Microwave & Magnetic (M2) Proteomics of a Mouse Model of Mild Traumatic Brain Injury

Abstract

Short-term increases in oxidative stress and decreases in motor function, including debilitating effects on balance and motor control, can occur following primary mild traumatic brain injuries (mTBI). However, the long-term effects on motor unit impairment and integrity as well as the molecular mechanisms underlying secondary injuries are poorly understood. We hypothesized that changes in central nervous system-specific protein (CSP) expression might correlate to these long-term effects. To test our hypothesis, we longitudinally assessed a closed-skull mTBI mouse model, vs. sham control, at 1, 7, 30, and 120 days post-injury. Motor impairment was determined by rotarod and grip strength performance measures, while motor unit integrity was determined using electromyography. Relative protein expression was determined by microwave & magnetic (M²) proteomics of ipsilateral brain tissue, as previously described. Isoprostane measurements were performed to confirm a primary oxidative stress response. Decoding the relative expression of 476 ± 56 top-ranked proteins for each specimen revealed statistically significant changes in the expression of two well-known CSPs at 1, 7 and 30 days post-injury: P < 0.001 for myelin basic protein (MBP) and P < 0.05 for myelin associated glycoprotein (MAG). This was confirmed by Western blot. Moreover, MAG, αII-spectrin (SPNA2) and neurofilament light (NEFL) expression at 30 days post-injury were directly related to grip strength (P < 0.05). While higher-powered studies of larger cohorts merit further investigation, this study supports the proof-of-concept that M² proteomics is a rapid method to quantify putative protein biomarkers and therapeutic targets of mTBI and suggests the feasibility of CSP expression correlations to long-term effects on motor impairment.

Keywords: MAG; MBP; biomarkers; central nervous system-specific protein; magnetic; microwave; mild traumatic brain injury; motor impairment; proteins; proteomics.

Figure 1. Imaging measures of mTBI

A) Structural MRI of WT mouse brain 3 days post-TBI. Red Square encompasses area of direct impact. N=3 B) Nissl stain(40× magnification, line=500µm) of sham and TBI treated WT mouse cortex and hippocampus (Ca1 and Ca2 regions) 24 hours post-TBI. N=4 C) DAPI (Blue), NeuN (Green) and GFAP (Red) (20× magnification, line=100µm) used to label nuclei, neurons and astrocytes in the ipsilateral (Injured) cortexand hippocampus of Sham and TBI animals 24 hours post-TBI. N=3

Figure 2. Body weight, apnea and righting reflex measures of mTBI (**p<0.01, *p<0.02)

A) mTBI causes a significant reduction in bodyweight loss up to 7 days post-injury B) Time spent apneic following mTBI is significantly increased as compared to sham controls C) Increased time to right self (righting reflex) following TBI is also significantly increased.

Figure 3. Motor impairment measures of mTBI (*p<0.05)

A) Significantly reduced rotarod performance at 7 days B) 30 days and C) 90 days post-injury (p<0.05) D) Grip strength was decreased at 2 days post-injury and significantly reduced at 7 days post-injury.

Figure 4. Motor unit integrity measures of mTBI (**p<0.01)

A) Representative tracings of Electromyography Abnormality Scoring used in the current study B) Electromyography of hind limb muscles indicate significant abnormality in the form of denervation potentials at all time points tested post-injury. C) Changes in muscle mass at 4 months post-injury D) Muscle mass normalized to body weight.

Figure 5. Isoprostane measures of mTBI (****p<0.0001, **p<0.001)

Significantly increased A: F2-Isoprostanes and B: F4-Isoprostanes 48 hours following mTBI as compared to Sham.

Figure 6. M² proteomics measures of mTBI (***p<0.001, **p<0.05)

A) Significantly decreased MBP expression up to 30 days post-injury as compared to sham controls at 180 days of age. B) Significantly increased MAG expression up to 30 days post-injury. Western blots of C) MBP (18–23 kDa) and D) MAG (63 kDa) with Beta-Actin (45 kDa) as a loading control were used to confirm M² proteomics results.

Figure 7. A representative MS/MS spectrum for MBP121-132 (TQDENPVVHFFK)

This spectrum shows the probability-based protein database search assignment of 19/23 amino acid sequence-specific b- and y-type ions (expectation = 5.8E-7), with a zoomed-in view of the TMT126-131 reporter ions used for quantification of this peptide in specimens from individual mice (inset). The post-injury time point (0,1,7,30 and 120 days) for each reporter ion is also shown, where ref = the pooled reference used to normalize relative expression across all specimens.

Figure 8. M² proteomics measures of mTBI correlated to grip strength (*p<0.05)

Decreased expression of A) SPNA2 and B) NEFL directly correlated to decreased grip strength at 30 days post-injury, as shown in C) and D), respectively.