Expanded genetic screening in Caenorhabditis elegans identifies new regulators and an inhibitory role for NAD+ in axon regeneration.

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.

Intermediate filament accumulation can stabilize microtubules in Caenorhabditis elegans motor neurons.

Neural circuits utilize a coordinated cellular machinery to form and eliminate synaptic connections, with the neuronal cytoskeleton playing a prominent role. During larval development of Caenorhabditis elegans, synapses of motor neurons are stereotypically rewired through a process facilitated by dynamic microtubules (MTs). Through a genetic suppressor screen on mutant animals that fail to rewire synapses, and in combination with live imaging and ultrastructural studies, we find that intermediate filaments (IFs) stabilize MTs to prevent synapse rewiring. Genetic ablation of IFs or pharmacological disruption of IF networks restores MT growth and rescues synapse rewiring defects in the mutant animals, indicating that IF accumulation directly alters MT stability. Our work sheds light on the impact of IFs on MT dynamics and axonal transport, which is relevant to the mechanistic understanding of several human motor neuron diseases characterized by IF accumulation in axonal swellings.

Differential regulation of polarized synaptic vesicle trafficking and synapse stability in neural circuit rewiring in Caenorhabditis elegans.

Neural circuits are dynamic, with activity-dependent changes in synapse density and connectivity peaking during different phases of animal development. In C. elegans, young larvae form mature motor circuits through a dramatic switch in GABAergic neuron connectivity, by concomitant elimination of existing synapses and formation of new synapses that are maintained throughout adulthood. We have previously shown that an increase in microtubule dynamics during motor circuit rewiring facilitates new synapse formation. Here, we further investigate cellular control of circuit rewiring through the analysis of mutants obtained in a forward genetic screen. Using live imaging, we characterize novel mutations that alter cargo binding in the dynein motor complex and enhance anterograde synaptic vesicle movement during remodeling, providing in vivo evidence for the tug-of-war between kinesin and dynein in fast axonal transport. We also find that a casein kinase homolog, TTBK-3, inhibits stabilization of nascent synapses in their new locations, a previously unexplored facet of structural plasticity of synapses. Our study delineates temporally distinct signaling pathways that are required for effective neural circuit refinement.

Neural circuit rewiring: insights from DD synapse remodeling.

Nervous systems exhibit many forms of neuronal plasticity during growth, learning and memory consolidation, as well as in response to injury. Such plasticity can occur across entire nervous systems as with the case of insect metamorphosis, in individual classes of neurons, or even at the level of a single neuron. A striking example of neuronal plasticity in C. elegans is the synaptic rewiring of the GABAergic Dorsal D-type motor neurons during larval development, termed DD remodeling. DD remodeling entails multi-step coordination to concurrently eliminate pre-existing synapses and form new synapses on different neurites, without changing the overall morphology of the neuron. This mini-review focuses on recent advances in understanding the cellular and molecular mechanisms driving DD remodeling.

Dynamic microtubules drive circuit rewiring in the absence of neurite remodeling.

A striking neuronal connectivity change in C. elegans involves the coordinated elimination of existing synapses and formation of synapses at new locations, without altering neuronal morphology. Here, we investigate the tripartite interaction between dynamic microtubules (MTs), kinesin-1, and vesicular cargo during this synapse remodeling. We find that a reduction in the dynamic MT population in motor neuron axons, resulting from genetic interaction between loss of function in the conserved MAPKKK dlk-1 and an α-tubulin mutation, specifically blocks synapse remodeling. Using live imaging and pharmacological modulation of the MT cytoskeleton, we show that dynamic MTs are increased at the onset of remodeling and are critical for new synapse formation. DLK-1 acts during synapse remodeling, and its function involves MT catastrophe factors including kinesin-13/KLP-7 and spastin/SPAS-1. Through a forward genetic screen, we identify gain-of-function mutations in kinesin-1 that can compensate for reduced dynamic MTs to promote synaptic vesicle transport during remodeling. Our data provide in vivo evidence supporting the requirement of dynamic MTs for kinesin-1-dependent axonal transport and shed light on the role of the MT cytoskeleton in facilitating neural circuit plasticity.

Spatial and temporal dynamics of neurite regrowth.

Injury to mature neurites triggers a series of events that have both growth promoting and inhibitory roles. Recent evidence from a variety of experimental models has revealed new neuronal re-growth modulators. The action of these modulators must be precisely regulated both in time and space, and involves multiple cellular processes including retrograde signaling and local translation in the injured neurite. New genetic techniques, in combination with pharmacological approaches, have served to advance mechanistic dissection of neuronal response to injury. Better understanding of the spatio-temporal cues would greatly aid in the development of effective regenerative therapies.