Abstract
Nogo-A has been extensively studied as a myelin-associated neurite outgrowth inhibitor in the lesioned adult central nervous system. However, its role in the intact central nervous system has not yet been clarified. Analysis of the intact adult nervous system of C57BL/6 Nogo-A knock-out (KO) versus wild-type (WT) mice by a combined two-dimensional gel electrophoresis and isotope-coded affinity tagging approach revealed regulation of cytoskeleton-, transport-, and signaling growth-related proteins, pointing to regulation of the actin cytoskeleton, the neuronal growth machinery, and in particular the Rho-GTPase/LIMK1/cofilin pathway. Nogo-A KO adult neurons showed enlarged, more motile growth cones compared with WT neurons. The phenotype was reproduced by acute in vitro neutralization of neuronal Nogo-A. LIMK1 phosphorylation was increased in Nogo-A KO growth cones, and its reduction caused the decrease of KO growth cone motility to WT levels. Our study suggests that in the unlesioned adult nervous system, neuronal Nogo-A can restrict neuronal growth through negative modulation of growth cone motility.
Highlights
We compared the adult central nervous system (CNS) of C57BL/6 naïve, unlesioned, Nogo-A KO and wildtype (WT) mice, and using a combination of approaches, we report that the depletion of Nogo-A or functional blockade of neuronal Nogo-A in the adult as well as postnatal intact nervous system causes the reorganization of the cytoskeletal growth cone machinery at both the molecular and morphological levels and that the LIMK1/cofilin phosphorylation state is critical for this process
We found that a large proportion of differentially expressed proteins in the Nogo-A KO intact adult nervous system are components of the cytoskeleton, cytoskeleton-binding proteins, signaling molecules involved in cytoskeleton remodeling and growth, early neurite growth markers, and axonal transport constituents
Summary—This study provides mechanistic insights into the function of neuronal Nogo-A in the unlesioned CNS and the related molecular signaling pathways
Summary
Animals—Male C57BL/6 Nogo-A KO mice with a strain purity of Ͼ99.98% and back-crossed for Ͼ10 generations [12] and male C57BL/6 WT mice were used. Two-dimensional Gel Electrophoresis—Lumbar spinal cords of three adult Nogo-A KO and WT mice were dissected and transferred to CHAPS lysis buffer (50 mM NaH2PO4 (pH 8.0), 150 mM NaCl, 0.5% CHAPS, protease inhibitor mixture (Roche Applied Science)) on ice. Tissues were disrupted using a rotorstator homogenizer. The parameters of neuronal morphology for P0 dissociated DRG were measured using ImageJ software in at least 10 neurons per coverslip in four coverslips (experimental replicates) in two separate experiments for a total of at least 80 neurons. Nogo-A Neutralization—Highly purified mouse anti-Nogo-A monoclonal antibody 11C7 or a control highly purified mouse IgG antibody (3 g/ml) was applied to Nogo-A KO and WT adult low density dissociated DRG neurons at plating time. The percentage of enlarged growth cones was calculated for the total number of neurons in three independent experiments.
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