Profilin 1 delivery tunes cytoskeletal dynamics toward CNS axon regeneration
Rita Pinto-Costa, Sara C. Sousa, Sérgio C. Leite, Joana Nogueira-Rodrigues, Tiago Ferreira da Silva, Diana Machado, Joana Marques, Ana Catarina Costa, Márcia A. Liz, Francesca Bartolini, Pedro Brites, Mercedes Costell, Reinhard Fässler, Mónica M. Sousa
Rita Pinto-Costa, Sara C. Sousa, Sérgio C. Leite, Joana Nogueira-Rodrigues, Tiago Ferreira da Silva, Diana Machado, Joana Marques, Ana Catarina Costa, Márcia A. Liz, Francesca Bartolini, Pedro Brites, Mercedes Costell, Reinhard Fässler, Mónica M. Sousa
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Research Article Cell biology Neuroscience

Profilin 1 delivery tunes cytoskeletal dynamics toward CNS axon regeneration

Abstract

After trauma, regeneration of adult CNS axons is abortive, causing devastating neurologic deficits. Despite progress in rehabilitative care, there is no effective treatment that stimulates axonal growth following injury. Using models with different regenerative capacities, followed by gain- and loss-of-function analysis, we identified profilin 1 (Pfn1) as a coordinator of actin and microtubules (MTs), powering axonal growth and regeneration. In growth cones, Pfn1 increased actin retrograde flow, MT growth speed, and invasion of filopodia by MTs, orchestrating cytoskeletal dynamics toward axonal growth. In vitro, active Pfn1 promoted MT growth in a formin-dependent manner, whereas localization of MTs to growth cone filopodia was facilitated by direct MT binding and interaction with formins. In vivo, Pfn1 ablation limited regeneration of growth-competent axons after sciatic nerve and spinal cord injury. Adeno-associated viral (AAV) delivery of constitutively active Pfn1 to rodents promoted axonal regeneration, neuromuscular junction maturation, and functional recovery of injured sciatic nerves, and increased the ability of regenerating axons to penetrate the inhibitory spinal cord glial scar. Thus, we identify Pfn1 as an important regulator of axonal regeneration and suggest that AAV-mediated delivery of constitutively active Pfn1, together with the identification of modulators of Pfn1 activity, should be considered to treat the injured nervous system.

Authors

Rita Pinto-Costa, Sara C. Sousa, Sérgio C. Leite, Joana Nogueira-Rodrigues, Tiago Ferreira da Silva, Diana Machado, Joana Marques, Ana Catarina Costa, Márcia A. Liz, Francesca Bartolini, Pedro Brites, Mercedes Costell, Reinhard Fässler, Mónica M. Sousa

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Figure 1

Active Pfn1 is increased after conditioning lesion (CL).

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Active Pfn1 is increased after conditioning lesion (CL). (A) Representat...
(A) Representation of SCI and of the CL paradigm (left and right to dashed line, respectively). In CL, a sciatic nerve injury (1) is performed 1 week prior to SCI (2), potentiating regeneration of central DRG axons (right green line, rostral to SCI). Western blot analyses of the dorsal SCI site and of DRG (blue rectangles) were performed. (B) Live-cell imaging of LifeAct-GFP in growth cones of naive and conditioned adult DRG neurons. Scale bars: 4 μm. (C) Kymographs related to B. (D) Quantification of actin retrograde flow and (E) growth cone area related to B. Data represent mean ± SEM (n = 12–13 growth cones/condition). **P < 0.01 by Student’s t test. (F) Pfn1 staining in growth cones of cultured naive and conditioned DRG neurons. Scale bars: 5 μm. (G) Quantification of line scans of Pfn1 fluorescence in relation to distance from growth cone leading edge related to F. Data represent mean ± SEM (n = 48–57 neurons/condition). ***P < 0.001 by 2-way ANOVA with Sidak’s post hoc test. (H) Western blot and (I) respective quantification showing Pfn1 levels in DRG of rats with SCI or CL. Vinculin was used as control. Data represent mean ± SEM (n = 4 animals/condition). *P < 0.05 by Student’s t test. (J) Western blot and (K) respective quantification showing Pfn1, Pfn1 p-S138, ROCK1, and Pfn2 levels in samples from the dorsal SCI site (horizontal blue rectangle in A), 1 week after SCI or CL. HPRT and vinculin were used as controls. Data represent mean ± SEM (n = 4–7 animals/condition). *P < 0.05, **P < 0.01 by Student’s t test. NS, not significant. (L) Pfn1 immunofluorescence (red) in sensory SCG10-positive axons (green) in a CL spinal cord. Arrowheads highlight growth cones. Scale bars: 20 μm.