Figure 8.

Inhibition of retrograde stress signaling in motor neurons is sufficient to block activation of P-c-Jun and to rescue motor neurons from mSOD1-induced toxicity. A, E14 embryonic rat motor neurons were grown for 3 weeks in compartmentalized Campenot chambers. CM-Dill tracer was added to the distal chambers to label the cell bodies of neurons that extend axons into the distal compartment. Glial cells were added to the distal compartments; both MN and glia cells were infected with HSV-mSOD1 or HSV-wtSOD1. Viable CM-Dill-positive MN cell bodies were counted after 2 (for basal number) and 6 d and percentages of survival after 6 d were scored. B, In coculture, expression of mSOD1 leads to significant cell death (∼50%) after 6 d. The addition of specific inhibitors to JNK, caspase-8, and p75 to the distal chambers in the presence of dynamitin overexpression to inhibit retrograde stress signaling led to a significant increase in cell survival (*p < 0.01). C, D, Immunostaining of MN cell bodies against the downstream target of JNK activation, the transcription factor P-c-Jun, shows activation in mSOD1-expressing cells in vitro. Partial inhibition of dynein transport by overexpression of the dynamitin (p50) subunit of dynactin prevents the upregulation of P-c-Jun (*p < 0.001) after 3 d but not after 5 d (C). However, adding a mixture of inhibitors (to JNK, p75, and caspase-8) prevents the upregulation of P-c-Jun (*p < 0.001) after 6 d (D). The experiments were repeated twice each time with four chambers. A total of more than 1000 cells for each condition was scored. MT, Microtubule. Scale bar, 20 μm. E, Western blot analysis of 85 d spinal cord extracts shows upregulation of P-c-Jun in mSOD1 mice (S) compared with n.Tg control (n.Tg). The Western blot analysis was repeated three times with similar results and quantified using ImageJ.