The neurite outgrowth inhibitor Nogo-A promotes denervation in an amyotrophic lateral sclerosis model

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss and muscle wasting. In muscles of ALS patients, Nogo-A-a protein known to inhibit axon regeneration-is ectopically expressed at levels that correlate with the severity of the clinical symptoms. We now show that the genetic ablation of Nogo-A extends survival and reduces muscle denervation in a mouse model of ALS. In turn, overexpression of Nogo-A in wild-type muscle fibres leads to shrinkage of the postsynapse and retraction of the presynaptic motor ending. This suggests that the expression of Nogo-A occurring early in ALS skeletal muscle could cause repulsion and destabilization of the motor nerve terminals, and subsequent dying back of the axons and motor neurons.

Figure 1

Genetic ablation of Nogo-A in Sod1(G86R) mice. (A) Cumulative probability of survival of G86R/Nogo-A^+/+ and G86R/Nogo-A^−/− mice (P=0.04, n=16–17 mice, log-rank test). (B) Cell counts in the ventral horns of the lumbar spinal cord of G86R/Nogo-A^+/+ and G86R/Nogo-A^−/− mice. Toluidine-blue-stained cells were divided into two groups of 300–600μm² and >600 μm², the latter clearly representing motor neurons. No differences were observed in the number of cells between non-Cu/Zn-superoxide dismutase 1 (Sod1)(G86R) Nogo-A-knockout and wild-type mice (data not shown; ^*P<0.05 versus corresponding +/+, n=3 mice, Student's t-test). (C) Ubiquitin immunoreactivity in large motor neurons (cross-sectional area >600 μm²; arrowheads) of the lumbar spinal cord from G86R/Nogo-A^+/+ and G86R/Nogo-A^−/− mice. Scale bar, 30 μm. (D) Messenger RNA levels of acetylcholine receptor α-subunit (AChRα) and muscle-specific receptor tyrosine kinase (MuSK) in the soleus muscle of wild-type (WT; open bars) and Sod1(G86R) mice (filled bars) containing (+/+) or not containing (−/−) Nogo-A (^***P<0.001 versus wild type, ^#P<0.05 versus +/+, n=4–5 mice, analysis of variance followed by Tukey's test). (E) Levels of MuSK protein as determined by western blot of immunoprecipitated MuSK from muscle extracts of Sod1(G86R) mice containing (+/+) or not containing (−/−) Nogo-A. MW, molecular weight markers. In all cases, symptomatic animals reached the end point of the disease. Sod1, Cu/Zn-superoxide dismutase 1.

Figure 2

Overexpression of Nogo-A in muscle causes the disassembly of the neuromuscular junction. Electroporation of the soleus muscle with expression constructs for Nogo-A and nuclear-localized green fluorescent protein (GFP) results in muscle fibres that express high levels of Nogo-A (red) and GFP (green) after 2 weeks. (A) Whole-mount and (B) cross-section of the soleus muscle. Representative pictures of neuromuscular junctions (NMJs), 6 weeks after electroporation of empty vector (control; C) or Nogo-A expression constructs (D–F). The postsynaptic apparatus was visualized by rhodamine-labelled α-bungarotoxin (acetylcholine receptor; red in merge). Presynaptic nerve terminals were labelled by yellow fluorescent protein (YFP; green in merge) and transfected muscle fibres by nuclear localization signal–GFP (GFP, green in merge). For clarification, GFP-positive myonuclei are indicated by arrowheads. In control transfections, both pre- and postsynaptic structures look normal (C). By contrast, overexpression of Nogo-A often results in smaller NMJs (D) and fragmentation of the postsynaptic structure including partial (E; hollow arrows) or total (F; asterisks) retraction of the presynaptic nerve terminal. Scale bar, 25 μm.