show the enlarged images. The asterisks (*) represent difference between groups *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. (not significant). Errors bars = 20µm.

Figure 2.

Exposure to traumatic injury in Drosophila leads to ubiquitinated and Ref(2)P-positive RIN-GFP. (A) Brains of RIN-GFP third instar larvae exposed to traumatic injury (0 hit or 8 hits at 60°) showed ubiquitin positive RIN-GFP puncta (arrows, yellow) after trauma and 24 hours’ post-injury. (B) Box-and-whisker plots showed a significantly higher number of ubiquitin puncta in animals exposed to trauma. (C) Quantification of the percentage (%) of ubiquitin-positive RIN-GFP puncta showed a significantly higher number after injury (n = 7). (D) Ubiquitin-positive SGs co-localized with Ref(2)P (arrows) after injury. (E) Quantitative analysis of ubiquitin-Ref(2)P-positive RIN-GFP puncta showed a significant increase post-injury (n = 8). (F) Western blot analysis of trauma (+ TBI) and non-trauma (−TBI) control or RIN-GFP flies showed elevated levels of ubiquitin and Ref(2)P 24 h after injury. Significant differences were observed in the levels of ubiquitin and Ref(2)P 24 h after trauma (n = 3). The insets show the enlarged images. The asterisks (*) represent difference between groups *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. (not significant). Errors bars = 20µm.

Figure 3.

Traumatic injury transiently promotes TBPH association with SGs in Drosophila brain. (A) Endogenous Drosophila TAR DNA-binding protein-43 homolog (TBPH, red) co-localized (merged, arrow) with RIN-GFP (green) within larval brain post-injury (8 hits at 60°). (B) Quantitative analysis of TBPH-positive SGs showed a significant increase after injury, which significantly decrease 24 hours’ post-trauma (n = 5). (C) Brains of adult flies exposed to trauma (10 hits at 70°) showed TBPH and RIN-GFP co-localization (yellow, arrows). (D) Quantification of TBPH-positive RIN-GFP SGs showed a significant increase after trauma, as well as a significant decrease 24 hours’ post-injury (n = 6). (E) Larvae brains from w1118 control animals exposed no hit (CTRL) or 8 hits at different degrees (30°, 50°, 70° or 90°) stained with anti-TBPH and anti-lamin. (F) Quantification of the number of TBPH puncta shows that 8 hits at 50° (P < 0.05), 70° (P < 0.001) or 90° (P < 0.001) but not 30° (P = n.s.) were significantly higher when compared with control (CTRL). The insets show the enlarged images. The asterisks (*) represent difference between groups *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant). Error bars = 20µm.

Figure 4.

Traumatic injury in Drosophila models of ALS enhances mortality and locomotive dysfunction. Adult flies expressing wild-type FUS (FUS WT), mutant FUS (FUS R518K or FUS P525L), C9ORF72 (C9ORF72–3 repeats and C9ORF72–30 repeats) in neurons, and w1118 control exposed to 10 hits at 70° (+TBI, blue) or no trauma (−TBI, red). Mortality index showed higher levels of death in ALS-expressing flies (A) 24 hours’ or (B) 3 days’ post-injury when compared with their respectively uninjured control. Climbing assay in control (−TBI) and injured (+TBI) flies expressing ALS-causing genes or control showed significant reduction in locomotion ability (C) 24 hours’ or (D) 10 days’ post-injury. (E) Immunofluorescence of 3-day-old EGFP or FUS (FUSWT, FUSR518K or FUSP525L) expressing fly brains that were exposed to no trauma (0 hit) or trauma (10 hits at 70°) stained with antibody against FUS and lamin. Arrows show FUS mislocalization in a subset of cells. (F) Quantification of the % of cells with FUS mislocalization showed no significant changes between trauma and non-trauma flies. The asterisks (*) represent difference between groups *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant). Values are mean ± S.E.M.

Figure 5.

Ref(2)P levels are altered in Drosophila expressing ALS-associated proteins. Ectopic expression of red fluorescence protein (RFP) alone or RFP-tagged FUS (WTFUS-RFP or P525LFUS-RFP) combined with RIN-GFP. Brains of adult flies expressing RFP alone (A), WTFUS-RFP (B) or P525L FUS-RFP (C) exposed no trauma (0 hit) or trauma (10 hits at 70°) and stained with Ref(2)P and lamin antibody. Arrows shows Ref(2)P puncta accumulating with FUS (red) and RIN-GFP (green). (D) Western blot analysis show high levels of ubiquitin and Ref(2)P in flies expressing RFP alone, WTFUS-RFP or P525L FUS-RFP combined with RIN-GFP 24 h after injury (+TBI) compared with uninjured control (−TBI). (E) Western blot of Ref(2)P levels in control, FUS (FUSWT, FUSR518K and FUSP525L) or C9ORF72 (C9ORF72–3 repeats and C9ORF72–30 repeats) expressing flies with (+) or without (−) trauma 24 h or day 5 post-trauma. (F) Ref(2)P levels significantly increase in control, FUSWT, FUSR518K, C9ORF72–3R, C9ORF72–30R (P < 0.001) and FUSP525L (P < 0.05) expressing flies 24 h after exposure to trauma when compared with their respective non-trauma control (n = 3). (G) Quantification of the same cohort of control, FUS (FUSWT, FUSR518K and FUSP525L) or C9ORF72 (C9ORF72–3 repeats and C9ORF72–30 repeats) expressing flies 5 days post-injury showed significantly higher levels of Ref(2)P in control, FUSWT, FUS-P525L, C9ORF72–30R (P < 0.001) but not FUSR518K or C9ORF72–3R (P = n.s.) when compared with their respective no trauma control. Tubulin was used as a control and Ref(2)P levels were normalized to tubulin. The asterisks (*) represent difference between groups *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant). Values are mean ± S.E.M.

Figure 6.

Rapamycin promotes SG disassembly and increases eclosion after injury. (A) Brains of RIN-GFP larvae subjected to 8 hits at 60° and fed rapamycin drug (0.5, 1, 10, 50, 100 or 200 µM) or dimethyl sulfoxide (DMSO) only for 24 h. Larval brains are stained with lamin (red) antibody. The insets show the enlarged images. (B) Quantification of the number of RIN-GFP puncta 24 hours’ post-trauma showed significant decrease in animals fed 0.5, 1, 10, 50, 100 or 200 µM rapamycin compared with DMSO-treated animals (P < 0.001, n = 10–25). (C) Quantification of the % of flies that eclosed after larvae was exposed to trauma and fed 1, 10, 50, 100 and 200 µM but not 0.5 µM rapamycin drug showed a significant increase when compared with DMSO-treated animals (P < 0.001). (D) Expression of autophagy-related protein 8a (Atg8a) significantly increases eclosion post-trauma when compared with animals expressing EGFP alone (P < 0.05). The asterisks (*) represent difference between groups, **P < 0.01, ***P < 0.001 and n.s. (not significant). Errors bars = 20µm.