Differential neuroproteomic and systems biology analysis of spinal cord injury

Acute spinal cord injury (SCI) is a devastating condition with many consequences and no known effective treatment. Although it is quite easy to diagnose traumatic SCI, the assessment of injury severity and projection of disease progression or recovery are often challenging, as no consensus biomarkers have been clearly identified. Here rats were subjected to experimental moderate or severe thoracic SCI. At 24h and 7d postinjury, spinal cord segment caudal to injury center versus sham samples was harvested and subjected to differential proteomic analysis. Cationic/anionic-exchange chromatography, followed by 1D polyacrylamide gel electrophoresis, was used to reduce protein complexity. A reverse phase liquid chromatography-tandem mass spectrometry proteomic platform was then utilized to identify proteome changes associated with SCI. Twenty-two and 22 proteins were up-regulated at 24 h and 7 day after SCI, respectively; whereas 19 and 16 proteins are down-regulated at 24 h and 7 day after SCI, respectively, when compared with sham control. A subset of 12 proteins were identified as candidate SCI biomarkers - TF (Transferrin), FASN (Fatty acid synthase), NME1 (Nucleoside diphosphate kinase 1), STMN1 (Stathmin 1), EEF2 (Eukaryotic translation elongation factor 2), CTSD (Cathepsin D), ANXA1 (Annexin A1), ANXA2 (Annexin A2), PGM1 (Phosphoglucomutase 1), PEA15 (Phosphoprotein enriched in astrocytes 15), GOT2 (Glutamic-oxaloacetic transaminase 2), and TPI-1 (Triosephosphate isomerase 1), data are available via ProteomeXchange with identifier PXD003473. In addition, Transferrin, Cathepsin D, and TPI-1 and PEA15 were further verified in rat spinal cord tissue and/or CSF samples after SCI and in human CSF samples from moderate/severe SCI patients. Lastly, a systems biology approach was utilized to determine the critical biochemical pathways and interactome in the pathogenesis of SCI. Thus, SCI candidate biomarkers identified can be used to correlate with disease progression or to identify potential SCI therapeutic targets

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

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 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. 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 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.</jats:p

The developing landscape of diagnostic and prognostic biomarkers for spinal cord injury in cerebrospinal fluid and blood

Review articleSTUDY DESIGN: Review study. OBJECTIVES: The identification of prognostic biomarkers of spinal cord injury (SCI) will help to assign SCI patients to the correct treatment and rehabilitation regimes. Further, the detection of biomarkers that predict permanent neurological outcome would aid in appropriate recruitment of patients into clinical trials. The objective of this review is to evaluate the current state-of-play in this developing field. SETTING: Studies from multiple countries were included. METHODS: We have completed a comprehensive review of studies that have investigated prognostic biomarkers in either the blood or cerebrospinal fluid (CSF) of animals and humans following SCI. RESULTS: Targeted and unbiased approaches have identified several prognostic biomarkers in CSF and blood. These proteins associate with cellular damage following SCI and include components from neurons, oligodendrocytes and reactive astrocytes, that is, neurofilament proteins, glial fibrillary acidic protein, Tau and S100 calcium-binding protein β. Unbiased approaches have also identified microRNAs that are specific to SCI, as well as other cell damage-associated proteins. CONCLUSIONS: The discovery and validation of stable, specific, sensitive and reproducible biomarkers of SCI is a rapidly expanding field of research. So far, few studies have utilised unbiased approaches aimed at the discovery of biomarkers within the CSF or blood in this field; however, some targeted approaches have been successfully used. Several studies using various animal models and some with small human patient cohorts have begun to pinpoint biomarkers in the CSF and blood with putative prognostic value. An increased sample size will be required to validate these biomarkers in the heterogeneous clinical setting