Proteomic Technologies for Biomarkers of TBI

Neuroproteomic technologies are uniquely suited for the discovery of otherwise unnoticed TBI biomarkers and are being used for mapping changes in proteins after injury. This will be very useful for developing diagnostic predictors after CNS injury and to identify new therapeutic targets. SDS-PAGE prior to in vitro proteolysis and capillary LC-MS is a promising strategy for the rapid discovery of putative protein biomarkers associated with TBI without a priori knowledge of the molecules involved. Large-scale neuroproteomic research in CNS injury is being applied for immediate biomarker discovery and the systems biology approach is used for understanding of how the brain responds to trauma. Eventually, the knowledge gained through neuroproteomics could lead to improvement in diagnostics and therapeutics of CNS injury.

After TBI, brain cells can deteriorate following more than one pathway, and many genes and proteins may be involved. Cell death following TBI often has the morphological appearance of apoptosis. Studies of apoptosis pose special challenges since there are multiple apoptotic pathways, and apoptosis is extremely sensitive to a number of variables including injury type and magnitude, cell type and stimulation/antagonism of specific receptors. The molecular events occurring after TBI are just beginning to be understood. Elevated neuronal calcium levels activate a number of calcium-dependent enzymes such as phospholipases, kinases, phosphatases, and proteases, all of which can modulate post-TBI cytoskeletal protein loss. Caspase-3 is a member of the caspase family of cysteine proteases. Activated caspase-3 has many cellular targets that, when severed and/or activated, produce the morphologic features of apoptosis. Calpains are calcium-activated, neutral cysteine proteases with relative selectivity for proteolysis of a subset of cellular pro?teins. Calpain activation has been implicated in different models of apoptosis and in different cell types, including neurons. Understanding of the contributions of calpains and caspases to cell injury/death following TBI may have important diagnostic and therapeutic implications. In vivo studies have provided evidence of cas- pase-3 activation following TBI. aII-spectrin is a major substrate for both calpain and caspase-3 cysteine proteases and considerable laboratory data exists on the potential utility of aII-spectrin degradation as a biomarker for TBI, including the ability to detect differences in severity of injury based on aII-spectrin detection in spinal fluid after TBI.

Initial research focused on proteolytic processing of cytoskeletal proteins such as lower molecular weight neurofilament 68 protein (NF-68) highlights their potential to provide useful information on activity of specific proteases such as p-calpain and m-calpain. Importantly, 2D GE studies suggested dephosphorylation of NF-68 may be associated with NF protein loss following TBI, a post translational modification that could have significance for biomarker development. This important biomarker could provide important information on the pathophysiology of both dendritic and axonal damage after TBI. Importantly, NF-68 has been used to quantify axonal injury in closed head injury models. Since diffuse axonal injury is currently considered one of the most common types of primary lesions in patients with severe closed TBI, a biomarker that provides information on axonal injury could potentially have clinical utility. Banyan Biomarkers Inc. is focusing on diagnostics for TBI and has 42 novel protein biomarkers in its pipeline that can provide important information on the severity of injury, the location of the injury in the brain and the biochemical mechanisms underlying this injury.

Single Molecule Array technology (SiMoAâ„¢, Quanterix) is used to develop tests based on protein biomarkers of TBI in blood. Sports-related concussion in professional ice hockey players, which is associated with acute axonal and astroglial injury, can be monitored using tau, S-100, calcium-binding protein B, and neuron-specific enolase concentrations in serum, which may be developed into clinical tools to guide sport physicians in the medical counseling of athletes in return-to-play decisions (Shahim et al. 2014).

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