Influence of Paramagnetic Changes in Cytochrome c on T2-weighted MRI of G93A-SOD1 Transgenic ALS Mice

Introduction. Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease characterized by selective degeneration of motor neurons in the brain stem, spinal cord, and motor cortex. Approximately 5–10% of ALS cases are familial, and 15–20% of these are linked to a mutation in Cu/Zn superoxide dismutase (SOD1) gene. Mice expressing mutant G93A-SOD1 (glycine to alanine substitution in SOD1) develop symptoms and pathology similar to those in human ALS. In this study, low temperature X-band EPR spectroscopy was used to monitor the alterations in paramagnetic heme protein (e.g., cytochrome c) in brain stem, spinal cord and muscle tissues in G93A-SOD1 mice. The effect of EPR paramagnetic changes on alterations in T2-weighted MRI of brain stem in G93A ALS mice will be reported. The onset and progression of neurodegenerative diseases (e.g., ALS, Parkinson’s, Alzheimer’s) is characterized by mitochondrial dysfunction. Mitochondria are abundant in paramagnetic metal-rich Fe-S clusters, heme and binuclear Cu(II) and Fe(III)-Cu(II) present at the active sites of the electron-transport chain. We used low temperature EPR spectroscopy to investigate specific paramagnetic changes to heme proteins of G93A-SOD1 transgenic ALS mice. We hypothesized that alterations in paramagnetism of endogenous heme proteins in normal and pathological tissues will influence T1 and T2 values, leading to tissue hyperor hypointensity in MRI. We monitored T2-weighted MRI (T2-W MRI) of G93A-SOD1 transgenic mice at 60, 80 and 115 days. The objectives here were to investigate the following: 1) the paramagnetic alterations in heme protein (e.g., cytochrome c) during ALS progression in G93A mice using low temperature EPR, and 2) the effect of the paramagnetic changes on T2-W MRI. Methods. Transgenic mice: B6SJL (G93A-SOD1) mice (Jackson Laboratories, Bar Harbor, USA) were crossed with non-transgenic B6SJL mice. They were identified by polymerase chain reaction (PCR). Age-matched non-transgenic litter mates were used as controls. The X-band EPR of tissues of normal and G93A-SOD1 mice were recorded at liquid helium temperatures on a Bruker E500 ELEXYS spectrometer with a 100 kHz field modulation, and equipped with an Oxford Instruments ESR-9 helium flow cryostat [1]. Temperature and microwave power dependence EPR spectra were obtained over the range of 4-50 K and 0.1-159 mW. EPR spectra were analyzed using the equation: