Mild traumatic brain injury in Drosophila melanogaster alters reactive oxygen and nitrogen species in a sex-dependent manner

Abstract

Traumatic brain injury (TBI) is an outside force causing a modification in brain function and/or structural brain pathology that upregulates brain inducible nitric oxide synthase (iNOS), instigating increased levels of nitric oxide activity which is implicated in secondary pathology leading to behavioral deficits (Hall et al., 2012; Garry et al., 2015; Kozlov et al., 2017). In mammals, TBI-induced NO production activates an immune response and potentiates metabolic crisis through mitochondrial dysfunction coupled with vascular dysregulation; however, the direct influence on pathology is complicated by the activation of numerous secondary cascades and activation of other reactive oxygen species. Drosophila TBI models have demonstrated key features of mammalian TBI, including temporary incapacitation, disorientation, motor deficits, activation of innate immunity (inflammation), and autophagy responses observed immediately after injury (Katzenberger et al., 2013; Barekat et al., 2016; Simon et al., 2017; Anderson et al., 2018; Buhlman et al., 2021b). We hypothesized that acute behavioral phenotypes would be associated with deficits in climbing behavior and increased oxidative stress. Because flies lack mammalian-like cardiovascular and adaptive immune systems, we were able to make our observations in the absence of vascular disruption and adaptive immune system interference in a system where highly targeted interventions can be rapidly evaluated. To demonstrate the induction of injury, ten-day-old transgenic flies received an injury of increasing angles from a modified high impact trauma (HIT) device where angle-dependent increases occurred for acute neurological behavior assessments and twenty-four-hour mortality, and survival was significantly decreased. Injury caused sex-dependent effects on climbing activity and measures of oxidative stress. Specifically, after a single 60-degree HIT, female flies exhibited significant impairments in climbing activity beyond that observed in male flies. We also found that several measures of oxidative stress, including Drosophila NOS (dNOS) expression, protein nitration, and hydrogen peroxide production were significantly decreased in female flies. Interestingly, protein nitration was also decreased in males, but surpassed sham levels with a more severe injury. We also observed decreased autophagy demand in vulnerable dopaminergic neurons in female, but not male flies. In addition, mitophagy initiation was decreased in females. Collectively, our data suggest that TBI in flies induces acute behavioral phenotypes and climbing deficits that are analogous to mammalian TBI. We also observed that various indices of oxidative stress, including dNOS expression, protein tyrosine nitration, and hydrogen peroxide levels, as well as basal levels of autophagy, are altered in response to injury, an effect that is more pronounced in female flies.

Keywords: Apoptosis; Drosophila; Inflammation; Innate immunity; Mitochondria; Neurodegeneration; Oxidative stress; Secondary injury; Traumatic brain injury; dNOS.

Figure 1.. HIT causes acute behavioral deficits and increased mortality.

Individual vials containing eight 10-day post-eclosion flies each were loaded into a modified HIT device and exposed to a single HIT at 30, 45, or 60-degree pendulum angles. Sham flies were loaded into the device but not exposed to injury. (A) Wing splay is the percentage of flies with abnormal wing position in shams or immediately after injury. (B) Representative sham flies with typical wing position. (C) Injured flies with splayed wings (black arrows). (D) The percentage of flies that were temporarily incapacitated following injury. (E) The percentage of dead flies was recorded twenty-four hours after injury. (F) The percentage of dead flies was recorded at least twice per week following injury until all flies expired. Kruskal-Wallis tests were performed to determine the effect of injury for A, D, and E. Each data point represents one vial (n ≥ 8; p < 0.05 for “*”, p < 0.01 for “**”, p < 0.001 for “***”, and p < 0.001 for “****.”). Error bars represent standard error of the mean. For survival analyses, we obtained the 95% prediction limits for sham flies and calculated the corresponding EC₅₀ values to set the limits for determining statistical significance. Injury curve EC₅₀ values that of the upper prediction limit were reported to be significantly lower than sham (n ≥ 53; p < 0.05). (B,C) Reproduced from Buhlman, Krishna et al. Drosophila as a model to explore secondary injury cascades after traumatic brain injury. Biomedicine & Pharmacotherapy 2021; 142(112079):1–17. Copyright © 2023. published by Elsevier Masson SAS. All rights reserved.

Figure 2.. Female climbing behavior is more sensitive to HIT compared with males.

(A) Immediately after HIT at 30- or 60-degree angles (or placement in the vial for sham), individual flies were loaded, and activity was recorded in the MB5 Multibeam Drosophila Activity Monitor for four hours. Total activity was decreased after 60-degree HIT, and only females were affected when exposed to a 30-degree HIT. Total moves for the duration of the experiment is calculated using the area under the curve (AUC). (B) Female climbing attempts decreased after injury, while male climbing attempts were only affected by 60-degree HIT. (C) Female average height climbed was decreased with injury and recovered four hours post-injury. Male average height climbed was unaffected. Each data point represents data from one fly (n ≥ 7). Kruskal-Wallis, Welch’s ANOVA, or one-way ANOVA tests were performed to determine the effect of injury. p values are reported for each comparison. Error bars represent standard error of the mean.

Figure 3.. dNOS levels in the central brain increase with age.

(A) Representative panels of female and male central brains expressing dNOS-driven GFP on day 0 – 5, 10 – 15, and 30 – 35 post-eclosion. Central brain regions are indicated by black outlines, and yellow “isosurfaces” indicate GFP expression above background. Scale bar represents 200 μm. (B) Total fluorescence volume and average intensity of central brain GFP emission were calculated. Each data point represents one brain (n ≥ 18). Kruskal-Wallis, Welch’s ANOVA, or one-way ANOVA tests were performed to determine the effect of age on dNOS levels. Error bars represent standard error of the mean. p values are reported for each comparison.

Figure 4.. dNOS levels decrease in the female central brain after HIT.

Brains were dissected from HIT-injured female and male flies expressing dNOS-driven GFP, and total volume and intensity of central brain GFP emission were calculated. (A) Representative panels of sham and injured female and male central brain images. Central brain regions are indicated by black outlines, and yellow “isosurfaces” indicate GFP expression above background. Black arrows indicate low expression in injured flies. Scale bar represents 200 μm. (B) Total fluorescence volume and average intensity. Each point represents data from one fly (n ≥ 7). Welch’s ANOVA or one-way ANOVA tests were performed to determine the effect of injury on dNOS levels. Error bars represent standard error of the mean. p values are reported for each comparison.

Figure 5.. Drosophila brain tyrosine nitration is altered after HIT in a severity- and sex-dependent manner.

Brains were dissected from single (1x) or double (2x) HIT-injured female and male flies expressing dNOS-driven GFP. Dissected brains were labeled with anti-tyrosine nitration antibody, and total volume of central brain fluorescence emissions were calculated. (A) Representative images of sham and injured female and male central brain tyrosine nitration. Central brain regions are indicated by black outlines, and blue “isosurfaces” indicate antibody labeling above background. Scale bar represents 200 μm. (B) Each point for total fluorescence volume graphs represents data for one brain (n ≥ 7). Kruskal-Wallis tests were performed to determine the effect of injury on tyrosine nitration. Error bars represent standard error of the mean. p values are reported for each comparison.

Figure 6.. HIT reduces hydrogen peroxide levels in female, but not male dopaminergic neuron mitochondria.

Brains were dissected from sham and injured flies expressing mito-roGFP2-Orp1, and ratios of total volumes of oxidized to non-oxidized fluorophore emissions were calculated for one PPL1 region per brain. (A) Representative panels showing raw PPL1 images (top row, where blue indicates TH antibody labeling, and red and green indicate oxidized and non-oxidized roGFP2, respectively). Automatically selected volume “isosurfaces” of oxidized (red, middle row) and non-oxidized (green, bottom row) mito-roGFP2-Orp1 above threshold are also shown. Scale bar represents 10 μm. (B) Each data point represents the ratio from one PPL1 region (n ≥ 8). Unpaired t tests were performed to determine the effect of injury. Error bars represent standard error of the mean, and p values are reported for each comparison.

Figure 7.. HIT reduces autophagosome formation and mitophagy initiation in female, but not male dopaminergic neurons.

Brains of injured flies expressing the mitoGFP and mCherry-Atg8a constructs in TH-producing cells were dissected, stained for TH (blue). (A) Representative panels show raw images (top row, where blue, red, and green indicate TH antibody labeling, mCherry-Atg8a, and mito-GFP, respectively). Selected volume “isosurfaces” are shown for green (mitochondrial), red (autophagosomes), and yellow, (mito-autophagosome colocalizations). Scale bar represents 10 μm. (B) The number of autophagosomes, and (C) GFP-mCherry colocalizations within TH-positive cell bodies was divided by the number of TH-positive cells. (D) Autosome-mitochondria recruitment efficiency was calculated by dividing the number of colocalizations by the number of autophagosomes per dopaminergic region. Mann-Whitney, Welch’s, and unpaired t-tests were performed to determine the effect of injury. p values are reported for each comparison. Data points indicate the number of brain regions analyzed (n ≥ 9), and error bars represent standard errors of the mean.