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Regulation of axon regeneration by the RNA repair and splicing pathway

Nature Neuroscience volume 18, pages 817–825 (2015)Cite this article

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Abstract

Mechanisms governing a neuron's regenerative ability are important but not well understood. We identify Rtca (RNA 3′-terminal phosphate cyclase) as an inhibitor of axon regeneration. Removal of Rtca cell-autonomously enhanced axon regrowth in the Drosophila CNS, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair and splicing pathway important for stress-induced mRNA splicing, including that of Xbp1, a cellular stress sensor. Drosophila Rtca and Archease had opposing effects on Xbp1 splicing, and deficiency of Archease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. Our study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma.

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Figure 1: Drosophila Rtca loss of function enhances axon regeneration in the CNS and PNS.
Figure 2: Drosophila Rtca overexpression reduces axon regeneration in the PNS.
Figure 3: Drosophila Rtca overexpression reduces motor axon regeneration.
Figure 4: The expression pattern of Rtca in Drosophila.
Figure 5: The interaction of Drosophila Rtca with known regeneration regulators.
Figure 6: The Drosophila Rtca pathway in regulating axon regeneration.
Figure 7: Rtca is an inhibitory factor for axon growth in vitro and RGC regeneration in vivo in rodents.

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Acknowledgements

We thank S. Younger, S.K. Ultanir, S. Yadav, D. Wang, S. Barbel, M. Tynan-La Fontaine and J. Sacramento for technical assistance; J. Martinez for advice on the Rtca pathway; I. Hariharan (University of California, Berkeley), T. Xu (Yale) and H.D. Ryoo (New York University) for fly lines; the Bloomington Stock Center, Kyoto Drosophila Genetic Resource Center (DGRC), Vienna Drosophila Resource Center (VDRC) and Exelixis for fly stocks; DGRC for plasmids; UCSF ES Cell Targeting Core for generating the knockout mouse; and members of the Jan laboratory for discussions. Y.S. is a recipient of a National Institute of Neurological Disorders and Stroke (NINDS) Pathway to Independence Award. This work was supported by a NINDS K99/R00 award (1K99NS088211-01) to Y.S., a National Science Foundation Graduate Research Fellowship (1144247) to S.M., a UC Irvine-Roman Reed Spinal Cord Injury grant (P0045665) to Y.N.J., a NIH grant (2R37NS040929) to Y.N.J., a NIH grant (EY010688-19) to D.S. and a Research to Prevent Blindness grant to D.S. L.Y.J. and Y.N.J. are investigators of Howard Hughes Medical Institute.

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Author notes

  1. Jim Berg

    Present address: Present address: Allen Institute for Brain Science, Seattle, Washington, USA.,

  2. Chun Han

    Present address: Present address: Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, USA.,

Authors and Affiliations

  1. Howard Hughes Medical Institute, University of California, San Francisco, California, USA

    Yuanquan Song, Jim Berg, Xi Huang, Tong Cheng, Xin Xiong, Shan Meltzer, Chun Han, Lily Yeh Jan & Yuh Nung Jan

  2. Department of Physiology, University of California, San Francisco, California, USA

    Yuanquan Song, Jim Berg, Xi Huang, Tong Cheng, Shan Meltzer, Chun Han, Lily Yeh Jan & Yuh Nung Jan

  3. Department of Ophthalmology, University of California, San Francisco, San Francisco, California, USA

    David Sretavan & Trong-Tuong Nguyen

  4. Brain and Spinal Injury Center, University of California, San Francisco, California, USA

    Ernesto A Salegio, Jacqueline C Bresnahan & Michael S Beattie

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Contributions

Y.S. carried out most of the experiments and performed the data analysis. D.S. performed the optic nerve crush experiment; E.A.S. performed β-galactosidase staining; J.B. performed Rtca expression analysis; X.H. contributed to the neuronal culture experiment; T.C. contributed to the RGC axon regeneration analysis; X.X. performed the motor axon regeneration assay; S.M. contributed to the stress assay; C.H. made the UAS-Rtca fly strain; T.-T.N. contributed to the optic nerve crush experiment; J.C.B. and M.S.B. contributed to the mouse axon regeneration experiment; and Y.S., L.Y.J. and Y.N.J. together conceived the research and wrote the manuscript.

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Correspondence to Yuh Nung Jan.

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Integrated supplementary information

Supplementary Figure 1 A schematic diagram depicting the axotomy protocol of da neuron axons in the PNS and CNS.

The axons in the PNS and CNS are labeled. Dashed line indicates VNC boundary. The axon terminal within the VNC of one representative class IV da neuron is shown in green.

Supplementary Figure 2 Drosophila Rtca loss of function, as in RtcaNP5057, which alters transcript expression, enhances axon regeneration in the VNC without affecting axon terminal patterning.

(a) P element insertion in dRtcaNP5057 disrupts mRNA splicing and reduces transcript level. Arrows indicate the primer locations. Two pairs of primers were used and are color coded in green and turquoise. The PCR products are color coded in the same way according to the primer position. Blots Gels were cropped and full-length images are presented in Supplementary Figure 13. (b) The overall patterning of class IV da neuron axons in the VNC (labeled by ppk-CD4tdGFP) is not affected after dRtca LOF. (c) Single clone analysis shows that dRtca LOF does not affect gross patterning of the nerve terminals. The axon terminal of a single class IV da neuron is shown in whole animal dRtca LOF mutants (upper panels), or in animals with dRtca RNAi in all class IV da neurons (lower panels). Scale bar = 20 μm.

Supplementary Figure 3 Quantification of sensory axon regeneration.

(a) For axon regeneration in the VNC, “Terminal branching” counts the number of axons that regenerate, reach the commissure bundle and appear to form elaborate branches, shown in red. “Commissure regrowth” counts the number of axons that have regenerated to connect the boundaries of commissure segments, longitudinally or laterally, shown in blue. (b) For axon regeneration in the PNS, “Regeneration index” is calculated as an increase of “axon length”/“distance between the cell body and the axon converging point (DCAC)”.

Supplementary Figure 4 Analyses of the CNS regeneration phenotype in Drosophila Rtca and Pten pathway mutants, as well as PNS axon regeneration after Rac1DN overexpression.

(a, b) Double mutants of dRtca and Pten do not further promote axon regeneration in the VNC. (a) Regeneration percentage is not significantly different between dRtca single mutants and dRtca Pten double mutants (N = 46, 16, 12 lesioned segments; ***P < 0.001, Fisher’s exact test, P < 0.0001, P = 0.0005, P = 0.1746). (b) Terminal branching and Commissure regrowth are comparable between dRtca single mutants and dRtca Pten double mutants (N = 28, 8, 6 larvae; *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Holm-Sidak's multiple comparisons test and Tukey's multiple comparisons test, respectively). (c, d) The regeneration phenotype in dRtca LOF mutants appears to be comparable if not stronger than Pten mutants (PtenMGH6, a hypomorphic allele) or Akt overexpression (ppk-Gal4>Akt) (N = 46, 17, 13, 16, 14 lesioned segments; **P < 0.01, ***P < 0.001, Fisher’s exact test in c, P = 0.0027, P = 0.0002, P = 0.0001, P < 0.0001; N = 29, 9, 7, 8, 7 larvae; *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Holm-Sidak's multiple comparisons test and Dunn’s multiple comparisons test, respectively, in d). (e–g) Rac1DN overexpression in class IV da neurons does not affect axon regeneration in the periphery (N = 27, 20 neurons; P > 0.05, Fisher’s exact test in e, P = 0.7365, and two-tailed unpaired Student’s t-test in f and g, P = 0.3533, P = 0.6708). Data are expressed as mean ± s.e.m. in bar plots or as median with minimum and maximum values in box-and-whisker plots, and box limits represent interquartile range.

Supplementary Figure 5 The proposed model of the Rtca-Archease-Xbp1 pathway in the regulation of regeneration.

Axon damage results in some type of cellular stress and the repair/splicing of mRNA substrates, such as Xbp1. The 3′-phosphate of the spliced RNA is converted into 2′,3′-cyclic phosphate by Rtca, whereas the RNA ligase Rtcb reverses this reaction and re-ligates the processed RNA, with the help of its catalyst Archease. Thus Rtca and Archease may have opposing functions on RNA repair/splicing and on regeneration.

Supplementary Figure 6 Drosophila ArcheasePBc01013 leads to altered mRNA splicing and reduced transcript expression, and is thus a loss of function allele.

Arrows indicate the primer locations. Two pairs of primers were used and are color coded in green and turquoise. The PCR products are color coded in the same way according to the primer position. Blots Gels were cropped and full-length images are presented in Supplementary Figure 14.

Supplementary Figure 7 Xbp1s overexpression increases class III da neuron axon regeneration in the periphery.

Dashed circle marks the injury site and arrowheads show the regenerating axon (N = 19, 24 neurons; *P < 0.05, Fisher’s exact test, P = 0.0261 and two-tailed unpaired Student’s t-test, P = 0.0384). Scale bar = 20 μm. Data are expressed as median with minimum and maximum values in box-and-whisker plots, and box limits represent interquartile range.

Supplementary Figure 8 The Rtca mutant mouse.

(a) A knockout-first line with a LacZ cassette inserted into the Rtca locus was used to generate the Rtca mutant mouse. The resulting mouse RtcaLacZ_loxP (RtcaIns/Ins) has a lacZ cassette inserted in-between the 3rd and 4th exons of Rtca, meanwhile, the 4th exon is flanked by loxP sites. (b) The lacZ insertion disrupts Rtca splicing and dramatically reduces transcript level, and thus results in a hypomorphic allele. Arrows indicate the primer locations. Two pairs of primers were used and are color coded in green and turquoise. The PCR products are color coded in the same way according to the primer position. Blots Gels were cropped and full-length images are presented in Supplementary Figure 15. Quantification of Rtca mRNA shows about 81% reduction (N = 4 experiments; the expression level of each data point was normalized to the average value of WT; **P < 0.01, two-tailed unpaired Student’s t-test, P = 0.0035). (c) Western blot analysis using retina tissue was performed. Anti-Rtca HPA027982 recognizes amino acids 111-193. The antibody cleanly labels the Rtca protein in WT, which is ∼39 KD, and shows a dramatic reduction of the protein after the insertion site in RtcaIns/Ins mutants, confirming the reduction of the Rtca full-length protein. The green line indicates the antigen region. Quantification of Rtca protein shows about 82% reduction (N = 3 experiments; the expression level of each data point was normalized to the average value of WT; *P < 0.05, two-tailed unpaired Student’s t-test, P = 0.0252). Data are expressed as mean ± s.e.m.

Supplementary Figure 9 Rtca LOF during development does not affect adult RGC number or survival after injury.

(a–c) Three weeks after optic nerve crush of sibling controls (Rtca+/+ and RtcaIns/+) and RtcaIns/Ins mutant mice, retina sections from both the control eye (uninjured nerve) and injured eye (crushed nerve) were immunostained for Tuj1 (βIII tubulin) to label RGC and DAPI to label nuclei. Confocal imaging of retina sections from mutant animals, normalized to siblings, shows no differences in basal RGC number in the contralateral uninjured retinas in (a, N = 3 siblings and 3 RtcaIns/Ins mice) or in RGC survival three weeks after optic nerve crush (b, N = 4 siblings and 5 RtcaIns/Ins mice; P > 0.05, two-tailed unpaired Student’s t-test, P = 0.6776, P = 0.9249). Representative confocal images of the retina sections immunostained for Tuj1 and DAPI are shown in (c). Scale bar = 20 μm. Data are expressed as mean ± s.e.m.

Supplementary Figure 10 Rtca is expressed in retinal ganglion cells.

(a–f) Positive LacZ staining was observed in the RGC layer of RtcaIns/+ mice (a–c, arrow) but not in WT littermates (d–f). (g) β-Galactosidase staining in RtcaIns/+ mice shows distinct expression of the LacZ reporter within NeuN+ neurons in the retinal ganglion cell layer of the retina, which is absent in WT littermates, indicating that Rtca is expressed by RGCs. Scale bar = 20 μm.

Supplementary Figure 11 Rtca reduction enhances RGC axon regeneration after optic nerve crush.

(a–d) Three weeks after optic nerve crush in 8–12 weeks old adult siblings (Rtca+/+ and RtcaIns/+), and RtcaIns/Ins mutant mice, regenerating fibers were labeled by anti-NF200 immunostaining. (a, b) Tissue sections through the optic nerve of a RtcaIns/Ins mutant mice show regenerating axons up to 300 μm distal to the lesion site, whereas no obvious axon regrowth beyond the crush site is seen in similar sections from a sibling control (RtcaIns/+, asterisk marks the crush site). Boxed regions are shown at higher magnification. Curving, turning and looping of axons were observed, indicative of new axon growth (arrowheads). (c) The furthest distance that axons travelled beyond the injury site is about 3.5 times longer in the mutants compared to siblings. (d) Regenerating fibers were counted at specified distances from the lesion site. More fibers regenerate in RtcaIns/Ins mice compared to sibling controls (N = 7 siblings and 4 RtcaIns/Ins mice; ***P < 0.001, two-tailed unpaired Student’s t-test in c; **P < 0.01, two-way ANOVA in d, P = 0.0012). (e) The optic nerve crush site is marked by the presence of ED1 staining, which labels the infiltrating macrophages. Whereas axons (labeled by NF200) rarely grow through the ED1+ region in the sibling controls (RtcaIns/+), a large number of axons are seen hundreds of microns beyond the ED1+ region in RtcaIns/Ins mutants. Asterisks mark the crush site. Scale bar = 100 μm. Data are expressed as mean ± s.e.m. in bar plots or as median with minimum and maximum values in box-and-whisker plots, and box limits represent interquartile range.

Supplementary Figure 12 Full-length DNA agarose gel for the analysis of Xbp1 splicing.

Full length DNA agarose gel (corresponding to Figure 6g).

Supplementary Figure 13 Full-length DNA agarose gel for the analysis of Drosohila Rtca expression.

Full length DNA agarose gel (corresponding to Supplementary Figure 2a).

Supplementary Figure 14 Full-length DNA agarose gel for the analysis of Drosophila Archease expression.

Full length DNA agarose gel (corresponding to Supplementary Figure 6).

Supplementary Figure 15 Full-length DNA agarose gel for the analysis of mouse Rtca expression.

Full length DNA agarose gel (corresponding to Supplementary Figure 8b).

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Song, Y., Sretavan, D., Salegio, E. et al. Regulation of axon regeneration by the RNA repair and splicing pathway. Nat Neurosci 18, 817–825 (2015). https://doi.org/10.1038/nn.4019

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