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.

Functional Selection of Tau Oligomerization‐Inhibiting Aptamers

“…Subjecting mice to exposure of mono‐specific BW1C‐Cy5.5 (BW) and bispecific BW1c‐TfR‐Cy5.5 (BWT) Tau aptamer . Mice, both naïve and subjected to controlled cortical impact (CCI), [31] were exposed to BW aptamer and BWT aptamer. We injected 8.6 nmol of either the BW1c aptamer labeled with Cy5 (BW) or the circular bispecific BW1c‐TfR aptamer labeled with Cy5.5 (BWT) (10 μL in phosphate‐buffered saline) via the retro‐orbital intravenous route.…”

GFAP as Astrocyte-Derived Extracellular Vesicle Cargo in Acute Ischemic Stroke Patients—A Pilot Study

“…Recently, its fragmentation patterns have been characterized in a rodent model of TBI and CSF samples from severe TBI patients. First, GFAP was fragmented at both the C- and N-terminals by calpain, resulting in GFAP BDPs ranging from 40 to 46 kDa, then 38 kDa [ 68 ]. The 38 kDa GFAP band marks the limit of calpain digestion and is a remarkably stable form of GFAP in dead or dying astrocytes [ 69 ].…”

“…When only caspases were activated but not calpain, an N-terminal BDP of 22 kDa and a C-terminal BDP of 20 kDa were generated from full-length GFAP. In CSF samples of TBI patients collected within 24 h, an increase of 38 kDa BDPs was observed [ 68 ]. A previous study also noted the calpain-mediated fragmentation of GFAP and the resulting BDPs after a TBI event [ 70 ].…”

Regional variances depict a unique glial-specific inflammatory response following closed-head injury

“…However, emerging research, as seen in the present study, shows changes within the contralateral side also ensuing (Grant et al, 2018;Yuan et al, 2020). A study by Yang et al (2022) utilized CCI to understand regional impact trauma. They observed significant histological alterations of GFAP when comparing the ipsilateral side of the cortex and regions of the hippocampus compared to the sham group.…”