Profilin 1 delivery tunes cytoskeletal dynamics toward CNS axon regeneration
Rita Pinto-Costa, … , Reinhard Fässler, Mónica M. Sousa
Rita Pinto-Costa, … , Reinhard Fässler, Mónica M. Sousa
Published January 16, 2020
Citation Information: J Clin Invest. 2020;130(4):2024-2040. https://doi.org/10.1172/JCI125771.
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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 5

S138A Pfn1 enhances MT dynamics via direct MT binding and formins.

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S138A Pfn1 enhances MT dynamics via direct MT binding and formins. (A) C...
(A) Crystal structure of hPfn1 (PDB code: 1cf0). Residues G118 (MT-binding), H134 (poly-proline-binding), and S138 (ROCK phosphorylation site, mediating inactivation of Pfn1-related functions) are highlighted. Actin-, poly-proline–, and PI(4,5)P2-binding regions of Pfn1 are shadowed in light yellow, gray, and red, respectively (adapted from ref. 66). (B) Live-cell imaging of EB3-GFP in hippocampal neurons transfected with EB3-GFP and either a control empty vector (Ctrl) or plasmids expressing S138A hPfn1 or S138A Pfn1 mutants (G118V/S138A or H134S/S138A hPfn1); Ctrl and S138A hPfn1 treated with SMIFH2 are also shown. Scale bars: 2 μm. (C) Kymographs related to B. (D) Analysis of MT growth speed and (E) EB3 comet invasion frequency per filopodia. In D and E, data represent mean ± SEM (n = 7–11 [D] and n= 3–7 [E] growth cones/condition). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant in relation to Ctrl. #P < 0.05, ##P < 0.01, ####P < 0.0001; #NS, not significant in relation to S138A hPfn1. (F) GFP+ hippocampal neurons transfected with either a control empty vector (Ctrl) or plasmids expressing different hPfn1 mutants, either untreated or treated with SMIFH2, whenever indicated. Scale bars: 30 μm. (G) Quantification of axonal length related to F. Data represent mean ± SEM (n =18–33 neurons/condition; representative of 3–5 independent experiments/condition). *P < 0.05, ****P < 0.0001; NS, not significant in relation to Ctrl. ###P < 0.001 and ####P < 0.0001 in relation to S138A hPfn1; both by 1-way ANOVA with Tukey’s post hoc test.