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

Glial fibrillary acidic protein (GFAP) is an important clinical biomarker of traumatic brain injury (TBI), yet understanding the nature, timing, and impact of its degraded and modified products is needed for clinical utility. We report distinct GFAP breakdown products (BDPs) and post-translational modifications (PTMs) that are unique to TBI. Mapped PTMs provided fragment and patient-specific citrullination signatures known for destabilizing GFAP filaments. GFAP and its fragments from patients cerebrospinal fluid (CSF) and serum were sequenced by mass spectrometry (MS), identifying two TBI-specific coproducts covering coil1 (20-26kDa) and coil2 (15-19kDa) in the rod-domain. These coproducts were imbalanced in biofluids with coil1-BDPs enriched over coil2-BDPs detected by label-free quantitative MS and independently confirmed by immunoblot densitometry in twenty-three TBI patients. Ten-day temporal trajectories showed ongoing proteolysis, progressing from large to small fragments, with prolonged elevation of 37/39kDa fragments, and delayed rise of small products. For the first time, profiles of GFAP fragments, but not of uncleaved GFAP, predicted six-month outcome (Extended Glasgow Outcome Scale, GOSE), highlighting proteolytic processing as prognostic biomarker signature. These new TBI-GFAP-BDPs were independently substantiated in a human culture trauma model, offering mechanistic insights and linking biomarker data to injury-induced astrocytopathy. To this end, specific findings include: Coil1-BPDs were fluid-released, while coil2-BDPs remained cellular, shown by epitope-defined coil-specific GFAP antibodies. A new cleavage site between the two coils was identified by selective epitope loss. Coil2 cellular retention could be explained by incorporation of citrullinated coil2-BDPs into non-filamentous aggregates within pathological astrocytes after injury. Trauma-triggered proteolysis involved calpains and caspases in distinct astrocyte injury states documented using inhibitors and live-cell reporters. These novel degradation findings have significant translational relevance for monitoring disease progression in TBI patients and for linking biofluid GFAP fragments to a trauma-inflicted astroglial proteinopathy during neurodegeneration.