Adverse Impact of Female Reproductive Signaling on Age-Dependent Neurodegeneration After Mild Head Trauma in Drosophila
Environmental insults, including mild head trauma, significantly increase the risk of neurodegeneration. However, it remains challenging to establish a causative connection between early-life exposure to mild head trauma and late-life emergence of neurodegenerative deficits, nor do we know how sex and age compound the outcome. Using aDrosophilamodel, we demonstrate that exposure to mild head trauma causes neurodegenerative conditions that emerge late in life and disproportionately affect females. Increasing age-at-injury further exacerbates this effect in a sexually dimorphic manner. We further identify Sex Peptide (SP) signaling as a key factor in female susceptibility to post-injury brain deficits. RNA sequencing highlights changes in innate immune defense transcripts specifically in mated females during late life. Our findings establish a causal relationship between early head trauma and late-life neurodegeneration, emphasizing sex differences in injury response and the impact of age-at-injury. Finally, our findings reveal that reproductive signaling adversely impacts female response to mild head insults and elevates vulnerability to late-life neurodegeneration. Overall design: To gain molecular insights into the sexually dimorphic responses to mild head trauma and the progressive development of late-life neurodegeneration, we conducted RNA sequencing on freshly dissected brain tissue from flies with and without vmHT exposure. Here, we focused on flies injured on day 3 after eclosion (D3Inj) since under this injury condition, only females mated with wildtype males exhibited behavioral and pathological deficits late in life (on D45, see Figure 3).  Four distinct D3Inj groups were chosen for RNA extraction and sequencing: wildtype males, females mated with wildtype males, virgin females, and females mated with SP0 males (Figure 8A). We assessed transcriptomic changes in the brain at two timepoints; 1-day post-injury (1dpi) for acute changes and 6-weeks post-injury (6wkpi) for chronic changes. Principal Component Analyses revealed that sex accounted for a large portion of variance in sequencing dataset, but the effects of vmHT were not immediately clear in either timepoints (Figure 8B). This observation reiterated the very mild nature of our injury paradigm. To further identify transcriptomic drivers of neurodegeneration after injury, we grouped the dataset by time post-injury, sex, and reproductive condition. Injured and sham flies were injured at 3 days post-eclosion. At 1 day and 6 weeks post-injury, brains from males, wildtype-mated females, virgin females, and SP0-mated females were collected, resulting in 16 conditions, each condition containing three independent cohorts of n=40 flies. RNA from brain tissue was extracted by TRIzol®/Chloroform extraction (ThermoFisher, 15596026) and further clean-up with the Zymo DNA Clean & Concentrator-5 kit (D4004) with on column DNase treatment (ThermoFisher, AM1907). Once obtained, RNA samples were transferred on dry ice to Admera Health for library preparation and sequencing. Total RNA integrity was checked by BioAnalyzer. QIAseq FastSelect rRNA Fly + NEB Ultra II Directional kit was used to deplete rRNA and prepare stranded cDNA libraries. Samples were sequenced on NextSeq High Output Flow Cell (Illumina) for 150 cycles to generate a total of 60M paired-ended, 101 base-pair reads.