EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment

Abstract

Central nervous system (CNS) axons lose their intrinsic ability to regenerate upon maturity, whereas peripheral nervous system (PNS) axons do not. A key difference between these neuronal types is their ability to transport integrins into axons. Integrins can mediate PNS regeneration, but are excluded from adult CNS axons along with their Rab11 carriers. We reasoned that exclusion of the contents of Rab11 vesicles including integrins might contribute to the intrinsic inability of CNS neurons to regenerate, and investigated this by performing laser axotomy. We identify a novel regulator of selective axon transport and regeneration, the ARF6 guanine-nucleotide-exchange factor (GEF) EFA6 (also known as PSD). EFA6 exerts its effects from a location within the axon initial segment (AIS). EFA6 does not localise at the AIS in dorsal root ganglion (DRG) axons, and in these neurons, ARF6 activation is counteracted by an ARF GTPase-activating protein (GAP), which is absent from the CNS, ACAP1. Depleting EFA6 from cortical neurons permits endosomal integrin transport and enhances regeneration, whereas overexpressing EFA6 prevents DRG regeneration. Our results demonstrate that ARF6 is an intrinsic regulator of regenerative capacity, implicating EFA6 as a focal molecule linking the AIS, signalling and transport.This article has an associated First Person interview with the first author of the paper.

Keywords: Axon initial segment; Axon regeneration; Axon transport; Integrin; Neuronal polarisation; Recycling endosome.

Fig. 1.

EFA6 is enriched in the AIS. (A) Immunolabelling of EFA6 and neurofascin in cortical neurons (E18, plus 4 to 21 DIV). EFA6 is expressed at low levels throughout the cell at an early stage (E18, 4 DIV). Expression increases with maturity, and EFA6 is enriched at the AIS from 7 DIV onwards. EFA6 is in green colour; pan-neurofascin is red (to identify the AIS). Arrows indicate the AIS. Spectrum colouring indicates the highest signal in the AIS. (B) Quantification of mean fluorescence intensity of EFA6 in the AIS, axon, initial dendrite and dendrite. n=45 neurons from three separate experiments. ***P<0.0001; n.s., not significant (ANOVA and Bonferroni's comparison test). All error bars are s.e.m.

Fig. 2.

EFA6 activates ARF throughout mature CNS axons. (A) Active ARF proteins (GGA3–ABD–GST, green) in proximal axons (neurofascin in blue), and distal axons (axon neurofilaments, red) at 14 DIV. The ellipse indicates the initial segment; arrows indicate a distal axon. (B) In young neurons (4 DIV), active ARF is detected in sparse tubulovesicular structures throughout developing axons, which diminish at the growth cone. (C) In differentiated neurons (14 DIV) active ARF (GGA3–ABD–GST+anti-GST) is distributed uniformly throughout axons (as indicated by immunolabelling with SMI312 for axonal neurofilaments). (D) Active ARF and total ARF6 in axons of neurons expressing either control shRNA or shRNA targeting EFA6 (red). Active ARF or ARF6 is shown in green, as indicated. Arrows indicate axons. Quantification of mean axonal ARF activity and total (mean) ARF6 in neurons expressing either control or EFA6 shRNA. n=61 and 64 neurons, respectively (for active ARF), n=50 for total ARF6 quantification. ***P<0.0001 (two-tailed Student's t-test). All error bars are s.e.m.

Fig. 3.

Depletion of EFA6 promotes axon transport of α9 and β1 integrins. (A) Kymographs showing dynamics of α9 integrin–GFP in the AIS, proximal and distal axon of neurons expressing control or EFA6 shRNA. (B) Quantification of α9 integrin–GFP axon transport; n=12 AIS control, 19 AIS experimental, 12 proximal control, 24 proximal experimental, 23 distal control and, 24 distal experimental, total 1024 vesicles. *P<0.05; **P<0.01; ***P<0.001 (ANOVA and Bonferroni's comparison test). (C) Immunolabelling of β1 integrin in neurons expressing control or EFA6 shRNA. Arrows indicate axons. (D) Quantification of axon-to-dendrite ratio for endogenous β1 integrin after EFA6 silencing; n=67 neurons from three experiments. ***P<0.0001 (Student's t-test). (E) Quantification of mean axon fluorescence intensity of endogenous β1 integrin after EFA6 silencing; n=67 neurons from three experiments. ***P<0.0001 (two-tailed Student's t-test). All error bars are s.e.m.

Fig. 4.

Depletion of EFA6 promotes axon transport of Rab11, and not APP. (A) Kymographs showing dynamics of Rab11–GFP in the AIS, and proximal and distal section of the axon of neurons expressing control or EFA6 shRNA. (B) Quantification of Rab11–GFP axon transport; n=27 control proximal, 19 experimental proximal, 31 control distal, 38 experimental distal. *P<0.05; ***P<0.001 (ANOVA and Bonferroni's comparison test). (C) Immunolabelling of Rab11 in neurons expressing either control or EFA6 shRNA. Arrows indicate axons. (D) Quantification of the axon-to-dendrite ratio for endogenous Rab11 after EFA6 silencing. n=71 neurons from three experiments. ***P<0.0001 (Student's t-test). Also see the associated Fig. S2. (E) Quantification of mean axon fluorescence intensity of endogenous β1 integrin after EFA6 silencing. n=71 neurons from three experiments. ***P<0.0001 (two-tailed Student's t-test). (F) Kymographs showing dynamics of APP–GFP in the proximal and distal axons of neurons expressing either control shRNA or shRNA targeting EFA6. (G) Quantification of APP–GFP vesicle movements in the proximal and distal axon n=12, 13, 17 and 17. No statistical difference was found between neurons expressing control or EFA6 shRNA using ANOVA and Bonferroni's comparison test. All error bars are s.e.m.

Fig. 5.

Depleting EFA6 promotes axon regeneration in CNS neurons. (A) Example of a neuron used for CNS axotomy experiments, indicating the site chosen for laser ablation (typically >1000 µm distal, on an unbranched section of axon). The fluorescent signal is RFP expressed with control shRNA from a single plasmid. (B) Example of regeneration failure, and formation of a stump. (C) Example of post-axotomy end bulb formed after axotomy. (D) Neuron expressing control shRNA, showing axotomy followed by regeneration. Note the small growth cone (typically <20 µm² and regeneration of <100 µm in 14 h). (E) Neuron expressing EFA6 shRNA, showing axotomy followed by regeneration >100 µm in 14 h, with growth cones typically >40 µm². (F–J) Quantification of regenerative response of cut axons of neurons expressing either control or EFA6 shRNA. (F) Percentage of axons regenerating within a 14 h period. ***P<0.001 (Fisher's exact test). n=59 (control shRNA) and 63 neurons (EFA6 shRNA). (G) Percentage of failed axons with a bulb versus with a stump. **P<0.01 (Fisher's exact test). (H) Distance grown after regeneration. *P<0.05 (two-tailed t-test). n=17 (control shRNA) and 34 (EFA6 shRNA). (I) Area of regenerated growth cones. ***P<0.0001 (two-tailed t-test). (J) Time taken to establish a growth cone and regenerate >50 µm. n.s., not significant (two-tailed t-test). All error bars are s.e.m.

Fig. 6.

ARF6 is regulated differently in CNS versus PNS neurons. (A) Cortical neurons and adult DRG neurons immunolabelled for EFA6 (upper panels) or ACAP1 (lower panels). Both neuronal types were labelled and imaged identically to allow comparison of the fluorescence signal. Images represent two independent immunolabelling experiments. (B) Axons of DRG and cortical neurons (10 DIV) labelled with GGA3–ABD–GST to detect active ARF protein; spectrum colouring of the highlighted section indicates the highest signals. The asterisk and red colour in the CNS panel indicate neurofascin labelling of the AIS to identify the axon. Graph shows quantification of mean±s.e.m. ARF protein activation in the two axon types. n=58 (DRG) and 60 (cortical). ***P<0.001 (two-tailed t-test).

Fig. 7.

EFA6 inhibits regeneration of adult DRG axons through its ARF6-activating region. (A) Cut DRG axon expressing GFP. (B) Cut DRG axon expressing EFA6–GFP. (C) Axon from panel A showing regeneration. (D) Axon from panel B showing failure to regenerate. (E) Quantification of axon regeneration of DRG neurons expressing either GFP (n=48), EFA6–GFP (n=44) or EFA6 E242K (EFA6 lacking the ability to activate ARF6) (n=31). *P<0.05; ***P<0.0001; ns, not significant (Fisher's exact test). All error bars are s.e.m.