Myelin-Associated Inhibitors Regulate Cofilin Phosphorylation and Neuronal Inhibition through LIM Kinase and Slingshot Phosphatase

Nogo induces cofilin dephosphorylation in cell bodies and neurites. A, Total cofilin and phospho-cofilin levels in P8 rat cerebellar neurons treated with AP-Nogo-66 or AP. Scale bar, 200 μm. B, Higher magnification view of cerebellar neurons treated with AP-Nogo-66 for 0, 0.5, or 60 min. Each image is accompanied by a false color image [colder colors (blue) representing low intensity to warmer colors (red) representing high intensity] to demonstrate concentrations of cofilin phosphorylation at neurite tips only 0.5 min after AP-Nogo-66 treatment. Green arrowheads signify neurite tips. C, Western blot of nonstimulated (–) or calyculin-stimulated (+) cerebellar lysates probed with anti-cofilin (Cof) or anti-phospho-cofilin antibody. D, E, Quantification of total cofilin or phospho-cofilin fluorescence intensity over cell bodies and neurites together (D) or independently (E) using ImageJ analysis software. Fluorescence intensity was normalized to control levels (0 min) for each experiment. Determinations are from three experiments with a minimum of 50 neurons per data point. *p < 0.01 compared with controls by Student's t test. Error bars represent SEM.

DN-LIMK1 blocks myelin-dependent growth cone collapse. A, Detection of recombinant proteins in a low-magnification view of DRG explants by immunofluorescence in HSV-, V5DN-LIMK1-, V5WTSSH1-, or V5SSH1(CS)-infected E13 chick DRG explants. Scale bar, 100 μm. B, Detection of recombinant protein in a high-magnification view of axons and growth cones from a V5DN-LIMK1-infected DRG explant. Scale bar, 40 μm. C, Growth cone collapse in nonstimulated (control) or AP-Nogo-66- or myelin-stimulated DRG explants infected with HSV, V5DN-LIMK1, V5WTSSH1, or V5SSH1(CS). Arrows indicate spread growth cones. Arrowheads indicate collapsed growth cones. Scalebar, 40 μm. D, Quantification of growth cone collapse from DRG explants infected with HSV, V5DN-LIMK1, V5WTSSH1, or V5SSH1(CS). Determinations are mean ± SEM from four experiments, each performed in duplicate. *p < 0.01 compared with HSV infection by Student's t test. E, F, Quantification of growth cone collapse from noninfected DRG explants (Control) or explants infected with HSV or V5DN-LIMK1 and treated with AP or AP-Nogo-66 (E) or myelin (F). Determinations are mean ± SEM from at least three experiments, each performed in duplicate. *p < 0.05 compared with no treatment by Student's t test. Error bars represent SEM.

DN-LIMK1 circumvents myelin-dependent neurite outgrowth inhibition. A, Neurite outgrowth from E13 chick dissociated DRG neurons infected with HSV, V5DN-LIMK1, V5SSH1(CS), or V5WTSSH1 on laminin (control) or myelin substrates. Scale bar, 100 μm. B, Quantification of neurite outgrowth/cell for each experiment. Determinations are from a minimum of three experiments. *p < 0.01 by Student's t test compared with 0 μg of treatment for each virus. Error bars represent SEM.

DN-LIMK1 and SSH1(CS) attenuate Nogo-dependent changes in cofilin phosphorylation. A, B, Phospho-cofilin levels in AP-Nogo-66-stimulated PC12 cells transfected with pcDNA, V5DN-LIMK1, or SSH1 (CS) (A) or dissociated P4 rat DRG neurons infected with HSV, HSVV5SSH1(CS), or HSVV5DN-LIMK1 (B). C, D, Quantification of phospho-cofilin levels in AP-Nogo-66 stimulated PC12 cells or DRG neurons. For DRG neurons, values were normalized to 0 min treatment for each virus. Determinations are from a minimum of three experiments. *p < 0.01 by Student's t test compared with 0 μg of treatment. Error bars represent SEM.