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Selective rab11 transport and the intrinsic regenerative ability of CNS axons

  1. Hiroaki Koseki
  2. Matteo Donegá
  3. Brian YH Lam
  4. Veselina Petrova
  5. Susan van Erp
  6. Giles SH Yeo
  7. Jessica CF Kwok
  8. Charles ffrench-Constant
  9. Richard Eva  Is a corresponding author
  10. James W Fawcett  Is a corresponding author
  1. University of Cambridge, United Kingdom
  2. University of Edinburgh, United Kingdom
  3. Faculty of Biological Sciences, University of Leeds, United Kingdom
  4. Institute of Experimental Medicine, Czech Academy of Sciences, Czech Republic
Research Article
Cite this article as: eLife 2017;6:e26956 doi: 10.7554/eLife.26956
10 figures, 3 videos, 1 table and 1 data set

Figures

Figure 1 with 3 supplements
Electrical maturation of neurons.

(A) Resting membrane potential of maturing neurons. The average membrane potential from at least three patching sessions is shown. At least 10 neurons per session. One-way ANOVA followed by Bonferroni’s post hoc test. (B) Membrane capacitance of maturing neurons. One-way ANOVA followed by Games-Howell post hoc test. (C) Percentage of patched neurons capable of firing action potentials when depolarizing currents were applied. The average percentage from at least three patching sessions is shown. At least 10 neurons/session. At 24 DIV, all neurons fired action potentials. Representative responses are shown in (E). (D) Percentage of neurons showing spontaneous activity. Fisher’s exact test. Error bars represent s.e.m. Patch clamp results from at least three independent sessions were accumulated. Sample numbers are shown in the figure. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

https://doi.org/10.7554/eLife.26956.003
Figure 1—figure supplement 1
Electrical and anatomical characterisation of maturing neurons.

(A,B,C) Quantification of spike properties plotted by DIV. Spike amplitude (A), width (B), and frequency (C) were measured. A: One-way ANOVA followed by Bonferroni’s post hoc test. B,C: Kruskal-Wallis test. (D,E) Spike threshold and input resistance of maturing neurons. D: One-way ANOVA followed by Games-Howell post hoc test. E: Kruskal-Wallis test. (F) Excitatory and inhibitory synapse formation at 10 DIV and 20 DIV. Excitatory synapses were marked with VGLUT1 and PSD95, and inhibitory synapses were marked with VGAT and gephyrin. Error bars represent s.e.m. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

https://doi.org/10.7554/eLife.26956.004
Figure 1—figure supplement 2
Characterisation of mRNA and protein expression changes with maturity.

(A) Staining of cultures at 3,7 and 14 days for two maturity markers, doublecortin and low molecular weight neurofilament. (B,C) Plots of the intensity of immunofluorescence and the mRNA levels of the two maturity markers. ANOVA followed by Bonferroni’s post hoc test. Error bars represent S.E.M.. *p<0.05, **p<0.01. (F) mRNA levels of rab11a and rab11b from the mRNA profile. (D,E) Analysis of genes that showed large expression changes in their mRNA profiles from ingenuity analysis.

https://doi.org/10.7554/eLife.26956.005
Figure 1—figure supplement 3
Confirmation of axon initial segment development and neuronal viability.

(A) Live neurofascin staining at 4 and 16 DIV to show staining of the axon initial segment. (B) caspase 3 staining of a GFP transfected 24 DIV culture, showing no signs of apoptosis and staining of a staurosporine treated culture as a positive control. Student’s t-test. Error bars represent S.E.M.. *p<0.05.

https://doi.org/10.7554/eLife.26956.006
Characterisation of the initial response following axotomy.

(A,B) Representative images of death, branch loss, bulb formation. Axotomy location is indicated by the yellow star. (C) Ratio of bulb formation, branch loss and cell death categorized by DIV. Fisher’s exact test with Bonferroni correction.

https://doi.org/10.7554/eLife.26956.007
Regeneration after axotomy.

(A) An axon before cutting, followed by retraction bulb formation and regeneration. (B) A further example of regeneration after axotomy and an example of ectopic growth. (C) The overall proportion of axons showing the different forms of behaviour after axotomy at different stages of maturity. Fisher’s exact test with Bonferroni correction. Error bars represent S.E.M.. *p<0.05, ****p<0.0001.

https://doi.org/10.7554/eLife.26956.008
Figure 4 with 1 supplement
Maturation related changes in retraction and regeneration.

(A) Relative frequency plot of retraction distance categorized by DIV. Average distance comparison between 4 DIV and 24 DIV: Welch’s t-test. (B) Retraction distance categorized separately by DIV, retraction, and axotomy distance. 16 DIV: One-way ANOVA followed by Bonferroni’s post hoc test, 24 DIV: Welch’s t-test. (C) Regeneration ratio categorized by DIV, retraction, and axotomy distance. 16 DIV: Fisher’s exact test with Bonferroni correction, 16 DIV proximal axotomy vs 24 DIV proximal axotomy: Fisher’s exact test, 24 DIV: Fisher’s exact test. (D,E,F) Regeneration initiation time, regeneration length and growth cone area categorized by DIV. One-way ANOVA followed by Games-Howell post hoc test. (G) Plot of regeneration success against log retraction distance. Error bars represent s.e.m. Axotomy results were accumulated from at least three independent axotomy sessions. Retraction distance was converted by log10. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

https://doi.org/10.7554/eLife.26956.012
Figure 4—figure supplement 1
Further analysis of changes in retraction and regeneration with maturity.

(A,B) Retraction distance and retraction bulb formation time categorized by DIV. Kruskal-Wallis test. (C) Correlation between retraction distance and retraction bulb formation time. (D,E) Retraction distance plotted against axotomy distance. Retraction distance was categorized by DIV and retraction group. (F,G,H) Regeneration initiation time (F), regeneration length (G), and growth cone area (H) of regenerating 16 DIV neurons categorized by retraction and axotomy distance. Categorization by retraction distance groups did not lead to any obvious trends. One-way ANOVA followed by Bonferroni’s post hoc test. Regeneration length showed a declining trend with axotomy distance (p=0.0618, Student’s t-test). Error bars represent s.e.m. Axotomy results were accumulated from at least three independent sessions. Sample numbers are described in the figure. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

https://doi.org/10.7554/eLife.26956.013
Decline in regeneration is not due to the mature environment.

(A) GFP transfected E18 cortical neurons were plated on either PDL glass or 21 DIV cultures, and cultured for 4 days. (B,C) Quantification of axon length (B) and branch numbers (C) 4 days after plating on PDL or 21 DIV cultures. n = 3 independent cultures, at least 20 neurons/culture. Student’s t-test. (D) Relative frequency plot of retraction distance categorized by DIV and plating surface. Distal axotomy results were compared. 4 DIV on 25 DIV vs 24 DIV on PDL: Student’s t-test. (E) Regeneration ratio categorized by DIV and plating surface. Distal axotomy results were compared. Fisher’s exact test. One-way ANOVA followed by Games-Howell post hoc test. Error bars represent s.e.m. Retraction distance was converted by log10. *p<0.05, **p<0.01 and ****p<0.0001.

https://doi.org/10.7554/eLife.26956.014
Endogenous rab11 becomes restricted to the somatodenritic domain with maturation.

(A) Immunofluorescence staining of rab11 in 4 and 24 DIV neurons. Neurons were co-stained with beta-3 tubulin or GFP for neuronal outline, and SMI312 or Ankyrin-G for axon marking. (B,C,D) Quantification of rab11 immunofluorescence intensity. Immunofluorescence intensities of the cell body, axon stem and proximal dendrite were measured, and ratios were calculated. Staining conditions were kept consistent and images were acquired under the same exposure. n = 3 independent cultures. ANOVA followed by Bonferroni’s post hoc test. Error bars represent S.E.M. ***p<0.001.

https://doi.org/10.7554/eLife.26956.015
Figure 7 with 1 supplement
Overexpressed rab11 mis-traffics into axons and is removed by predominantly retrograde transport.

(A,B) Axon stem/proximal dendrite ratio (A) and axon stem signal intensity (B) of total rab11 (both transfected and endogenous) in cytosolic GFP or rab11a WT overexpressed neurons. Transfected neurons were immunofluorescently labelled with rab11 antibody and quantified. n = 3 independent staining, Welch’s t-test. (C) Axon stem/proximal dendrite immunofluorescence intensity ratio of transfected neurons. Neurons were transfected with cytosolic GFP or fluorescence-tagged rab11a forms, and the fluorescent protein was probed by immunofluorescence staining. (D) Comparison of fluorescence signal between proximal and distal axon. Distal axon intensity was divided by axon stem intensity. (E) Representative kymographs of fluorescence-tagged rab11a forms in axon stems and proximal dendrites. (F) Quantification of vesicular movements in neurons overexpressing fluorescence-tagged rab11a forms. Images were acquired every second for 3 min, and kymographs were produced from a 30 μm region of the axon stem or proximal dendrite. Vesicle movement direction (F) and average vesicle velocity (retrograde and anterograde Figure 7—figure supplement 1A,B) were quantified. Error bars represent s.e.m. rab11 overexpression results were accumulated from at least three independent transfected cultures. Sample numbers are described in the figure. (B ~F) One-way ANOVA followed by Bonferroni’s post hoc test. Black asterisks: axon and dendrite comparison, blue asterisks: rab11 type comparison. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

https://doi.org/10.7554/eLife.26956.016
Figure 7—figure supplement 1
Quantification of vesicular movements in neurons overexpressing fluorescence-tagged rab11 constructs.

(A,B) Images were acquired every second for 3 min, and kymographs were produced from a 30 μm region of the axon stem or proximal dendrite. Average vesicle velocity was quantified. Error bars represent s.e.m. Sample numbers are described in the figure. Two-way ANOVA followed by Bonferroni’s post hoc test. Black asterisks: axon and dendrite comparison, blue asterisks: rab11 type comparison. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

https://doi.org/10.7554/eLife.26956.017
Rab11 overexpression increases intra-axonal rab11 and enhances regeneration.

(A) Accumulation of overexpressed rab11 WT at the retraction bulb. Note how rab11a accumulation can start earlier than GFP. (B,C) Retraction distance and retraction bulb formation time correlate (B), and the frequency plot of retraction distance takes a biphasic distribution (C) in rab11 transfected neurons. (D,E,F) Retraction distance (F,G) and regeneration ratio (H) of rab11 overexpressed neurons categorized by retraction group and form. F, G: One-way ANOVA followed by Dunnett’s post hoc test. H: Fisher’s exact test with Bonferroni correction. (G,H,I) Regeneration initiation time, regeneration length and growth cone area of rab11 overexpressed neurons categorized by rab11 type. One-way ANOVA followed by Dunnett’s post hoc test. Error bars represent s.e.m. Axotomy results were accumulated from at least three independent sessions. Retraction distance was converted by log10. Sample numbers are described in the figure. *p<0.05, **p<0.01 and ***p<0.001.

https://doi.org/10.7554/eLife.26956.018
Rab11 forced into axons by overexpression carries α5 integrin with it.

DIV 10 neurons were transfected with either rab11-GFP or control GFP, then fixed and immunolabelled on DIV16. (A) left shows that in control cells GFP enters dendrites and the axon identified by neurofascin staining, while α5 integrin is excluded. On the right, rab11-GFP is transfected, and mistrafficks into the proximal axon. The axon now contains plentiful α5 integrin. The distribution of α5 integrin between axons and dendrites is quantified in (B) and (C) as fluorescence intensity levels and axon/dendrite intensity ratio. Bar = 25 μm, ***=P < 001 by student's t test.

https://doi.org/10.7554/eLife.26956.020
Age-dependent decline in axonal regeneration of hESC-derived neurons is partially rescued by increasing intra-axonal rab11 levels.

(A) hESC-derived neurons show an age-related decrease in regeneration following laser-mediated axotomy. Overexpression of rab11-WT improves the percentage of regenerated axons in d45-55 neurons. n = 4 independent experiments per time point. (B,C) Axonal levels of rab11-GFP decline in mature hESC-derived neurons as compared to dendritic levels. Axons were identified using SMI-312 immunolabeling and intensity of GFP immunolabeling was measured. n > 3 independent stainings. (D) Regeneration length measured at 2 hr after initiation of regeneration is increased in rab11 overexpressing hESC-derived neurons. n = 16 for both for GFP and rab11 collected from four independent experiments. (E) Retraction distance distribution is not affected by rab11 overexpression in hESC-derived neurons. n = 22 and 23 for GFP and rab11 +GFP respectively, collected from four independent experiments. In all graphs, error bars represent S.E.M. *p<0.05. Unpaired t-test, Welch’s correction.

https://doi.org/10.7554/eLife.26956.021

Videos

Video 1
shows successful regeneration of an axon from an immature neuron, cut at 4 DIV.
https://doi.org/10.7554/eLife.26956.010
Video 2
shows failure of regeneration of an axon from a mature neuron, cut at 30 DIV.
https://doi.org/10.7554/eLife.26956.011
Video 3
shows an example of a neuron transfected with rab11 DN-RFP and GFP, showing that rab11 appears in the stump soon after axotomy and is present in the growth cone throughout regeneration and growth.

Table

https://doi.org/10.7554/eLife.26956.019

Tables

Table 1
Definition of the measures of regeneration
https://doi.org/10.7554/eLife.26956.009
Regeneration factorRegeneration factors were measured as below when a neuron was categorized as regeneration
Retraction distanceThe length of axon that was lost between the location of axotomy and the initial retraction bulb
Retraction bulb formation timeThe time it took to form the initial retraction bulb, measured from the time point of axotomy
Regeneration ratioThe number of neurons that regenerated, over the total neurons which formed a retraction bulb
Regeneration initiation timeThe time between the retraction bulb formation and the start of a steady extension lasting more than 1 hr and leading to regeneration
Regeneration lengthThe length of axon that extended within 2 hr after regeneration initiation time
Growth cone areaThe average of the extending tip area measured at 20, 40, and 60 min after regeneration initiation

Data availability

The following data sets were generated
    1. Lam B
    2. Koseki H
    (2016) Gene expression profiling of cultured embryonic rat cortical neurons
    Publicly available at NCBI Gene Expression Omnibus (accession no: GSE92856).

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