Figure 6.
Alterations in the retrograde transport and localization of signaling molecules in neurons expressing mSOD1. A–C, Double ligation assays of sciatic nerves of 85-d-old SOD1G93A (mSOD), 6-month Loa, and 1-year Tgdynamitin overexpression mice (p50) and age-matched control mice were performed, and segments immediately proximal and distal to the ligation site were subjected to Western blot analysis. Stress/death factors like p75 cleavage fragments (A), JNK (B), and activated caspase-8 (actv-casp8) (C) showed alterations in retrograde axonal transport only in the mSOD1 mice. P, Proximal segment; D, distal segment. The bars represent the average intensity ± SEM relative to GAPDH from three different experiments. D–G, Rat E14 embryonic motor neurons were infected with either HSV-wtSOD or HSV-SOD1G85R and stained with antibodies to P-Trk (D) or the intracellular domain of p75 (E). Motor neuron cultures expressing mSOD1 displayed less P-Trk and more p75 localization to processes compared with neurons expressing wtSOD1 (boxed regions are shown at higher magnifications in the insets). Scale bar: (in D) D, E, 5 μm. G, Quantification of staining indicates significantly higher levels of p75 and lower levels of pTrk (n > 30; *p < 0.01) in mSOD1 neurites than in processes from neurons expressing wtSOD1. The number of immunoreactive puncta per unit length was also quantitated. There were fewer P-Trk-positive and more p75-positive puncta along the neurites of mSOD1-expressing cells (n > 30; *p < 0.01). The graphs shown represent average ± SEM. Line scan analyses along neurites in the indicated regions are also shown (F). H, I, In vivo mouse immunostaining of p75 and P-Trk motor neurons from the sternomastoid muscles additionally shows less P-Trk expression and more p75 in mSOD1 axons. Scale bars: H, 50 μm; I, 5 μm. J, Localization along the axon. Representative line scans along axons from mSOD1 and wtSOD1 mice confirm the altered patterns of P-Trk and p75. The experiment was repeated twice with similar results.