Journal: bioRxiv

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

doi: 10.1101/2025.08.01.668181

Figure Lengend Snippet: A) TBI patient CSF immunoblots of a 15% gel show GFAP detected by a polyclonal (top) and a monoclonal (clone 3E10, bottom) antibody (postinjury days, i+1 through 1+3) of an 84 year old male sustaining facial fractures, subarachnoid and intraventricular hemorrhage and intraparenchymal hematomas from a fall. Observed GFAP band molecular weights were bands (II.) 36-39kDa, bands (III.) 20.4-23kDa, and bands (IV.) 17-19kDa, missed by clone 3E10. B) MS peptide maps of immunoprecipitated GFAP breakdown products (BDPs) of four cut out gel band sets (I.-IV., Suppl. Fig. 1) from six CSF TBI samples of three patients. Percent of fragment sequence coverage (left) by high confidence (green, FDR≤1%) and repeatedly identified medium confidence (yellow FDR≤5%) peptides. Bands (I.) had 359 amino acids (aa) with calculated molecular weight of 41.7kDa and suggested new N- and C-termini of serine 53 (S53) and lysine 411 (K411). Bands (II.) spanned 349 aa from glycine 56 (G56) to leucine (L404) within which 37/38kDa fragments with variable endings between TBI samples were located (Suppl.Fig.1). Bands (III.) contained two GFAP BDPs, 92% covered coil1-containing product of 185 aa, calculated molecular weight of 21.8kDa; 77% covered coil2-containing product of 151 aa and peptides, estimated molecular weight of 17.5kDa. Bands (IV.) 96% covered 181 aa coil1 product with size between 21-21.6kDa -±PTMs; and 77% covered coil2 product of 130 aa with estimated molecular weight of 15-15.3kDa. C) Predicted endings of the two sets of novel TBI patients’ GFAP-BDPs. Coil1 products N-termini were S53 (III.) and alanine, A55 (IV.) and new C-termini were arginine 239 (R239) C-terminus (III.) and A233 (IV.), both stretching beyond previously described caspase 6 cleavage around VELD225 . Coil2 products had consistent N-terminus phenylalanine 261 (F261), see reported caspase 3 cleavage RSKFA↓DLTS , and variably ended with K411 (III.) and with R390 (IV.).

Article Snippet: Pan caspase inhibitor ZVAD-FMK (Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (Enzo/ThermoFisher) is a cell-permeable, irreversible pan-caspase inhibitor effective against a spectrum of caspases (1-10, except 2; Invitrogen) .

Techniques: Western Blot, Immunoprecipitation, Sequencing, Molecular Weight

Journal: bioRxiv

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

doi: 10.1101/2025.08.01.668181

Figure Lengend Snippet: A) Unstretched or stretch-injured human astrocytes were treated with calpain inhibitor calpeptin (C, 13mM), caspase inhibitor Z-VAD-FMK (V, 11mM), both (CV), or no drug (−) for 48 hours. Adherent cells were lysed (Whole Cell lysate, WCL), centrifuged fluid pellet contained lifted material (Lifted Cell Pellet) and fluids were concentrated (Conditioned medium, CM). Top: 2sec exposures for 50kDa GFAP and 42-47kDa BDPs. Mid: 1min exposures for 38-25kDa BDP bands. Bottom: 20min (WCL, CM) and 5min (Lifted cell pellet) exposures for 17-25kDa BDPs (See Suppl.Fig.7 for 5hr blot and 48h densitometry). B+C) Live imaging of distinct cerebral human astrocyte morphotypes and their acute trauma stages defined by membrane leak, calpain and caspase activities during 5-6 hours postinjury . B) . Left : Elongated ( Top ) and bushy ( bottom ) morphotypes and injury shapes on phase contrast. Right : Fluorescence images for plasma membrane permeability (5min uptake assay of propidium iodide, [PI+], red), calpain activity (CMAC-tBOC-leucyl-methionine, [CMAC-tBLM], converted substrate, blue) and caspase activity ([NucView488] converted substrate, green). Top : Control fibrous astrocytes display intact processes, acutely stretched cells had thinned, frequently beaded clasmatodendrotic processes (arrows). [PI−] astrocytes were calpain active. [PI+], leaky cells presented variable caspase activity (yellow/orange), lacking calpain-activity. Bottom : Bushy control and stretched astrocytes were calpain active (blue). The majority of stretched bushy astrocytes were calpain and caspase active (teal). [PI+], leaky bushy cells either retained both activities (yellow/white), or lost these protease activities (red, arrows) displaying necrotic, atrophic morphology. Phase-dense vacuoles were unstained. C) Enlarged from B: Individual fibrous (top) and bushy (bottom) astrocytes showing distinct structural and functional features of intact, injured and necrotic astrocyte phenotypes: Intact [PI−], calpain[+]; Wounded : [PI−/+], cytoplasmic and nuclear caspase[+]; Dying: [PI+] nuclear caspase [−/+], calpain[−]. Suppl.Fig.8 for separate fluorescence channels. Scale bars: 100µm.

Article Snippet: Pan caspase inhibitor ZVAD-FMK (Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (Enzo/ThermoFisher) is a cell-permeable, irreversible pan-caspase inhibitor effective against a spectrum of caspases (1-10, except 2; Invitrogen) .

Techniques: Imaging, Membrane, Fluorescence, Clinical Proteomics, Permeability, Activity Assay, Control, Functional Assay