Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal heterogeneous neurodegenerative disease that causes motor neuron (MN) loss and skeletal muscle paralysis. It is uncertain whether this degeneration of MNs is triggered intrinsically and is autonomous, or if the disease initiating mechanisms are extrinsic to MNs. We hypothesized that skeletal muscle is a primary site of pathogenesis in ALS that triggers MN degeneration. Some inherited forms of ALS are caused by mutations in the superoxide dismutase-1 (SOD1) gene, that encodes an antioxidant protein, so we created transgenic (tg) mice expressing wild-type-, G37R-, and G93A-human SOD1 gene variants only in skeletal muscle. Presence of human SOD1 (hSOD1) protein in skeletal muscle was verified by western blotting, enzyme activity gels, and immunofluorescence in myofibers and satellite cells. These tg mice developed limb weakness and paresis with motor deficits, limb and chest muscle wasting, diaphragm atrophy, and age-related fatal disease with a lifespan shortening of 10–16%. Brown and white adipose tissue also became wasted. Myofibers of tg mice developed crystalline-like inclusions, individualized sarcomere destruction, mitochondriopathy with vesiculation, DNA damage, and activated p53. Satellite cells became apoptotic. The diaphragm developed severe loss of neuromuscular junction presynaptic and postsynaptic integrity, including decreased innervation, loss of synaptophysin, nitration of synaptophysin, and loss of nicotinic acetylcholine receptor and scaffold protein rapsyn. Co-immunoprecipitation identified hSOD1 interaction with rapsyn. Spinal cords of tg mice developed gross atrophy. Spinal MNs formed cytoplasmic and nuclear inclusions, axonopathy, mitochondriopathy, accumulated DNA damage, activated p53 and cleaved caspase-3, and died. Tg mice had a 40–50% loss of MNs. This work shows that hSOD1 in skeletal muscle is a driver of pathogenesis in ALS, that involves myofiber and satellite cell toxicity, and apparent muscle-adipose tissue disease relationships. It also identifies a non-autonomous mechanism for MN degeneration explaining their selective vulnerability as likely a form of target-deprivation retrograde neurodegeneration.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes skeletal muscle paralysis, respiratory failure, and death generally within 3–5 years after symptom onset [1, 2]
This study shows that aged tg mice expressing wild-type, G37R, and G93A-human superoxide dismutase-1 (SOD1) gene variants only in skeletal muscle develop a fatal ALS-like disease phenotype involving prominent skeletal muscle and spinal cord pathology, suggesting a skeletal muscle disease triggered, motor neuron (MN) non-autonomous degeneration in ALS driven by DNA damage and retrograde neurodegeneration
In the mitochondrial-enriched fraction of ∼15month-old mice, human SOD1 (hSOD1) was mostly present in an oligomer form (Figure 1B, arrow). hSOD1 was found as monomer and oligomer in the nuclear compartment (Figure 1B) as was seen in 8–12-month-old mice [72]
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes skeletal muscle paralysis, respiratory failure, and death generally within 3–5 years after symptom onset [1, 2]. Heterozygous mutations in the superoxide dismutase (SOD1) gene account for ∼20% of all fALS cases (∼2% of all ALS cases) [5, 6]. SOD1 (copper/zinc SOD) is ubiquitous in most tissues [7] and is a metalloenzyme that functions as a ∼32 kDa non-covalently assembled homodimer of ∼16 kDa subunits that bind one copper ion and one zinc ion [8]. This enzyme detoxifies and maintains intracellular superoxide anion (O2−) concentration in the low femtomolar range by catalyzing the dismutation of O2− to molecular oxygen and hydrogen peroxide [8]. SOD1 mutants appear to gain a toxic property or function, rather than losing O2− scavenging activity [5, 9, 10], and wildtype SOD1 can become toxic through oxidative post-translation modification and zinc deficiency [11,12,13,14]
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