Absence of SOD1 leads to oxidative stress in peripheral nerve and causes a progressive distal motor axonopathy

Abstract

Oxidative stress is commonly implicated in the pathogenesis of motor neuron disease. However, the cause and effect relationship between oxidative stress and motor neuron degeneration is poorly defined. We recently identified denervation at the neuromuscular junction in mice lacking the antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD1) (Fischer et al., 2011). These mice show a phenotype of progressive muscle atrophy and weakness in the setting of chronic oxidative stress. Here, we investigated further the extent of motor neuron pathology in this model, and the relationship between motor pathology and oxidative stress. We report preferential denervation of fast-twitch muscles beginning between 1 and 4 months of age, with relative sparing of slow-twitch muscle. Motor axon terminals in affected muscles show widespread sprouting and formation of large axonal swellings. We confirmed, as was previously reported, that spinal motor neurons and motor and sensory nerve roots in these mice are preserved, even out to 18 months of age. We also found preservation of distal sensory fibers in the epidermis, illustrating the specificity of pathology in this model for distal motor axons. Using HPLC measurement of the glutathione redox potential, we quantified oxidative stress in peripheral nerve and muscle at the onset of denervation. SOD1 knockout tibial nerve, but not gastrocnemius muscle, showed significant oxidation of the glutathione pool, suggesting that axonal degeneration is a consequence of impaired redox homeostasis in peripheral nerve. We conclude that the SOD1 knockout mouse is a model of oxidative stress-mediated motor axonopathy. Pathology in this model primarily affects motor axon terminals at the neuromuscular junction, demonstrating the vulnerability of this synapse to oxidative injury.

Figure 1

Denervation in Sod1−/− hind limb muscles preferentially affects fast twitch muscles. A. Representative confocal projections of tibialis anterior (fast twitch), medial gastrocnemius (fast twitch), and soleus (slow twitch) from 18 month-old Sod1−/− mice and Sod1+/+ littermates, with motor axons in green (YFP) and endplates in red (bungarotoxin). Tibialis anterior and medial gastrocnemius muscles have undergone extensive denervation, but the majority of neuromuscular junctions in soleus remain innervated. B.–C. Percent innervated, intermediate, and denervated endplates in tibialis anterior (B) and soleus muscle (C) from 1 to 18 months of age. Denervation in the soleus muscle is milder than in TA and does not begin until 12 months of age (n=3–7 animals per group, *p<0.05, **p<0.01, ***p<0.001 vs. Sod1+/+).

Figure 2

Loss of SOD1 causes morphologic abnormalities at the neuromuscular junction. A. Long terminal sprouts (arrow) arising from innervated junctions. B. A terminal sprout (arrow) that has reinnervated a nearby endplate (*). C. Percent of endplates in TA showing terminal sprouts. By 1 month of age, prior to the onset of denervation, a significant percent of NMJs already show sprouting. D. Percent of endplates in soleus muscle with terminal sprouts. Again, significant sprouting is seen by 4 months, in advance of denervation (In C–D, n=3–7 animals per group; **p<0.01, ***p<0.001 vs. Sod1+/+ and +/−). E. A terminal axon swellings (arrow), along with smaller varicosities involving the intramuscular nerve bundle (*). F. Terminal axon swellings show dense accumulation of phosphorylated neurofilament (SMI-31). All images in A–F from Sod1−/− tibialis anterior, 4 months of age.

Figure 3

Sod1−/− mice lack pathologic involvement of lumbar spinal cord and proximal axons. A. Morphology of Nissl-stained motor neurons in Sod1−/− lumbar spinal cord is identical to wild type at 4 and 18 months of age (scale bar = 25 μm). B. No degenerating axons are seen in Sod1−/− L4 ventral root and tibial nerve at 18 months of age (scale bar = 10 μm). C. Immunohistochemical staining shows little evidence for pathologic involvement of Sod1−/− lumbar spinal cord (representative ventral horns are shown from n=3 mice examined at each stage; motor neuron cell bodies are not counterstained in these sections). Phosphorylated NF-H/NF-M does not accumulate in motor neuron cell bodies (*) or proximal axons (SMI-31). A mild increase in GFAP-positive astrocytes in Sod1−/− mice is seen at both 4 and 18 months of age, but no apparent microgliosis (Iba1). No ubiquitin- or TDP-43-positive inclusions are present, and nuclear TDP-43 localization appears similar in Sod1−/− mice and controls (scale bars = 25 μm).

Figure 4

Distal sensory fibers do not degenerate in Sod1−/− mice. A. PGP9.5-labelled epidermal nerve fibers from the plantar footpad of Sod1−/− mice remain intact at 4 and 18 months of age. A subtle increase in intra-epidermal branching (arrow) is present in 18 month-old Sod1−/− mice (scale bar = 20 μm). B. Epidermal nerve fiber density, expressed as the number of fibers/mm of epidermis, is equivalent in Sod1−/− mice and controls (number of animals is indicated on bar graph).

Figure 5

Loss of SOD1 leads to a more oxidized redox state in peripheral nerve, but not muscle, by 4 months of age. A–C. GSH (A) and GSSG (B) levels from tibial nerve of 4 month-old mice, measured by HPLC, and the calculated redox potential (E_h) (C). Eh is approximately 20 mV higher (more oxidized) in tibial nerve of 4 month-old Sod1−/− mice compared to controls (*p<0.05, ***p<0.001). D–F. GSH (D), GSSG (E), and E_h (F) from gastrocnemius muscle show no difference between Sod1−/− and controls (same animals as in A–C). For all panels, n=7 (Sod1+/+), n=13 (Sod1+/−), n=14 (Sod1−/−).

Figure 6

Poor axon outgrowth in Sod1−/− primary motor neurons is rescued by antioxidant treatment. A. Primary motor neurons at 24 hrs in culture, labeled with phosphorylated neurofilament (green) and DAPI (blue). Sod1−/− motor neurons show significantly shorter axons than Sod1+/+ and Sod1+/− neurons at 24 hrs. Treatment with 5 mM NAC restores Sod1−/− axons to wild-type length (scale bar = 20 μm). B.–C. Mean axon length at 24 hrs ± SEM from 4 independent experiments is shown. 500–600 neurons per group were measured (***p<0.001 vs. Sod1+/+ and +/− (B); or vs. Sod1−/− (C)).