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

Abstract

Background: Traumatic brain injury (TBI) is a major public health concern that demands effective patient monitoring to mitigate secondary complications and improve recovery. TBI increases the risk for neurodegeneration by destabilizing proteostasis that increases protein degradation and proteinopathy. Despite the clinical importance of noninvasive TBI biomarkers, such as glial fibrillary acidic protein (GFAP), their mechanistic and cytological underpinnings remain poorly understood, hence are addressed here.

Methods: Deep proteomic profiling of GFAP used mass spectrometry (MS) sequencing of immunopurified GFAP breakdown products (BDPs) in TBI patients' cerebrospinal fluid (CSF) and serum. A unique trauma culture model used human neocortical astrocytes for GFAP epitope mapping and GFAP-BDP compartmental localization. Immunofluorescence, protease inhibitors (calpeptin, ZVAD-FMK), and live protease reporters document GFAP proteolysis and citrullination in dye-uptake-identified membrane-wounded astrocytes. Temporal profiles of GFAP and GFAP-BDPs were measured in TBI patients' CSF via calibrated, sub-saturated immunoblot densitometry. Fragment-specific prediction of six-month recovery used the Extended Glasgow Outcome Scale (GOSE).

Results: GFAP-BDP sequence sets with mapped posttranslational modifications (PTMs, citrullinations and acetylations) were identified in TBI patients' biofluids. Citrullinated GFAP profiles were distinct from unmodified GFAP in TBI patients' CSF. Novel TBI-specific cleavage fragments differed from those of Alzheimer's and Alexander diseases.Antibody mapping in the culture trauma model confirmed GFAP-BDPs, identified a new trauma cleavage region and revealed GFAP rod-domain harbored coil1-BDPs were selectively released into fluids, while coil2-BDPs remained as intracellular aggregates. Citrullinated coil2-BDPs formed non-filamentous aggregates in dystrophic astrocytes. Calpains and caspases contributed to trauma-generated GFAP-BDPs that were active in distinct astrocyte morphotypes and injury states.TBI patient GFAP proteolysis trajectories show that full-length GFAP and 45-49kDa fragments peaked acutely, then declined, whereas calpain-generated 37-39kDa BDPs remained elevated. Small GFAP-BDP-levels were imbalanced from expected proportions, with 20-26kDa products exceeding 15-19kDa products in CSF, consistent with preferential coil1-BDP release and coil2 retention shown in vitro. GFAP fragment profiles, but not those of uncleaved GFAP, predicted good versus poor outcome of TBI patients.

Conclusion: These findings highlight the translational importance of novel proteomic GFAP proteolysis profiles that could serve in neurocritical care monitoring. GFAP use as fluid biomarker, tied to astroglial proteinopathy, is relevant to post-traumatic neurodegeneration.