GFAP Proteolysis in TBI: Linking Novel Modified Cleavage Products to Astrocyte Pathology and Patient Outcomes

Wanner, I. B.; Halford, J.; Lopez, J.; Shen, S.; Chen, Y.; Zhao, H.; Salas, C. I.; Loo, R. R.; Robicsek, S.; Ellingson, B. M.; Gornbein, J.; Van Meter, T. E.; Shaw, G.; Loo, J. A.; Vespa, P. M.

doi:10.1101/2025.08.01.668181

bioRxiv · 10.1101/2025.08.01.668181

GFAP Proteolysis in TBI: Linking Novel Modified Cleavage Products to Astrocyte Pathology and Patient Outcomes

Wanner, I. B.·Halford, J.·Lopez, J.·Shen, S.·Chen, Y.·Zhao, H.·Salas, C. I.·Loo, R. R.·Robicsek, S.·Ellingson, B. M.·Gornbein, J.·Van Meter, T. E.·Shaw, G.·Loo, J. A.·Vespa, P. M.

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Abstract

Glial fibrillary acidic protein (GFAP) is a significant clinical biomarker of traumatic brain injury (TBI), yet understanding the nature, timing, and impact of its degraded and modified products would inform clinical utility. We report novel GFAP breakdown products (BDPs) and post-translational modifications (PTMs) that are unique to TBI including fragment- and patient-specific citrullination signatures that destabilize GFAP filaments. GFAP and its fragments were sequenced by mass spectrometry (MS) from severe TBI patients cerebrospinal fluid (CSF) and sera, identifying two distinct TBI-specific jointly generated product sets within the rod-domain covering coil1 (20-26kDa) and coil2 (15-19kDa). Their endings differed from GFAP fragments described in Alzheimers and Alexander disease. Label-free quantification showed biofluid co-product abundance differences with coil1-BDPs enriched over coil2-BDPs. Coil imbalance was independently confirmed by immunoblot densitometry in twenty-three TBI patients. Measurements over ten days showed injury day peaks of full-length and end-clipped GFAP fragments, while proteolytic 37/39kDa and small fragments remained elevated. Six-month outcome (Extended Glasgow Outcome Scale) correlated with GFAP fragments, whereas levels of uncleaved and end-clipped GFAP did not. A human astrocyte culture trauma model provided mechanistic insights linking fluid GFAP levels, subcellular localization, and injury-induced astrocytopathy. A new cleavage site between the two coils was identified through selective epitope loss. Coil1-BDPs were fluid-released post-injury, while coil2-BDPs remained intracellular. Their cellular retention was explained by non-filamentous aggregation of citrulline-modified coil2-BDPs in pathological astrocytes. Trauma-triggered proteolysis involved calpains and caspases in specific astrocyte injury states using protease inhibitors and live-dye-reporter imaging. These novel data link TBI biofluid GFAP fragments with assembly-defective GFAP aggregation in pathological astrocytes. Clinical TBI outcome correlated with GFAP degradation rather than with overall GFAP release. These translational findings indicate that TBI biomarker GFAP undergoes degradation and modification alongside astrocyte pathology, providing a new conceptual framework for investigating biomarker-associated astrocyte proteinopathy potentially linked to post-traumatic neurodegeneration.

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Wanner, I. B., Halford, J., Lopez, J., Shen, S., Chen, Y., Zhao, H., Salas, C. I., Loo, R. R., Robicsek, S., Ellingson, B. M., Gornbein, J., Van Meter, T. E., Shaw, G., Loo, J. A., Vespa, P. M.. 2025-08-01. GFAP Proteolysis in TBI: Linking Novel Modified Cleavage Products to Astrocyte Pathology and Patient Outcomes. https://doi.org/10.1101/2025.08.01.668181

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