Glial fibrillary acidic protein (GFAP) is the major intermediate filament III protein of astroglia cells and is upregulated in astrogliosis following traumatic brain injury (TBI). Here we reported that GFAP is truncated at both the C- and N-terminals by cytosolic protease calpain to GFAP breakdown products (GBDP) of 46-40 kDa then 38 kDa following pro-necrotic (A23187) and pro-apoptotic (staurosporine) challenges to primary cultured astroglia or neuron-glia mixed cells. In addition, with another pro-apoptotic challenge (EDTA) where caspases are activated but not calpain, GFAP was fragmented internally, generating C-terminal GBDP of 20 kDa. Following controlled cortical impact in mice, GBDP of 46-40K and 38K were formed at day 3 to 28 post-injury. Purified GFAP protein treated with calpain-1 and -2 revealed that they produce (i) major N-terminal cleavage site between A-56 and A-75, and (ii) major C-terminal cleavage sites between T-383 and Q-388, producing a limit fragment of 38K and concurrently releasing several small GFAP peptides. Caspase-6 treated GFAP was cleaved at D-78, R-79, D-266 and A-267, where GFAP was relatively resistant to caspase-3. We also derived a GBDP-38K N-terminal specific antibody which only labels injured astroglia cell body in both cultured astroglia and in mouse cortex and hippocampus after TBI. Lastly as clinical translation, we observed that CSF samples collected from severe human TBI have elevated levels of calpain-generated GBDP-38K. Thus by examining the various forms of GFAP breakdown products as biomarkers, one can gain mechanistic insights of tracking astroglia injury under conditions where calpain and/or caspase-6 are activated.

Endogenous peptides were collected from CSF samples with 5-10kDa MWCO spin columns prior to targeted LC/MS/MS measurements incorporating 13C-labeled heavy peptide internal standards, spiking studies, and controls.

Human Cerebrospinal Fluid Samples: The CSF samples from a severe TBI study were collected from consented adult subjects presenting to the Emergency Department of Ben Taub General Hospital, Baylor College of Medicine, (Houston, TX, USA). The study protocol was approved by the Baylor College of Medicine IRB. TBI subjects with a Glasgow coma scale 3-8 were enrolled and the CSF samples were collected as described in previous study(26) according to the hospital’s standard procedures. Timed CSF samples were collected within 24 hours of a head injury and underwent an immediately sample preparation as follows. The 5 to 10 mL CSF was centrifuged at 4,000 g at room temperature for 5 minutes to remove loose cells and debris. The cleared CSF (supernatant) samples were aliquoted into 1ml-vivals followed by snap-frozen and stored at -80°C ultralow freezer until use. 10 μL of each CSF sample was used for immunoblot analysis. The control CSF samples were purchased from Bioreclamation (Westbury, NY, USA)(27).