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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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 2

Pfn1 downregulation impairs axonal growth in vitro in different neuronal types and developmental stages.

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Pfn1 downregulation impairs axonal growth in vitro in different neuronal...
(A) Timeline of naive DRG neuron cultures. (B) GFP-expressing naive adult DRG neurons transfected with control empty (Ctrl) or Pfn1 shRNA plasmid. (C) Timeline of conditioned DRG neuron cultures. (D) GFP-expressing conditioned DRG neurons transfected with Ctrl or Pfn1 shRNA plasmid. Scale bars in B and D: 70 μm. (E) Total neurite length related to B and D. Data represent mean ± SEM (n = 3–6 independent samples/condition; 6–36 neurons/sample). *P < 0.05; ****P < 0.0001; by Student’s t test. NS, not significant. (F) Branching analysis related to E. Data represent mean ± SEM. *P < 0.05, **P < 0.01 refers to Ctrl versus Pfn1 shRNA of naive DRG neurons; ####P < 0.0001 refers to Ctrl versus Pfn1 shRNA of CL DRG neurons; 2-way ANOVA with Tukey’s post hoc test. (G) Timeline for Pfn1 downregulation in DIV3 hippocampal neurons using lentiviral infection. (H) βIII-tubulin in hippocampal neurons after lentiviral expression of control empty (Ctrl) or Pfn1 shRNA plasmid. Scale bars: 10 μm. (I) Timeline for Pfn1 downregulation in DIV0 hippocampal neurons. (J) βIII-tubulin in DIV4 hippocampal neurons expressing a control empty (Ctrl) or a Pfn1 shRNA plasmid. Middle panels (Pfn1 shRNA) show representative images of stage 1 to 3 hippocampal neurons. Scale bars: 30 μm (Ctrl and Pfn1 shRNA + WT hPfn1*) and 20 μm (Pfn1 shRNA). (K) Axonal length related to J. Data represent mean ± SEM (n = 3–5 independent samples/condition; 11–26 neurons/sample). *P < 0.05 by 1-way ANOVA Tukey’s post hoc test. NS, not significant. (L) Dendritic length of DIV7 hippocampal neurons expressing control empty (Ctrl) or Pfn1 shRNA plasmid. Data represent mean ± SEM (n = 4–5 independent samples/condition; 3–25 neurons/sample). *P < 0.05 by Student’s t test. All rescue experiments were performed using shRNA-resistant WT Pfn1 (WT hPfn1*).