Exposure of adult fruit flies to a very mild form of repetitive head impacts at different ages elicits minimal acute effects.

(A) Schematic illustration of the experimental design. Male and female flies were exposed to vmHT on D3, D17, or D31 post-eclosion (denoted as D3Inj, D17Inj, and D31Inj), whereas the sham groups did not receive vmHT. Sensorimotor behavior and brain pathology were assessed at 90 min and 1 day after vmHT exposure using mNGA and imaging-based quantification of vacuolization. (B) Survival curves showing no significant alteration in lifespan among different age-at-injury groups and between sexes. Kaplan-Meier p-values were determined using the Mantel-Cox log rank test with Bonferroni correction. Total N=871, n>50 flies in each condition. (C) Schematic diagram depicting mNGA where the Climbing Index (CI) at each second of the 10 second trial was calculated using the number of flies in each of the ten height bins (see Methods for details). (D-F) CI plots depicting acute effects of vmHT on the climbing behavior of D3Inj, D17Inj, and D31Inj cohorts. (D) Both male and female D3Inj flies showed no deficits in climbing at 90 min and 1-day post-injury. (E) Only female D17Inj flies exhibited climbing deficits at 90 min post-injury (*** p=5.75e-08) but they recovered 1-day post-injury. (F) Both male and female D31Inj flies exhibited marked decline in climbing ability at both time points (p values: 5.36e-08 and 6.2e-06 for female +90m and +1d; 0.00033 and 1.91e-07 for male +90m and +1d). A total of 9 videos were used for each condition: 3 experimental repeats of 3 trials, number of flies in each video n≥ 10. Error bar: ±se. Repeated measures ANOVAs were conducted to examine the effects of injury on climbing indices at each second.

Exposure to vmHT does not acutely increase vacuole formation in the brain.

(A-C) Two-photon whole brain imaging and quantification of vacuoles in D3Inj (A), D17Inj (B), and D31Inj (C) flies. Top panels: representative z-projected whole-brain images of different injury groups with their respective sham brains of both sexes. Vacuoles are highlighted by yellow color. Scale bar = 100 μm. Bottom: violin plots and boxplots represent quantification of vacuole number and total vacuole area in each condition. Boxplots whiskers correspond to the maximum 1.5 interquartile range. Two experimental replicates resulting in n>10 brains for all conditions. Stats: non-parametric Wilcoxon rank sum tests, p>0.05.

Exposure to vmHT results in late-life brain deficits and neurodegeneration.

(A) Exposure to vmHT at various ages altered negative geotaxis behavior when assessed on D45. A total of 9 videos were used for each condition: 3 experimental repeats of 3 trials, number of flies in each trial n≥ 10. Error bar: ±se. Repeated measures ANOVAs and Bonferroni post hoc tests were conducted to examine the effects of injury on climbing indices at each second. Overall, vmHT exposure reduced CI for females (*** p=2e-16) and males (*** p=1.2e-11). See Supplementary Data S1 for p values from pairwise comparisons by injury conditions and time. (B) Sex differences in vmHT-induced climbing impairment are associated with age-at-injury. D3Inj and D17Inj females exhibit a stronger decline in normalized accumulated CI compared to males. D31Inj females and males, on the other hand, suffered a similar substantial reduction in accumulated CI. Accumulated CI data of injury groups are normalized to their respective sham levels. (C) Representative z-projected whole images depicting vacuole formation in each condition (sex/ injury group). Scale bar = 100 μm. (D) Quantification of vacuole number and total vacuole area in each condition (Total N=352, n>30 in each condition). Here all sham groups were combined by sex. Boxplots whiskers correspond to the maximum 1.5 interquartile range. Statistics: non-parametric Wilcoxon rank sum tests. See Supplementary Data S1 for p values from pairwise comparisons by injury conditions. (E) Sex differences in the increase of brain vacuolation were partially dependent on age-at-injury. Vacuole number and total vacuole area of each injury condition were normalized to their respective sham groups. Overall, females exhibited a higher percentage of increase in vacuolation than males. (F) Quantification of the percentage of vacuoles in neuropil and cortex region of the brain in sham, D3Inj, and D31Inj groups of both sexes. Data was from two independent trials of n=15 brains in each condition. Two sample t-tests were used to compare the percentage of vacuoles in neuropil and cortical region between each injury condition. D31Inj females had significantly less percentage of vacuoles in the neuropil region compared to the sham group (*** p=2.19493e-6).

AI-assisted tracking and quantification of individual fly’s behavior provides insight into defects in the speed and direction of movement.

(A) Representative snapshot of idtracker.ai-generated video at T=6 seconds. Videos used for analyses were from D45 flies (see Supplemental video SV1). Each trail represents fly movement speed calculated from the last 30 frames. Color is auto assigned to individual flies in each trial. (B) Histogram plots of the climbing speed of individual flies at a temporal resolution of 1/60 sec. The y-axis depicts the percentage of flies with a specific speed (x-axis). (C) Angular histograms showing the angle distribution of individual flies during the first 3 secs of mNGA trial. Highlighted sections represent normal fly directional orientation (between -30° and 30° with 0° as vertical). Arrows indicate the increased incidents of climbing angles outside the normal range. (D) Average percentages and accumulated percentages of flies with abnormal directional movement (<-30° or >30°) during the first three seconds of NGA trial. Two sample t tests, p<0.001, ***. (E) Rose plot depicting overall frequency distribution of fly orientation during the first three seconds of NGA trial.

Virgin females do not exhibit neurodegenerative conditions regardless of age-at-injury.

(A) Comparison of size and mass of virgin female, mated female, and male flies. 3 separate trials of n>100 in each condition. Total mass in each trial was averaged over the number of flies to generate a weight range for each condition. (B) vmHT resulted in no significant change to the lifespan of virgin females. Kaplan–Meier p-values were determined using the Mantel-Cox log rank test with Bonferroni correction. Total N=271, n>30 flies in each condition. (C) vmHT did not affect climbing behaviors of virgin females on D45 regardless of the age-at-injury. A total of 9 videos were used for each condition: 3 experimental repeats of 3 trials, number of flies in each trial n≥ 10. Repeated measures ANOVAs were conducted to examine the effects of injury on climbing indices at each second (p>0.05). (D) vmHT did not increase vacuole number or total vacuole area on D45 regardless of the age-at-injury. Total N= 224, n>25 for each condition. Statistics: non-parametric Wilcoxon rank sum tests. (E) Plots depicting differences in vacuole formation between virgin and mated females. Vacuole number and total vacuole area of each injury condition were normalized to the respective sham controls. Mated females exhibited much higher percentage of increase in vacuolation than virgin females.

Eliminating SP signaling mitigates the emergence of neurodegenerative conditions in the female.

(A-C) Survival curves, mNGA quantification, and vacuole quantification of female flies mated to SP0-males, females with pan-neuronal RNAi knockdown of SPR mated to wildtype males, and females with SPSN-specific knockdown of SPR mated to wildtype males (n>30 in each condition). No difference was detected between sham and injured groups in all three genotypes.

Introducing SP-signaling to pre-injured virgin females reinstates neurodegenerative phenotypes.

(A) Diagram of mating schematic and relevant behavioral and pathological assays. Virgin female flies were subjected to vmHT on D3, whereas sham flies never receive an injury. On D10, these females were exposed to either wildtype or SP0 males for 24 hours to allow mating. mNGA and brain vacuolation were assessed on D45. (B) No change in the lifespan was observed for females that subjected to vmHT as virgin followed by post-injury mating with either male group. N= 182, n> 40 for each condition. (C) When assessed on D45, females subjected to post-injury mating with wildtype males exhibited significantly sensorimotor deficits (*** p=3.4e-15) and vacuole formation (vacuole number: p=0.0004536, vacuole area: p=0.0009563). A total of 9 NGA videos were used for each condition: 3 experimental repeats of 3 trials, number of flies in each trial n≥ 10. Error bar: ±se. Repeated measures ANOVAs and Bonferroni post hoc tests were conducted to examine the effects of injury on climbing indices at each second. For vacuole analyses, N= 149, n>30 in each condition. (D) When assessed on D45, females subjected to post-injury mating with SP0 male flies did not elicit sensorimotor deficits and vacuole formation. A total of 9 NGA videos were used for each condition: 3 experimental repeats of 3 trials, number of flies in each trial n≥ 10. For vacuole analyses, N= 105, n>20 in each condition.

RNA sequencing reveals sexually dimorphic and reproductive status-dependent transcriptomic responses in D3Inj fly brains.

(A) Timeline and design of RNA-seq experiment. Sham and D3Inj brains were collected at 1dpi and 6wkpi, in four different conditions (wildtype males, females mated with wildtype males, virgin females, and females mated with SP0 males), and in three biological replicates (n=40 brains per condition per replicate). Total sample number n= 48. (B) PCA analyses of samples at chronic and acute timepoints. (C, E) Volcano plots depicting gene expression at the acute and chronic timepoints. Upregulated genes are highlighted in green, downregulated genes are highlighted in red. (D, F) Venn diagram displaying the limited overlap of genes between the four conditions at the acute and chronic timepoints. (G) Venn diagram depicting no overlap between the acute and chronic gene changes in females mated to wildtype males. Upregulated genes are highlighted in green, downregulated genes are highlighted in red.

Exposure to vmHT on D3 does not elicit gross morphological alterations in PDF neurons and mushroom bodies.

(A-B) vmHT exposure on D3 did not significantly alter PDF neuron axonal projections or dendritic arborizations when assessed 24 hours or 6 weeks after injury. (C-D) vmHT exposure on D3 did not visibly alter mushroom body axon bundles when assessed 24 hours or 6 weeks after injury. Scale bar = 100 microns.

Exposure to vmHT does not elicit significant apoptosis.

(A-C) Representative images of TUNEL staining in Drosophila brains at 1 day post D3Inj, D17Inj, and D31Inj. There was limited TUNEL signal in both sexes and no differences in staining pattern and signal intensity were observed between sham and injured groups. A set of flies were subject to stabbing injuries at the same time and their brains were used as positive controls of apoptosis (right panels). TUNEL staining was visible in the optic lobes or other regions that were penetrated by the fly pin. (D) Representative images of TUNEL staining in sham and injured brains on D45. vmHT were delivered on D3, D17, or D31. Stabbing injuries were performed on D44 (24 hours prior to fixation). No differences in staining pattern and signal intensity were observed between sham and injured groups and between sexes. In comparison, brains of stabbed flies (right panels) exhibit much stronger TUNEL signal intensity and region-specific apoptosis near location of primary injury. Scale bar = 100 microns. Each condition contained n=15 flies.

Age-at-injury affects post-injury recovery after two weeks.

(A) CI plots depicting sensorimotor behavior assessed two weeks following D3Inj, D17Inj, and D31Inj. Female D3Inj groups exhibited a significant decrease in negative geotaxis when assessed on D17 (** p=0.00114), whereas males did not (p>0.05). Both female (*** p= 1.53e-10) and male (*** p= 2.47e-05) D17Inj groups suffered sensorimotor deficits when assessed on D31. D31Inj flies all exhibited significant detriment to climbing behavior when assessed on D45 (*** p= 2e-16 for female, *** p= 2.58e-07 for male). Repeated Measures ANOVA tests were used to calculate statistical significance. Total of three experimental repeats and a total of 9 videos were used in each condition. n>15 per condition per repeat. (B) In both male and female flies, older age-at-injury is associated with larger decreases in accumulated climbing indices normalized against the respective sham controls. (C-E) Quantification of vacuole formation at two weeks following D3Inj, D17Inj, and D31Inj. D3Inj increased vacuole number (** p=0.0017) and total vacuole area (*** p=0.00016) in female brains but not in male brains. D17Inj did not significantly alter vacuole formation in both sexes though there seems to be a trend towards injury increasing both vacuole size and vacuole number. D31Inj increased both vacuole formation in female and males. Two repeats resulting in n>12 brains per condition. Wilcoxon rank sum tests were used to calculate p values. From left to right, *** p = 2.6e-05, *** p=4.5e-05, *** p=8e-05, and *** p=0.00048. (F) Vacuole number and total vacuole number in each injury condition were normalized against their respective sham groups. Older age-at-injuries were associated with accelerated vacuole formation in males, but this association was not observed in females.

Exposure to reduced vmHT on D3 elicited similar late-life behavioral deficits and brain pathology as exposure to regular vmHT.

(A) CI plots depicting decreases in CI in both regular D3Inj and reduced D3Inj females when climbing behavior was assessed on D45. (B) Quantification of vacuole formation on D45. Vacuolation was similarly elevated in reduced D3Inj brains and in regular D3Inj brains. Only one experiment was performed for reduced D3Inj (n=15).