GFAP Degradation in TBI: Linking Novel Modified Products to Astrocyte Pathology and Patient Outcome

Monitoring patients with traumatic brain injury (TBI) is essential for mitigating secondary complications. TBI disrupts proteostasis, driving protein degradation and proteinopathy, which may heighten the risk of neurodegeneration. This underscores the need for early, noninvasive biomarker detection. Glial fibrillary acidic protein (GFAP), an astroglial intermediate filament, is a well-established TBI biomarker. Yet, GFAP biomarker-associated pathomechanisms are incompletely understood. An observational cohort of neurocritical care patients was used for detailed study of TBI biofluid-derived GFAP proteoforms, complemented by controlled experiments using a primary human astrocyte cell culture trauma model. This study defined for the first time the trauma-generated GFAP degradome with posttranslational modifications (PTMs) through mass spectrometry (MS) sequencing of immunopurified GFAP breakdown products (BDPs). Trauma-specific citrullinations and acetylations were validated via MS spectra. The TBI-GFAP fragments differed from those of Alzheimer’s and Alexander diseases, revealing novel coil1/coil2-harboring cleavage products in TBI CSF, while TBI serum samples had only coil1-products. Epitope mapping identified a novel trauma-specific cleavage site and revealed coil1-BDPs as selectively released into fluid, while coil2 fragments remained intracellular in human traumatized astrocytes. Signals of citrullinated GFAP-specific antibodies differed from those of unmodified GFAP both in TBI patients’ CSF and in the trauma model, where non-filamentous aggregates were detected in dystrophic astrocytes. TBI-GFAP fragments were generated by stepwise trauma-activated calpain and caspase activities shown by in vitro inhibitor studies. Live cell imaging with fluorescent protease reporters demonstrates coexisting calpain and caspase activities alongside membrane disruption, jointly identifying distinct astrocyte injury states. The trajectory of GFAP proteolysis was analyzed in CSF from 23 TBI patients over 10 postinjury days (PIDs), via calibrated, band-specific scaled immunoblot densitometry. Profiles covaried with patient age and body temperature. Full-length GFAP and 45-49kDa fragments peaked in CSF on injury day and declined over time, whereas calpain-generated 37-39kDa BDPs remained elevated. Small BDPs exhibited either delayed rises or remained undetected. Compared to the 20-26kDa BDPs, 15-19kDa products were notably underrepresented, consistent with coil1-BDPs being preferentially released from astrocytes and intracellular retention of coil2-BDPs. In the trauma culture model, fluid levels of calpain-generated GFAP fragment varied with time and injury severity. In TBI patients, CSF GFAP-BDP trajectories distinguished good from poor outcome on the Extended Glasgow Outcome Scale (GOSE), while uncleaved GFAP trajectories failed to predict six-month outcomes. These novel GFAP-BDP signatures support a potential context of use (COU) in neurocritical care TBI monitoring and offer preliminary insights into astroglial proteinopathy-potentially linked to neurodegeneration after TBI.