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. 2014 Feb 5;81(3):561-73.
doi: 10.1016/j.neuron.2013.11.019. Epub 2014 Jan 16.

Insulin/IGF1 signaling inhibits age-dependent axon regeneration

Alexandra B Byrne  1 , Trent Walradt  1 , Kathryn E Gardner  2 , Austin Hubbert  1 , Valerie Reinke  2 , Marc Hammarlund  3
Affiliations

Insulin/IGF1 signaling inhibits age-dependent axon regeneration

Alexandra B Byrne et al. Neuron. .

Abstract

The ability of injured axons to regenerate declines with age, yet the mechanisms that regulate axon regeneration in response to age are not known. Here we show that axon regeneration in aging C. elegans motor neurons is inhibited by the conserved insulin/IGF1 receptor DAF-2. DAF-2's function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16/FOXO. DAF-16 regulates regeneration independently of lifespan, indicating that neuronal aging is an intrinsic, neuron-specific, and genetically regulated process. In addition, we found that DAF-18/PTEN inhibits regeneration independently of age and FOXO signaling via the TOR pathway. Finally, DLK-1, a conserved regulator of regeneration, is downregulated by insulin/IGF1 signaling, bound by DAF-16 in neurons, and required for both DAF-16- and DAF-18-mediated regeneration. Together, our data establish that insulin signaling specifically inhibits regeneration in aging adult neurons and that this mechanism is independent of PTEN and TOR.

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Figures

Figure 1
Figure 1. Regeneration declines in aged animals
(A) GABA commissures are severed with a pulsed laser and scored for regeneration after 24 hours in young adult (1-day adult, L4 + 1 day at 25°C) and aged adult (5-day adult, L4 + 5 days at 25°C) animals. (B) GABA commissures 24 hrs after being severed in 1-day adult wild type and (C) 5-day adult wild type animals. (D) Comparison of axon regeneration frequency in young and aged wild type animals. Regeneration in 5-day adult animals is significantly reduced relative to 1-day adult animals (p<0.01, Fisher exact test). (E) Regenerating axons were binned into four categories (depicted in panel A) based on how far they had extended. Regeneration categories: ML-, did not extend past the midline; ML, extended to the midline; ML+, extended ¾ of the distance between the ventral and dorsal cords; Full, full regeneration to the dorsal cord. All animals contain Punc-47∷GFP, which drives GFP expression specifically in the GABA motor neurons. Error bars represent 95% confidence intervals.
Figure 2
Figure 2. daf-2 inhibits the number of regenerating axons and limits their extension during early senescence
(A-C) Regenerating axons were binned into four categories based on how far they had extended. (A) GABA commissures 24 hours after being severed in 5-day adult wild type and 5-day adult daf-2(e1370) animals. (B) Quantification of regeneration in 5-day adult wild type and daf-2(e1370) animals 24 hours after axotomy. Regeneration in 5-day adult daf-2(-) animals is significantly increased relative to 5-day adult wild type animals (p<0.01, Fisher exact test). (C) Regenerating axons were binned into categories according to how far they extended towards the dorsal nerve cord (categories described in Figure Legend 1). More severed axons in aged adult daf-2(-) animals extend significantly (ML+ and Full) compared to severed axons in aged adult wild type animals (p<0.01, Fisher exact test). (D) Axon regeneration frequencies in wild type and daf-2(e1370) animals of increasing age. (E) Lengths of commissural fragments 24 hours after axotomy in 5-day adults. Width represents commissure length prior to axotomy. For precision, axons were severed on the ventral side of the seam cells which are located at the midline. All animals express the Punc-47∷GFP reporter. Error bars represent 95% confidence intervals in B and D, and standard error in E.
Figure 3
Figure 3. daf-16 mediates age-dependent regeneration downstream of daf-2
(A) Comparison of axon regeneration frequencies in aged adult insulin pathway mutants, (B) binned into categories described in Figure 1. (C) 5-day adult and 10-day adult time points are indicated on the lifespans of wild type (blue), daf-2(e1370) (purple); daf-16(mu86) (green), and daf-2(e1370); daf-16(mu86) (orange) animals. (D) A simplified representation of the insulin/IGF1 signaling pathway. daf-16 regulates regeneration downstream of daf-2 in aged adults either (i) directly, or (ii) indirectly. Error bars represent 95% confidence intervals.
Figure 4
Figure 4. daf-16 mediates age-dependent regeneration independently of lifespan
(A-E) We assessed lifespan and regeneration in (A) aged daf-2(e1370); daf-16(mu86) worms expressing daf-16 in the intestine (Pges-1∷daf-16) and in the nervous system (Prgef-1∷daf-16). (B) Lifespans of daf-2(e1370); daf-16(mu86) (charcoal), with intestinal (blue) or neuronal (red) daf-16 expression. Duplicates represent separate extrachromosomal array-expressing lines. (C) Regeneration of aged daf-2(e1370); daf-16(mu86) worms expressing daf-16 in the nervous system (Prgef-1∷daf-16) and in the intestine (Pspl-1∷daf-16). (D) Regenerating axons were binned into categories described in Figure 1. Error bars represent 95% confidence intervals. See also Figure S1.
Figure 5
Figure 5. DAF-18/PTEN does not regulate regeneration via DAF-16/FOXO in aged animals
(A) Axon regeneration frequency of young and (B) aged animals. Error bars represent 95% confidence intervals. *, p<0.05; **, p<0.01, ***, p<0.001 relative to equivalently aged wild type, Fisher’s exact test. (C) Regenerating axons of young adult and (D) aged adult animals were binned into categories described in Figure 1. (E) Lifespan analysis of various daf-18 mutants. (F) Quantification of axon regeneration in aged animals of the indicated genotypes. (G) Axon regeneration frequency of aged wild type, daf-18(mg198), and daf-18(mg198) animals with extrachromasomal Punc-47∷daf-18cDNA, which is expressed in GABA neurons. *, p<0.05, relative to daf-18(mg198), Fisher exact test. (H) Model of interactions between components of the age-related regeneration response. In contrast to the mechanisms of lifespan determination, daf-18/PTEN regulates regeneration independently of daf-16/FOXO and daf-2. In all panels, daf-2(-), daf-16(-), and daf-18(-) represent daf-2(e1370), daf-16(mu86), and daf-18(mg198), respectively.
Figure 6
Figure 6. DAF-18/PTEN, but not insulin, regulates regeneration via TOR signaling
(A) Representative images of axon regeneration wild type and daf-18(mg198) animals aged on DMSO or rapamycin plates. (B) Axon regeneration frequencies of aged adult animals placed on rapamycin or DMSO as indicated. Error bars represent 95% confidence intervals. (*, p<0.05, relative to daf-18(mg198), Fisher exact test).
Figure 7
Figure 7. Insulin signaling regulates expression of DLK-1/DLK
(A) Axon regeneration frequency of aged animals of the indicated genotypes. Due to space restrictions, daf-2(-), daf-16(-), and daf-18(-) represent daf-2(e1370), daf-16(mu86), and daf-18(mg198) respectively. Error bars represent 95% confidence intervals. (B) Quantitative reverse transcription PCR analysis of DLK-1 pathway genes in aged daf-2(e1370) animals. All values were normalized to ama-1 expression levels and to relative mRNA expression in aged wild type animals. Error bars represent 95% confidence intervals. (C) daf-2(e1370); daf-16(mu86) animals expressing GFP-tagged DAF-16A fused to either an intestinal (spl-1) or a (D) neuronal (rgef-1) promoter. GFP∷DAF-16A expression was restricted to the intestinal nuclei (arrow, inset) when fused to the intestinal promoter and was restricted to neuronal nuclei (arrowhead, inset) when fused to the neuronal promoter. Background intestinal autofluorescence was observed in both strains. Scale bars represent 10um. (E) Tissue specific DAF-16A binding profiles of strains presented in C and D. Profiles span the dlk-1 locus (bordered by dashed lines) on chromosome 1 (LG 1). (F) Magnification of DAF-16A binding profiles at the dlk-1 locus. In neurons, two regions in the dlk-1 promoter/locus were significantly enriched for DAF-16A bound sequences (pink asterisks). In the intestine, three regions in the dlk-1 promoter/locus were bound by DAF-16A (green asterisks). See also Figure S2.
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References

    1. Antebi A. Genetics of aging in Caenorhabditis elegans. PLoS genetics. 2007;3:1565–1571. - PMC - PubMed
    1. Apfeld J, Kenyon C. Regulation of lifespan by sensory perception in Caenorhabditis elegans. Nature. 1999;402:804–809. - PubMed
    1. Araya CL, Kawli T, Kundaje A, Cayting P, Jiang L, Wu B, Niu W, Boyle A, Xie D, Ma M, Mace D, Gevirtzman L, Murray J, Reinke V, Waterston RH, Snyder M. A spatiotemporally-resolved cis-regulatory map of the C. elegans genome. to be re-submitted to Nature.
    1. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37:W202–208. - PMC - PubMed
    1. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. - PMC - PubMed

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