Figure 3.

Genetic knockdown of RyaR and IP₃R1 protects degeneration of mechanically or toxic injured axons. An siRNA pool was used to determine whether depletion of RyaR1 and RyaR3 (RyR1/3) or IP₃R1 from dissociated DRG cultures protect from axotomy and vincristine-triggered axonal degeneration. DRGs were transfected with the siRNA pool or a control (Ctrl) nontargeting siRNA. A, Transfected DRG neurons were cultured on transwell chambers, allowing the separation of axonal processes from cell bodies. After 72 h of culture, neuronal cell bodies were scraped away from the upper membrane surface. Control siRNA-transfected neurons showed almost complete degeneration of distal axon 12 h after transection, but RyaR1/3 and IP₃R1 siRNA transfected neurons showed preservation of axons over the same period of time. Scale bar, 50 μm. C, DRGs were transfected as in A and treated with vincristine (1 μm) to induce axonal degeneration. Both RyaR1/3 and IP₃R1 siRNA-transfected neurons showed preservation of axons 12 h after vincristine treatment. B, D, Quantification of injury-induced degeneration of A and C using the degeneration index (n = 9, 3 images per sample, 3 samples per condition, 3 repetitions; *p < 0.05 by Student's t test compared with nontarget siRNA; error bars indicate SEM). E, Reduction of RyaR1, RyaR3, and IP₃R1 mRNA after siRNA was assessed by qPCR in embryonic DRG neurons. E, F, Gel bands of each qPCR result. A significant decrease of RyaR1, RyaR3, and IP₃R1 mRNA was observed after siRNA (n = 3 per each group; *p < 0.05 by Student's t test compared with nontarget siRNA; error bars indicate SEM).