Characterization of Calpain and Caspase-6-Generated Glial Fibrillary Acidic Protein Breakdown Products Following Traumatic Brain Injury and Astroglial Cell Injury

Abstract

Glial fibrillary acidic protein (GFAP) is the major intermediate filament III protein of astroglia cells which is upregulated in 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 46-40K then 38K 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 a C-terminal GBDP of 20 kDa. Following controlled cortical impact in mice, GBDP of 46-40K and 38K were formed from day 3 to 28 post-injury. Purified GFAP protein treated with calpain-1 and -2 generates (i) major N-terminal cleavage sites at A-56*A-61 and (ii) major C-terminal cleavage sites at T-383*Q-388, producing a limit fragment of 38K. Caspase-6 treated GFAP was cleaved at D-78/R-79 and D-225/A-226, 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 mouse cortex and hippocampus after TBI. As a clinical translation, we observed that CSF samples collected from severe human TBI have elevated levels of GBDP-38K as well as two C-terminally released GFAP peptides (DGEVIKES and DGEVIKE). Thus, in addition to intact GFAP, both the GBDP-38K as well as unique GFAP released C-terminal proteolytic peptides species might have the potential in tracking brain injury progression.

Keywords: GFAP; astroglial injury; biomarkers; calpain; caspase; traumatic brain injury.

Figure 1

GFAP proteolysis versus αII-spectrin breakdown in cerebrocortical neuron-astroglial mixed culture (CTX) after subjecting to cytotoxin challenges (24 h). (A) CTX was subjected to A23187, STS, or NMDA treatment for 6 h. Cell lysate probed with αII-Spectrin or GFAP core Mb. (B) CTX treated with A23187, STS, and EDTA for 24 h in the absence or presence of calpain inhibitor MDL-28170 or pan-caspase inhibitor Z-D-DCB. Cell lysate probed with αII-Spectrin or GFAP core MAb or GFAP C-terminal PAb. Blots are representative of four separate experiments. CTX subjected to STS treatment in the absence or presence of caspase-6 inhibitor Z-IVED-FMK or with calpain inhibitor MDL-28170.

Figure 2

GBDP formation in astroglial cells and release into cell-conditioned media parallel cell injury in primary astroglial culture challenges with pro-necrotic (A23187) and pro-apoptotic cytotoxins (STS, EDTA). (A) Cell lysate or cell media probed with αII-Spectrin, GFAP core MAb or GFAP C-terminal PAb. (B,C) Quantification of GBDP formation and release parallel astroglial cell injury measured by LDH release with a time course of A23187, STS and EDTA treatment. Panel (D) quantification of LDH releases while (E,F) are GFAP-38K quantification in cell lysate and cell-conditioned media respectively. Shown are mean ± SEM (n = 4). * p < 0.05, ** p < 0.01 when compared to control (student t-test).

Figure 2

GBDP formation in astroglial cells and release into cell-conditioned media parallel cell injury in primary astroglial culture challenges with pro-necrotic (A23187) and pro-apoptotic cytotoxins (STS, EDTA). (A) Cell lysate or cell media probed with αII-Spectrin, GFAP core MAb or GFAP C-terminal PAb. (B,C) Quantification of GBDP formation and release parallel astroglial cell injury measured by LDH release with a time course of A23187, STS and EDTA treatment. Panel (D) quantification of LDH releases while (E,F) are GFAP-38K quantification in cell lysate and cell-conditioned media respectively. Shown are mean ± SEM (n = 4). * p < 0.05, ** p < 0.01 when compared to control (student t-test).

Figure 3

Digestion of purified GFAP by calpain-1 and caspase-6. Characterization with different GFAP antibodies and GBDP-specific antibodies by immunoblotting. (A) GFAP core Mab, (B) GFAP C-terminal Pab, (C) GBDP-specific antibody. (D) GFAP proteolysis with calpain, caspase-6, and predicted major fragment sizes. Data based on N-terminal sequencing. (E) Schematics of GFAP proteolytic fragment formation. The top panel shows GFAP linear model and major calpain/caspase-6 cleavage sites. The middle panel shows the positions of major fragments. The bottom panel shows the human GFAP amino acid sequence and the annotated cleavage sites (*) by calpain (blue), and by caspase-6 (red).

Figure 3

Digestion of purified GFAP by calpain-1 and caspase-6. Characterization with different GFAP antibodies and GBDP-specific antibodies by immunoblotting. (A) GFAP core Mab, (B) GFAP C-terminal Pab, (C) GBDP-specific antibody. (D) GFAP proteolysis with calpain, caspase-6, and predicted major fragment sizes. Data based on N-terminal sequencing. (E) Schematics of GFAP proteolytic fragment formation. The top panel shows GFAP linear model and major calpain/caspase-6 cleavage sites. The middle panel shows the positions of major fragments. The bottom panel shows the human GFAP amino acid sequence and the annotated cleavage sites (*) by calpain (blue), and by caspase-6 (red).

Figure 4

Characterization of calpain-derived GBDP (38K)-N-terminal specific antibody: immunoblotting of in vitro digest of GFAP in cerebrocortical neuron-astroglia mixture lysate with caspase-6 (A) vs. calpain-1 (B). Characterization of αII-Spectrin, GFAP core, and C-terminal antibodies and calpain-derived GBDP (38K)-N-terminal specific antibody. (C) Immunocytochemical characterization of rat primary astroglia cells labeled with total GFAP and GBDP-specific antibodies. Rat primary astrocytes subjected to A23187, STS, EDTA, or NMDA were stained with anti-GFAP core MAb, GBDP-N-terminal specific antibody. Yellow arrows show distinct GBDP-staining of the astrocyte cell body. Yellow brackets show degenerative astrocyte processes.

Figure 5

GFAP protein and GBDP are neurotoxic to rat cerebrocortical culture. Cultured cells were treated with 500 ng GFAP fragment (calpain or caspase-6 digestion) * compared with control p < 0.05, ** compared with control p < 0.01; # compared with intact GFAP treatment, p < 0.05.

Figure 6

Ipsilateral cortex GFAP and GFAP-breakdown product (GBDP) profile by immunoblotting at different time points after controlled cortical impact (CCI) injury in mice. Time points are D1, 3, 7, 14, or 28 after injury. (A) Representative blots for GFAP and housekeeping gene loading control (Carbonic anhydrase II, 29 kDa). (B) Quantification of GFAP and GBDP-44K (equalized by CA-II band intensity). * p < 0.05, when compared with naïve GFAP, # p < 0.05; ##, p < 0.01, when compared with naïve GBDP-44K. (C) GBDP-specific MAb did not detect naïve or sham hippocampus but sensitively detected the formation of GBDP-40K and GBDP-38K at D3 and D7 post- CCI samples.

Figure 7

Immunohistochemical staining with an injured mouse brain with total GFAP and anti-GBDP N-terminal specific antibodies. CCI mouse. Red: anti-GFAP, green: anti-GBDP. Shown are injured cortex (A), injured hippocampus [CA1 and CA3 cell layers (B) and dentate gyrus (C)] with total GFAP and anti-GBDP N-terminal specific antibody. 7 days post-CCI mouse brains are examined. Red: anti-GFAP, green: anti-GBDP. ICA1, ipsilateral CA1, CCA1, contralateral CA1, ICA3, Ipsilateral CA3, CCA3, contralateral CA3. Yellow arrows indicated GBDP Antibody labeled cells. (D) quantification of total GFA or GBDP positive cells from control rats (naïve) or rats 7 days after CCI, ipsilateral and contralateral cortex, and hippocampus (CA1, CA3, and dentate gyrus (DG)) were used. Shown are mean ± SEM (n = 4). * p < 0.05, ^# p < 0.05 when compared to control. (student’s t-test).

Figure 8

GFAP and 38K GBDP in human CSF samples within 24 h of severe TBI. (A) Representative blots showed αll-Spectrin fragment SBDP150, intact GFAP and GBDP-38K in human control and TBI CSF samples. (B) Scattered plot for control and TBI CSF (collected within 24 h post-admission). Groups are shown as the median and interquartile range (IQR), * p < 0.05. Control has N = 12, TBI N = 30. (C,D) ROC showed higher AUC for GFAP-38K (0.944) that intact GFAP (0.909).

Figure 9

Unbiased LC/MS/MS followed by (A,B) de novo sequencing or (C,D) database searching both revealed GFAP peptides from pooled human acute TBI CSF samples from n = 10 samples (6–12 h post-injury): (A,C) DGEVIKES and (B,D) DGEVIKE as shown by the annotated spectra. Note: I/L are isobaric, based on the amino acid sequence for human GFAP DGEVLKES and DGEVLKE (de novo sequencing) &DGEVIKES and DGEVIKE (database searching).

Figure 9

Unbiased LC/MS/MS followed by (A,B) de novo sequencing or (C,D) database searching both revealed GFAP peptides from pooled human acute TBI CSF samples from n = 10 samples (6–12 h post-injury): (A,C) DGEVIKES and (B,D) DGEVIKE as shown by the annotated spectra. Note: I/L are isobaric, based on the amino acid sequence for human GFAP DGEVLKES and DGEVLKE (de novo sequencing) &DGEVIKES and DGEVIKE (database searching).

Figure 10

Targeted LC/MS/MS extracted ion chromatograms for light and heavy versions of GFAP peptides in control and in acute and subacute time points: (A) DGEVIKES and (B) DGEVIKE. Heavy peptides were spiked in at 20 fmol/50 uL of pooled CSF before ultrafiltration with a 10 kDa membrane. Pooled CSF samples were generated by pooling 10 uL of each sample type (control, n = 14; acute TBI, n = 15; subacute TBI, n = 13) from the available CSF samples. The data shown are for a single analysis of each pooled sample type.