Most neurons can’t be renewed from stem cell populations. Continued functioning of neuronal circuits therefore requires neurons to survive and preserve their information-processing capabilities throughout the life of the organism. To accomplish this feat of survival, neurons must maintain their structure and repair it when damaged. By identifying critical components of neuronal maintenance and regeneration, we seek to provide a basis for a more complete understanding of the cellular mechanisms that keep neurons functional.
The nematode C. elegans is well-suited for the study of the mature nervous system for several reasons. First, the nervous system structure can be analyzed by light microscopic techniques. The worm is transparent throughout its life, and fluorescent markers can be used to visualize the nervous system with single-cell resolution. Second, the worm can survive even severe insults to nervous system function, allowing the analysis of critical processes in mature animals. Third, a wide variety of tools allow us to manipulate the worm's genome and to investigate the cell biology of neuronal functions. These advantages make C. elegans an ideal model organism for our research.
We found that axons in mutant animals that lack beta-spectrin break spontaneously and regenerate. Because axon morphology in these mutants depends on regeneration, this strain provides a sensitized background that enables us to identify factors required for regeneration. We have developed a suite of genetic tools to initiate and monitor regeneration. We also use femtosecond laser surgery to sever individual axons.
A paper describing axon breaking in beta-spectrin mutants was published in Journal of Cell Biology (PDF).
A paper describing the regeneration screen was published in Journal of Neuroscience (PDF)
MAP Kinase Signaling
We performed a genetic screen for regeneration genes and identified the dual-leucine zipper MAP3K DLK-1. Our work showed that DLK-1 and its downstream targets MKK-4 (MAP2K) and PMK-3 (p38 MAPK) are required for regeneration. When axons are severed in dlk-1, mkk-4, or pmk-3 mutants, the axon stump never initiates a growth cone. Importantly, these genes are not essential for axon outgrowth during development. It is only when axons are challenged to regenerate that DLK-1, MKK-4 and PMK-3 are required. Because loss of this pathway blocks regeneration at an early step (growth cone formation), DLK-1 activity appears to be a critical link between axon damage and initiation of regeneration.
A paper describing these findings was published in Science (PDF).
How individual neurons age, the cellular pathways that regulate this process, and the ultimate effects of aging on neuronal function, are poorly understood. We are investigating how aging affects axon regeneration in the GABA neurons. We find that aging in these cells can be decoupled from overall lifespan by manipulating the InsR/FOXO daf-2/daf-16 pathway.
A paper detailing the effects of aging on GABA neuron regeneration, and analyzing how these effects are regulated by insulin signaling, PTEN/Tor, and the dlk-1 MAP kinase pathway was published in Neuron (PDF).
The ability of neurons to regenerate depends in part on intrinsic regulatory pathways, yet only a few such pathways are known. Using single-neuron analysis of regeneration in vivo, we found that Notch/lin-12 signaling inhibits the regeneration of mature neurons. Notch signaling suppresses regeneration by acting autonomously in the injured cell to prevent growth cone formation. The metalloprotease and gamma-secretase cleavage events that lead to Notch activation during development are also required for its activity in regeneration. Furthermore, a small molecule that blocks Notch activation improves regeneration when applied immediately after nerve injury. Our results define a novel, post-developmental role for the Notch pathway as a repressor of axon regeneration in vivo, and suggest potential targets for therapy after nerve damage.
A paper describing these initial findings was published in Neuron (PDF).
We are currently working to define the activation mechanism and the relevant targets for Notch in regeneration.
We are working to develop new tools and techniques to enable novel approaches to studying neuronal function and disease. We adapted the optogenetic tool KillerRed, which generates reactive oxygen when stimulated with light, for use in C. elegans. This tool facilitates the study of the effects of ROS in neurons, and can also be used as a highly efficient way to disable specific neurons and analyze their contribution to circuit function and behavior.
A paper describing KillerRed was published in Cell Reports (PDF).
We developed a way to achieve gene knockdown only in designated neurons in response to feeding RNAi. This technique allows us to screen through thousands of genes that normally cause lethal or other pleiotropic phenotypes for their specific role in neurons.
A paper describing this approach and it use in a screen for GABA neuron function was published in PLoS Genetics (PDF).
We are currently working on developing additional new tools.