ROS-related mitochondrial dysfunction in skeletal muscle of an ALS mouse model during the disease progression

Abstract

In amyotrophic lateral sclerosis (ALS), mitochondrial dysfunction and oxidative stress form a vicious cycle that promotes neurodegeneration and muscle wasting. To quantify the disease-stage-dependent changes of mitochondrial function and their relationship to the generation of reactive oxygen species (ROS), we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1 and mt-cpYFP transgenes, in which mt-cpYFP detects dynamic changes of ROS-related mitoflash events at individual mitochondria level. Compared with wild type mice, mitoflash activity in the SOD1 (G93A) mouse muscle showed an increased flashing frequency prior to the onset of ALS symptom (at the age of 2 months), whereas the onset of ALS symptoms (at the age of 4 months) is associated with drastic changes in the kinetics property of mitoflash signal with prolonged full duration at half maximum (FDHM). Elevated levels of cytosolic ROS in skeletal muscle derived from the SOD1 mice were confirmed with fluorescent probes, MitoSOX Red and ROS Brite570. Immunoblotting analysis of subcellular mitochondrial fractionation of G93A muscle revealed an increased expression level of cyclophilin D (CypD), a regulatory component of the mitochondrial permeability transition pore (mPTP), at the age of 4 months but not at the age of 2 months. Transient overexpressing of SOD1 in skeletal muscle of wild type mice directly promoted mitochondrial ROS production with an enhanced mitoflash activity in the absence of motor neuron axonal withdrawal. Remarkably, the SOD1-induced mitoflash activity was attenuated by the application of cyclosporine A (CsA), an inhibitor of CypD. Similar to the observation with the SOD1 transgenic mice, an increased expression level of CypD was also detected in skeletal muscle following transient overexpression of SOD1. Overall, this study reveals a disease-stage-dependent change in mitochondrial function that is associated with CypD-dependent mPTP opening; and the ALS mutation SOD1 directly contributes to mitochondrial dysfunction in the absence of motor neuron axonal withdrawal.

Repeated intramuscular transplantations of hUCB-MSCs improves motor function and survival in the SOD1 G93A mice through activation of AMPK

“…One of the reasons for the difficulty in investigating and developing therapeutic agents is the complexity of the non-cell-autonomous status of the disease 3 . During ALS disease progression, the loss of neuromuscular junction and muscle atrophy are critically related to disease progression 4 . However, most studies have focused on the central nervous system, especially on the motor neurons, as the main targets to improve disease progression and survival [5][6][7] .…”

Antioxidant Alternatives in the Treatment of Amyotrophic Lateral Sclerosis: A Comprehensive Review

“…The transgenic mouse model of ALS (SOD1 G93A ) has enabled the identification of pathogenic mechanisms and the establishment of a "vicious cycle" between: OS, mitochondrial dysfunction and deterioration of the ETC (Jung et al, 2002;Menzies et al, 2002;Pfohl et al, 2015;Xiao et al, 2018). Most of the oxidative species that are formed in the CNS are secondary to oxidative phosphorylation (Brookes et al, 2004;Balaban et al, 2005;Karam et al, 2017) and mitochondrial dysfunction is the largest source of ROS production, so is a clear sign of affectation of motor neurons in the spinal cord and the motor cortex (Bendotti et al, 2001;Kawahara et al, 2004;Loizzo et al, 2010;Cozzolino and Carrì, 2012).…”

Recombinant human MG53 protein preserves mitochondria integrity in cardiomyocytes during ischemia reperfusion-induced oxidative stress

“…Transgenic mice with expression of mt-cpYFP provides assessment of the dynamic changes in superoxide signal from the mitochondria [15][16][17][18][19][20] Cardiomyocytes were isolated from the mt-cpYFP transgenic mice following the protocol of Wang et al [17]. The isolated cardiomyocytes were subjected to 3 hours of hypoxia and 2 hours of reoxygenation.…”

“…HL-1 cardiomyocytes (Sigma, SCC065) were cultured at low passages (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) according to established protocols [22,23]. For H2O2 damage, HL-1 cells were subjected to 300 μM H2O2 in unsupplemented Claycomb media (Sigma, 51800C) for 1 hour and recovered with either 10 µg/mL BSA or rhMG53 for 2 hours.…”

“…White scale bar = 50 μmWang et al[26] developed a novel indicator targeted to the mitochondrial matrix called circularly permuted yellow fluorescent protein (mt-cpYFP) that selectively fluoresce in the presence of O2 -, the main ROS created by the electron transport chain in the mitochondria. We have recently used this tool to study mitochondrial ROS generation in the mouse model of amyotrophic lateral sclerosis (ALS), termed "mitoFlash"[15,[18][19][20].…”