Modeling Sex Differences and Neurodegeneration in Repetitive Traumatic Brain Injury Using Drosophila

Traumatic brain injury (TBI) is a public health burden with short-term and long-term consequences for those who survive. Repetitive TBI (rTBI) is highly associated with neurodegenerative diseases such as chronic traumatic encephalopathy. Here, we present a force-adjustable, enhanced CO2-driven Drosophila model of rTBI to investigate locomotor, cognitive, proteomic, and neurodegenerative changes following rTBI. Injured males demonstrate dose-dependent locomotor deficits, while injured females display greater tolerance to rTBI. However, both sexes exhibit significant decision-making deficits. Proteomic analysis revealed changes in proteins linked with locomotory behavior, mitochondria, and energy production post-injury, correlating with observed behavioral phenotypes. Histological analysis revealed increased vacuolization area in rTBI flies. These results suggest that rTBI drives sex-specific behavioral deficits, proteomic disruption, and neurodegeneration. Overall, this enhanced Drosophila rTBI model provides a valuable tool for exploring sexually dimorphic outcomes and understanding the long-term pathophysiology of repetitive brain trauma in the context of neurodegenerative disease. Significance StatementRepetitive traumatic brain injury (rTBI) is linked to long-term cognitive decline and neurodegeneration, yet standardized, scalable models that capture sex-dependent outcomes are limited. We developed an automated, force-adjustable, closed-head Drosophila rTBI apparatus that delivers reproducible injuries without off-target damage, enabling precise control of injury severity across sexes. Using this platform, we show sexually dimorphic survival and locomotor vulnerability, persistent decision-making deficits, and progressive brain pathology marked by vacuolization and apoptosis. Proteomic profiling reveals time-dependent, sex-specific disruption of mitochondrial, oxidative stress, and locomotor-related protein networks, providing mechanistic entry points for discovering modifiers and therapies for repetitive brain trauma.