Target-selective vertebrate motor axon regeneration depends on interaction with glial cells at a peripheral nerve plexus.

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle, and then re-sort into target-specific bundles. Here, we examine this process at a plexus located at the base of the zebrafish pectoral fin, equivalent to tetrapod forelimbs. Using live cell imaging and sparse axon labeling, we find that regenerating motor axons from 3 nerves coalesce into the plexus. There, they intermingle and sort into distinct branches, and then navigate to their original muscle domains with high fidelity that restores functionality. We demonstrate that this regeneration process includes selective retraction of mistargeted axons, suggesting active correction mechanisms. Moreover, we find that Schwann cells are enriched and associate with axons at the plexus, and that Schwann cell ablation during regeneration causes profound axonal mistargeting. Our data provide the first real-time account of regenerating vertebrate motor axons navigating a nerve plexus and reveal a previously unappreciated role for Schwann cells to promote axon sorting at a plexus during regeneration.

A glia cell dependent mechanism at a peripheral nerve plexus critical for target-selective axon regeneration.

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle, and then re-sort into target-specific bundles. Here, we examine this process at a plexus located at the base of the zebrafish pectoral fin, equivalent to tetrapod forelimbs. Using live cell imaging and sparse axon labeling, we find that regenerating motor axons from three nerves coalesce into the plexus. There, they intermingle and sort into distinct branches, and then navigate to their original muscle domains with high fidelity that restores functionality. We demonstrate that this regeneration process includes selective retraction of mistargeted axons, suggesting active correction mechanisms. Moreover, we find that Schwann cells are enriched and associate with axons at the plexus, and that Schwann cell ablation during regeneration causes profound axonal mistargeting. Our data provide the first real time account of regenerating vertebrate motor axons navigating a nerve plexus and reveal a previously unappreciated role for Schwann cells to promote axon sorting at a plexus during regeneration.

Agrin/Lrp4 signal constrains MuSK-dependent neuromuscular synapse development in appendicular muscle.

The receptor tyrosine kinase MuSK, its co-receptor Lrp4 and the Agrin ligand constitute a signaling pathway that is crucial in axial muscle for neuromuscular synapse development, yet whether this pathway functions similarly in appendicular muscle is unclear. Here, using the larval zebrafish pectoral fin, equivalent to tetrapod forelimbs, we show that, similar to axial muscle, developing appendicular muscles form aneural acetylcholine receptor (AChR) clusters prior to innervation. As motor axons arrive, neural AChR clusters form, eventually leading to functional synapses in a MuSK-dependent manner. We find that loss of Agrin or Lrp4 function, which abolishes synaptic AChR clusters in axial muscle, results in enlarged presynaptic nerve regions and progressively expanding appendicular AChR clusters, mimicking the consequences of motoneuron ablation. Moreover, musk depletion in lrp4 mutants partially restores synaptic AChR patterning. Combined, our results provide compelling evidence that, in addition to the canonical pathway in which Agrin/Lrp4 stimulates MuSK activity, Agrin/Lrp4 signaling in appendicular muscle constrains MuSK-dependent neuromuscular synapse organization. Thus, we reveal a previously unappreciated role for Agrin/Lrp4 signaling, thereby highlighting distinct differences between axial and appendicular synapse development.

Vagus Topographic Map: Wandering through a gRAdient.

Topographic map development requires precise coordination between navigating axons and their targets in a dynamic environment. In this issue of Developmental Cell, Isabella et al. describe a mechanism in which a changing gradient of the morphogen retinoic acid regulates the expression of guidance factors to shape topographic axon targeting.

DLK Activation Synergizes with Mitochondrial Dysfunction to Downregulate Axon Survival Factors and Promote SARM1-Dependent Axon Degeneration.

Axon degeneration is a prominent component of many neurological disorders. Identifying cellular pathways that contribute to axon vulnerability may identify new therapeutic strategies for maintenance of neural circuits. Dual leucine zipper kinase (DLK) is an axonal stress response MAP3K that is chronically activated in several neurodegenerative diseases. Activated DLK transmits an axon injury signal to the neuronal cell body to provoke transcriptional adaptations. However, the consequence of enhanced DLK signaling to axon vulnerability is unknown. We find that stimulating DLK activity predisposes axons to SARM1-dependent degeneration. Activating DLK reduces levels of the axon survival factors NMNAT2 and SCG10, accelerating their loss from severed axons. Moreover, mitochondrial dysfunction independently decreases the levels of NMNAT2 and SCG10 in axons, and in conjunction with DLK activation, leads to a dramatic loss of axonal NMNAT2 and SCG10 and evokes spontaneous axon degeneration. Hence, enhanced DLK activity reduces axon survival factor abundance and renders axons more susceptible to trauma and metabolic insult.

The synaptic receptor Lrp4 promotes peripheral nerve regeneration.

Early during PNS regeneration, regenerating axons emerge from the proximal nerve stump, yet whether they extend simultaneously or whether pioneering axons establish a path for follower axons remains unknown. Moreover, the molecular mechanisms underlying robust regeneration are incompletely understood. Using live imaging, we demonstrate that in zebrafish pioneering axons establish a regenerative path for follower axons. We find this process requires the synaptic receptor lrp4, and in lrp4 mutants pioneers are unaffected while follower axons frequently stall at the injury gap, providing evidence for molecular diversity between pioneering and follower axons in regeneration. We demonstrate that Lrp4 promotes regeneration through an axon extrinsic mechanism and independent of membrane anchoring and MuSK co-receptor signaling essential for synaptic development. Finally, we show that Lrp4 coordinates the realignment of denervated Schwann cells with regenerating axons, consistent with a model by which Lrp4 is repurposed to promote sustained peripheral nerve regeneration via axon-glia interactions.

MAPK signaling promotes axonal degeneration by speeding the turnover of the axonal maintenance factor NMNAT2.

Injury-induced (Wallerian) axonal degeneration is regulated via the opposing actions of pro-degenerative factors such as SARM1 and a MAPK signal and pro-survival factors, the most important of which is the NAD+ biosynthetic enzyme NMNAT2 that inhibits activation of the SARM1 pathway. Here we investigate the mechanism by which MAPK signaling facilitates axonal degeneration. We show that MAPK signaling promotes the turnover of the axonal survival factor NMNAT2 in cultured mammalian neurons as well as the Drosophila ortholog dNMNAT in motoneurons. The increased levels of NMNAT2 are required for the axonal protection caused by loss of MAPK signaling. Regulation of NMNAT2 by MAPK signaling does not require SARM1, and so cannot be downstream of SARM1. Hence, pro-degenerative MAPK signaling functions upstream of SARM1 by limiting the levels of the essential axonal survival factor NMNAT2 to promote injury-dependent SARM1 activation. These findings are consistent with a linear molecular pathway for the axonal degeneration program.

Cytoskeletal disruption activates the DLK/JNK pathway, which promotes axonal regeneration and mimics a preconditioning injury.

Nerve injury can lead to axonal regeneration, axonal degeneration, and/or neuronal cell death. Remarkably, the MAP3K dual leucine zipper kinase, DLK, promotes each of these responses, suggesting that DLK is a sensor of axon injury. In Drosophila, mutations in proteins that stabilize the actin and microtubule cytoskeletons activate the DLK pathway, suggesting that DLK may be activated by cytoskeletal disruption. Here we test this model in mammalian sensory neurons. We find that pharmacological agents designed to disrupt either the actin or microtubule cytoskeleton activate the DLK pathway, and that activation is independent of calcium influx or induction of the axon degeneration program. Moreover, activation of the DLK pathway by targeting the cytoskeleton induces a pro-regenerative state, enhancing axon regeneration in response to a subsequent injury in a process akin to preconditioning. This highlights the potential utility of activating the DLK pathway as a method to improve axon regeneration. Moreover, DLK is required for these responses to cytoskeletal perturbations, suggesting that DLK functions as a key neuronal sensor of cytoskeletal damage.

Loss of the spectraplakin short stop activates the DLK injury response pathway in Drosophila.

The MAPKKK dual leucine zipper-containing kinase (DLK, Wallenda in Drosophila) is an evolutionarily conserved component of the axonal injury response pathway. After nerve injury, DLK promotes degeneration of distal axons and regeneration of proximal axons. This dual role in coordinating degeneration and regeneration suggests that DLK may be a sensor of axon injury, and so understanding how DLK is activated is important. Two mechanisms are known to activate DLK. First, increasing the levels of DLK via overexpression or loss of the PHR ubiquitin ligases that target DLK activate DLK signaling. Second, in Caenorhabditis elegans, a calcium-dependent mechanism, can activate DLK. Here we describe a new mechanism that activates DLK in Drosophila: loss of the spectraplakin short stop (shot). In a genetic screen for mutants with defective neuromuscular junction development, we identify a hypomorphic allele of shot that displays synaptic terminal overgrowth and a precocious regenerative response to nerve injury. We demonstrate that both phenotypes are the result of overactivation of the DLK signaling pathway. We further show that, unlike mutations in the PHR ligase Highwire, loss of function of shot activates DLK without a concomitant increase in the levels of DLK. As a spectraplakin, Shot binds to both actin and microtubules and promotes cytoskeletal stability. The DLK pathway is also activated by downregulation of the TCP1 chaperonin complex, whose normal function is to promote cytoskeletal stability. These findings support the model that DLK is activated by cytoskeletal instability, which is a shared feature of both spectraplakin mutants and injured axons.

Dysfunctional behavioral modulation of corticostriatal communication in the R6/2 mouse model of Huntington's disease.

BACKGROUND: In Huntington's disease (HD), motor symptoms develop prior to the widespread loss of neurons in striatum and cerebral cortex. The aim of this study was to examine dysfunctional patterns of corticostriatal communication during spontaneously occurring behaviors in a transgenic mouse model of HD. METHODOLOGY/PRINCIPAL FINDINGS: Local field potentials (LFPs) were recorded from two closely interconnected areas, motor cortex and dorsal striatum, in wild-type controls (WT, n = 14) and a widely used transgenic HD model (R6/2 mice, n = 12). All mice were between the ages of 7-9 weeks, a critical period of motor symptom development in R6/2s. Recordings were obtained while the mice were behaving freely in an open field. Specific LFP activity was extracted using timestamps for three increasingly demanding motor behaviors: 1) resting; 2) grooming; and 3) active exploration. Power spectral densities (PSD) were obtained for the cortical and striatal LFPs as well as coherence levels and relative phase across the frequency spectrum. In both brain regions, only R6/2s showed high frequency LFP oscillations during rest and grooming. As behavior increased from resting to exploring, corticostriatal synchrony at high frequencies declined in R6/2s, completely opposite to the WT pattern. R6/2s also exhibited nearly in-phase corticostriatal activity (cortex phase leads of ∼5°), while the WTs consistently showed cortical phase lags of ∼20° across all assessed behaviors, indicating a lead role for striatum. CONCLUSIONS/SIGNIFICANCE: Our results add to growing evidence for altered communication between cortex and striatum in HD and suggest more generally that increasingly demanding motor behaviors differentially modulate corticostriatal communication. Our data also suggest conduction delays in R6/2 corticostriatal transmission, leading to compensatory speeding of LFP activity, as evidenced by the presence of high frequency LFP oscillations.