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Expanded Genetic Screening in Caenorhabditis elegans Identifies New Regulators and an Inhibitory Role for NAD + in Axon Regeneration

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Expanded Genetic Screening in Caenorhabditis elegans Identifies New Regulators and an Inhibitory Role for NAD + in Axon Regeneration

Kyung Won Kim et al. Elife.

Abstract

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.

Keywords: C. elegans; Kelch-domain protein; NMNAT; axon reconnection/fusion; membrane contact site (MCS); membrane transporter; neuroscience; phospholipid metabolic enzyme.

Conflict of interest statement

KK, NT, CP, MA, SP, MZ, NK, SC, ZW, AC, YJ No competing interests declared

Figures

Figure 1.
Figure 1.. Overview and results of expanded axon regrowth screen.
(A) Pie chart showing fraction of genes screened displaying significantly reduced or increased regrowth at 24 hr. Right: representative inverted grayscale images of PLM 24 hr post-axotomy in wild type (black box), and mutants with reduced (red boxes) or increased regrowth (blue boxes). Orange arrowhead, site of axotomy. (B) Distribution of reduced/increased regrowth mutants among nine functional or structural gene classes, shown as percentage of genes in each class. See Figure 1—source data 1 for lists of genes in each class.
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. The overview and results of axon regrowth screen combined with our previous study.
(A) Pie chart showing combined genes from both screens (Chen et al., 2011)(this work) displaying significantly reduced or increased regrowth at 24 hr. (B) Distribution of reduced/increased regrowth mutants among nine functional or structural gene classes, shown as percentage of genes in each class.
Figure 1—figure supplement 2.
Figure 1—figure supplement 2.. Mutants affecting multiple biological processes required for normal axon regrowth.
(A–E) Normalized PLM axon regrowth 24 hr post-axotomy in mutants affecting: (A) components of basement membrane (BM) and ADAMTS, (B) components of trafficking and Annexins, (C) microtubule acetyltransferases, (D) microtubule isotypes, and (E) actin filament regulators. Statistics, Student’s t-test with same day controls. (F) Normalized PLM axon regrowth 24 hr post-axotomy in mutants affecting mfsd-6 mutants. Pan-neuronal expression of MFSD-6 [Pn::mfsd-6] in mfsd-6(tm3356) animals rescues axon regeneration. Statistics, one-way ANOVA followed by Tukey’s multiple comparison tests. Left: MFSD-6 protein structure. Loss-of-function alleles are indicated above (tm3356, deletion; ju833, missense point mutation). Blue rectangles represent transmembrane domains. Data are shown as mean ±SEM. n, number of animals shown within columns. ns, not significant. *p<0.05; **p<0.01; ***p<0.001. In Figure 1—figure supplement 2D, tba-1, tbb-4, and tbb-6 (Chen et al., 2011) are included for comparison.
Figure 2.
Figure 2.. NMNAT/NMAT-2 inhibits PLM axon regrowth via its catalytic domain.
(A) Overview of NAD+ salvage biosynthesis pathway. Top, key enzymes; Bottom, C. elegans orthologs (Shaye and Greenwald, 2011). (B) Top, nmat-2 gene structure and mutant alleles. NMAT-2 contains an NMNAT domain. nmat-2(ju1514) point mutation and nmat-2(ju1512) deletion alleles were generated using CRISPR-Cas9 genome editing. Bottom, sequence alignment of the active site of NMNAT domain of C. elegans NMAT-2 (accession number: NP_492480.1; amino acids 4–14) with human NMNAT1–3 (NP_073624.2, NP_055854, NP_001307441) and C. elegans NMAT-1 (NP_510010.2). Sequences were analyzed using Clustal Omega. (C) Normalized regrowth 24 hr post-axotomy in mutants lacking genes encoding enzymes in the NAD+ biosynthesis pathway. Statistics, Student’s t-test with same day controls. For the statistical test of transgene analysis, one-way ANOVA followed by Tukey’s multiple comparison test. (D) PLM axon regrowth 24 hr post-axotomy in transgenic animals expressing nmat-2(+) driven by tissue-specific promoters for mechanosensory neurons (Pmec-4), epidermis (Pcol-12) or intestine (Pmtl-2) in a nmat-2(ju1512) background. One-way ANOVA followed by Tukey’s multiple comparison test. Data are shown as mean ± SEM. n, number of animals shown within columns. ns, not significant; *p<0.05; **p<0.01; ***p<0.001.
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. nmat-1 show no defect in PLM axon regrowth even when its germline is defective.
Normalized PLM axon regrowth 24 hr post-axotomy in mutants affecting nmat-1 mutants when treated with 50 µg/ml FUdR. Data are shown as mean ± SEM. n, number of animals shown within columns. ns, not significant.
Figure 3.
Figure 3.. Select ER-PM membrane contact site proteins are required for axon regeneration and are sensitive to injury.
(A) Junctophilin-1 protein structure. From top to bottom: C. elegans JPH-1 (NP_492193.2), its Drosophila ortholog JP (NP_523525.2), and human ortholog JPH1 (NP_065698.1). Junctophilins contain N-terminal MORN (Membrane Occupation and Recognition Nexus) repeats (green) and a C-terminal transmembrane domain (blue). C. elegans deletion allele is indicated above (ok2823). (B) Percentage of axons that exhibit fusion between the regrowing axon and distal fragment 24 hr post-axotomy. Upper image shows a regrowing axon that has not fused with the distal fragment in a wild-type animal. Lower image shows fusion between the regrowing axon and the distal fragment in a jph-1(ok2823) animal. Fisher’s exact test. *p<0.05; **p<0.01. (C) Normalized regrowth 24 hr post-axotomy in mutants lacking selected genes encoding ER-PM MCS proteins. Data are shown as mean ± SEM. n, number of animals shown within columns. Student’s t-test with same day controls. ns, not significant; *p<0.05; **p<0.01. (D) E-Syt protein structure. From top to bottom: C. elegans ESYT-2 and its human orthologs E-Syt2, E-Syt3, and E-Syt1 (NP_065779.1, NP_114119.2, NP_056107.1, respectively). Amino acid length is indicated to the right of each protein. E-Syt proteins contain N-terminal hydrophobic regions (blue), SMP (Synaptotagmin-like Mitochondrial and lipid-binding Protein) domains (yellow), and C-terminal C2 domains (red). C. elegans deletion allele is indicated above (ju1409). (E) Images of the PLM cell body and surrounding neurites. Left, GFP::PISY-1 ER marker; Middle, mKate2::ESYT-2 driven by the mec-4 promoter; Right, Image overlays. Images show single slices taken at 1 μm intervals. (F) Representative inverted grayscale images of GFP::ESYT-2 in the axon of the PLM neuron before and immediately after axotomy (upper and lower panels, respectively). Site of laser axotomy indicated by asterisk; puncta indicated by arrowheads.
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. esyt-2 is widely expressed in the nervous system.
Confocal images of Pesyt-2::GFP transcriptional reporter showing widespread expression in the nervous system of L4 stage animals, including in head ganglia, ventral nerve cord, and tail ganglia. Maximum intensity projection.
Figure 4.
Figure 4.. PLM axon regeneration involves membrane lipid biosynthesis pathway.
(A) Normalized PLM axon regrowth 24 hr post-axotomy in mutants affecting neutral lipolysis. (B) Normalized PLM axon regrowth 24 hr post-axotomy in mutants affecting acid lipolysis. (C) Overview of C.elegans Kennedy pathway for de novo biosynthesis of PE and PC, the major phospholipids in the PM. (D) Normalized PLM axon regrowth 24 hr post-axotomy in mutants lacking select genes encoding enzymes in the Kennedy pathway. Data are shown as mean ±SEM. n, number of animals shown within columns. Student’s t-test with same day controls. ns, not significant; *p<0.05.
Figure 5.
Figure 5.. The Kelch-domain protein IVNS-1 inhibits axon regeneration.
(A) ivns-1 gene structure. Left: Loss-of-function alleles are indicated below (gk252 and ok3171). Right: Alignment of the C. elegans IVNS-1 (NP_510109.1) with its human ortholog IVNS1ABP (NP_006460.1) and mouse ortholog ND1-L (NP_473443.2). Number indicates percentage identity of protein sequences. Sequences were analyzed using Clustal Omega. (B) Normalized PLM axon regrowth 24 hr post-axotomy in mutants of Kelch-domain proteins. Data are shown as mean ±SEM. n, number of animals shown within columns. Student’s t-test with same day controls. ns, not significant; *p<0.05; **p<0.01; ***p<0.001. Right: representative inverted grayscale images of PLM 24 hr post-axotomy. Scale bar, 25 μm. (C) Normalized PLM axon regrowth 6 hr post-axotomy. Data are shown as mean ±SEM. One-way ANOVA followed by Tukey’s multiple comparison test. n, number of animals shown within columns. **p<0.01. (D) Percentage of axons with growth cones (GCs) 6 hr post-axotomy. n, Number of animals shown below columns. Fisher’s exact test. ns, not significant.

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