Proteolysis of multiple myelin basic protein isoforms after neurotrauma: characterization by mass spectrometry

Abstract

Neurotrauma, as in the case of traumatic brain injury, promotes protease over‐activation characterized by the select fragmentation of brain proteins. The resulting polypeptides are indicators of biochemical processes, which can be used to study post‐injury dynamics and may also be developed into biomarkers. To this end, we devised a novel mass spectrometry approach to characterize post‐injury calpain proteolytic processing of myelin basic protein (MBP), a biomarker of brain injury that denotes white matter damage and recovery. Our approach exceeds conventional immunological assays in its deconvolution of multiple protein isoforms, its absolute quantification of proteolytic fragments and its polypeptide selectivity. We quantified and characterized post‐injury proteolytic processing of all MBP isoforms identified in adult rat cortex. Further, the translation of calpain‐cleaved MBP into CSF was verified following brain injury. We ascertained that the exon‐6 sequence of MBP resulted in a characteristic shift in gel migration for intact and fragmented protein alike. We also found evidence for a second post‐TBI cleavage event within exon‐2 and for the dimerization of the post‐TBI 4.3 kDa fragment. Ultimately, the novel methodology described here can be used to study MBP dynamics and other similar proteolytic events of relevance to brain injury and other CNS processes.

Long-Term Consequences of Traumatic Brain Injury: Current Status of Potential Mechanisms of Injury and Neurological Outcomes

“…[159][160][161][162][163] Using both in vitro and in vivo models, calpain-specific breakdown products, including a-spectrin, are observed within axonal, dendritic, and somatic regions after injury, leading to transport disruption and secondary axotomy. 164 Recently, specific spectrin breakdown products have been reported to be present in CSF from traumatized animals and humans, representing a useful biomarker to assess injury severity and pathophysiological events.…”

“…164 Recently, specific spectrin breakdown products have been reported to be present in CSF from traumatized animals and humans, representing a useful biomarker to assess injury severity and pathophysiological events. 160,165 Indeed, direct relationships between calpain-mediated proteolysis and injury severity have been associated with neurodegenerative and restorative responses. 162 Multiple strategies targeting calpain breakdown have been used to target the acute and more chronic consequences of CNS injury.…”

Therapy Development for Diffuse Axonal Injury

“…[135][136][137][138] Also, a decrease and proteolytic breakdown of myelin basic protein may be an indicator for ongoing Wallerian degeneration and its associated axonal destruction. [137][138][139][140][141][142][143] The potential utility of these biomarkers will be influenced by their different expression profiles over time post-injury because of their differential roles in the initiation of axonal damage and its progression. Despite these limitations, however, these biomarkers offer the most promise for the detection of DAI.…”

Blood biomarkers for evaluation of perinatal encephalopathy: state of the art

“…Under normal conditions only a small amount is released into the blood stream, but in white matter brain injury the concentration of MBP in blood and CSF increases rapidly reflecting the severity of myelin damage which allows MBP to be used as a specific biomarker of white matter lesions or nerve fiber demyelination (Lv et al, 2015). MBP diminishes significantly in the contused rat cortex as early as 2 h after traumatic brain injury reaching its lowest level at 48 h (Ottens et al, 2008). Serum and CSF MBP has been studied as a biomarker for traumatic brain injury and for determination of outcome (Yokobori et al, 2013).…”