Paola Nix

Axon regeneration requires a conserved MAP kinase pathway.

Regeneration of injured neurons can restore function, but most neurons regenerate poorly or not at all. The failure to regenerate in some cases is due to a lack of activation of cell-intrinsic regeneration pathways. These pathways might be targeted for the development of therapies that can restore neuron function after injury or disease. Here, we show that the DLK-1 mitogen-activated protein (MAP) kinase pathway is essential for regeneration in Caenorhabditis elegans motor neurons. Loss of this pathway eliminates regeneration, whereas activating it improves regeneration. Further, these proteins also regulate the later step of growth cone migration. We conclude that after axon injury, activation of this MAP kinase cascade is required to switch the mature neuron from an aplastic state to a state capable of growth.

Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways

Signaling pathways essential for axon regeneration, but not for neuron development or function, are particularly well suited targets for therapeutic intervention. We find that the parallel PMK-3(p38) and KGB-1(JNK) MAPK pathways must be coordinately activated to promote axon regeneration. Axon regeneration fails if the activity of either pathway is absent. These two MAPKs are coregulated by the E3 ubiquitin ligase RPM-1(Phr1) via targeted degradation of the MAPKKKs DLK-1 and MLK-1 and by the MAPK phosphatase VHP-1(MKP7), which negatively regulates both PMK-3(p38) and KGB-1(JNK).

Targeted gene deletions in C. elegans using transposon excision.

We developed a method, MosDEL, to generate targeted knockouts of genes in Caenorhabditis elegans by injection. We generated a double-strand break by mobilizing a Mos1 transposon adjacent to the region to be deleted; the double-stranded break is repaired using injected DNA as a template. Repair can delete up to 25 kb of DNA and simultaneously insert a positive selection marker.

Axon Regeneration Genes Identified by RNAi Screening in C. elegans

Axons of the mammalian CNS lose the ability to regenerate soon after development due to both an inhibitory CNS environment and the loss of cell-intrinsic factors necessary for regeneration. The complex molecular events required for robust regeneration of mature neurons are not fully understood, particularly in vivo. To identify genes affecting axon regeneration in Caenorhabditis elegans, we performed both an RNAi-based screen for defective motor axon regeneration in unc-70/β-spectrin mutants and a candidate gene screen. From these screens, we identified at least 50 conserved genes with growth-promoting or growth-inhibiting functions. Through our analysis of mutants, we shed new light on certain aspects of regeneration, including the role of β-spectrin and membrane dynamics, the antagonistic activity of MAP kinase signaling pathways, and the role of stress in promoting axon regeneration. Many gene candidates had not previously been associated with axon regeneration and implicate new pathways of interest for therapeutic intervention.

The growth factor SVH-1 regulates axon regeneration in C. elegans via the JNK MAPK cascade

The ability of neurons to undergo regenerative growth after injury is governed by cell-intrinsic and cell-extrinsic regeneration pathways. These pathways represent potential targets for therapies to enhance regeneration. However, the signaling pathways that orchestrate axon regeneration are not well understood. In Caenorhabditis elegans, the Jun N-terminal kinase (JNK) and p38 MAP kinase (MAPK) pathways are important for axon regeneration. We found that the C. elegans SVH-1 growth factor and its receptor, SVH-2 tyrosine kinase, regulate axon regeneration. Loss of SVH-1–SVH-2 signaling resulted in a substantial defect in the ability of neurons to regenerate, whereas its activation improved regeneration. Furthermore, SVH-1–SVH-2 signaling was initiated extrinsically by a pair of sensory neurons and functioned upstream of the JNK-MAPK pathway. Thus, SVH-1–SVH-2 signaling via activation of the MAPK pathway acts to coordinate neuron regeneration response after axon injury.

Endocannabinoid-Goα signalling inhibits axon regeneration in Caenorhabditis elegans by antagonizing Gqα-PKC-JNK signalling

The ability of neurons to regenerate their axons after injury is determined by a balance between cellular pathways that promote and those that inhibit regeneration. In Caenorhabditis elegans, axon regeneration is positively regulated by the c-Jun N-terminal kinase mitogen activated protein kinase pathway, which is activated by growth factor-receptor tyrosine kinase signalling. Here we show that fatty acid amide hydrolase-1, an enzyme involved in the degradation of the endocannabinoid anandamide (arachidonoyl ethanolamide), regulates the axon regeneration response of γ-aminobutyric acid neurons after laser axotomy. Exogenous arachidonoyl ethanolamide inhibits axon regeneration via the Goα subunit GOA-1, which antagonizes the Gqα subunit EGL-30. We further demonstrate that protein kinase C functions downstream of Gqα and activates the MLK-1-MEK-1-KGB-1 c-Jun N-terminal kinase pathway by phosphorylating MLK-1. Our results show that arachidonoyl ethanolamide induction of a G protein signal transduction pathway has a role in the inhibition of post-development axon regeneration.

Axotomy-induced HIF-serotonin signalling axis promotes axon regeneration in C. elegans

The molecular mechanisms underlying the ability of axons to regenerate after injury remain poorly understood. Here we show that in Caenorhabditis elegans, axotomy induces ectopic expression of serotonin (5-HT) in axotomized non-serotonergic neurons via HIF-1, a hypoxia-inducible transcription factor, and that 5-HT subsequently promotes axon regeneration by autocrine signalling through the SER-7 5-HT receptor. Furthermore, we identify the rhgf-1 and rga-5 genes, encoding homologues of RhoGEF and RhoGAP, respectively, as regulators of axon regeneration. We demonstrate that one pathway initiated by SER-7 acts upstream of the C. elegans RhoA homolog RHO-1 in neuron regeneration, which functions via G12α and RHGF-1. In this pathway, RHO-1 inhibits diacylglycerol kinase, resulting in an increase in diacylglycerol. SER-7 also promotes axon regeneration by activating the cyclic AMP (cAMP) signalling pathway. Thus, HIF-1-mediated activation of 5-HT signalling promotes axon regeneration by activating both the RhoA and cAMP pathways.

Heterochronic Genes Turn Back the Clock in Old Neurons

A signaling pathway is implicated in the age-dependent decline of neuron regeneration. [Also see Report by Zou et al.] Although some neuron types regenerate better than others, all neurons lose the ability to regenerate with age. This intrinsic decline is the primary cause of regeneration failure even in permissive environments. This was shown in 1995 by comparing the regeneration ability of retinal neurons from different aged retinas growing into tectums of different ages (1). Embryonic retinal axons regrew into tectum of any age, including older tectum with an inhibitory glial environment, whereas postnatal day 2 or older retinal axons failed to regrow even into embryonic tectum. This indicated a “programmed” loss of axon regeneration ability with neuron age. Similarly, young hindbrain neurons transplanted into older spinal cords could regenerate axons into a normally inhibitory myelinated environment (2). Despite the clear therapeutic implications of these observations, the underlying molecular mechanisms controlling age-dependent regenerative capacity were unclear. On page 372 in this issue, Zou et al. (3) report that the highly conserved let-7–LIN-41 heterochronic signaling pathway is responsible for part of the age-related decline in axon regeneration in the worm Caenorhabditis elegans.