Brain and spinal cord neurons in mammals, including humans, do not regenerate when they are broken due to injury or disease. Yet, neurons have a tremendous capacity for regeneration in mammalian embryos and in some adult animals. The puzzle of why adult mammalian neurons lose their capacity to regenerate can be solved by using the simple model organism, C. elegans. A genetic mutation was discovered in C. elegans that causes axons to break spontaneously. In response to these spontaneous axon breaks, the neurons successfully regrow new axons back to their target muscles. This spontaneous axon break, followed by axon regeneration, will be used to test the function of each gene in the C. elegans genome for its effect on axon regeneration. This would be the first genetic screen to identify all genes involved in axon regeneration in any organism and should lead to a comprehensive and fundamental understanding of the signaling pathways controlling axon regeneration. It should also allow identification of the signaling changes that occur with age that prevent axon regeneration in most adult mammalian neurons. The similarity between genes in C. elegans and humans means that any knowledge gained from the screen should be applicable to human nerve cell regeneration and provide rational targets for new drug therapies. This research will also provide training in cutting edge research technologies for graduate students and undergraduates, and will provide research opportunities for high school students through the University of Utah Bio-Sciences program that places students in laboratories for 7 weeks each summer.
Diseases and injuries of the brain have a devastating affect on humans. Whether it is spinal cord injury, stroke, Alzheimer's, or other dementias it is clear that the inability of neurons to regenerate exacerbates all of these conditions. My lab made the interesting discovery that axons missing a structural molecule (spectrin) are very fragile and spontaneously break due to mechanical stress from nearby muscle contractions. We further observed that the broken axons regenerate in young animals, but not in old ones. This led us to propose an "unbiased" genetic screen for genes that affect axon regeneration in the genetic model system C. elegans. Our method was to knock out the function of a gene in the fragile axon animals and then look at the number of axons that were able to regenerate. If the gene functioned to improve axon regeneration then we would see fewer regenerating axons in the gene knockout, however, if the gene functioned to inhibit axon regeneration then we would see more regenerating axons in the gene knock out. We screened over 5000 of the C. elegans genes most closely related to human genes (about 25% of the entire C. elegans and human genomes) for their affects on axon regeneration. We identified 53 genes that act to either improve axon regeneration or make it worse. The schematic image of a regenerating neuron summarizes the functional classes of genes we discovered. The most important finding of our research was the discovery of the "injury signaling" molecules DLK and MLK. We were able to show that these signaling molecules and the MAPK pathways they activate are absolutely required for axon regeneration, but not for the normal development of neurons. We showed that over expression of DLK and MLK improves axon regeneration and delays the age related decline in regeneration ability. As in any rebuilding project the first step is to limit further damage, make a temporary repair, and then get rid of the damaged components. Several of the genes we found affecting axon regeneration fit into this category of "membrane repair and recycle". The next step in regeneration is to turn on the "transcription factors" that reprogram the neuron from its normal function of communication to regrowth. The transcription factors we identified are regulated by stress activated signaling pathways, most importantly the MAPK pathways, but also the starvation, heat shock, and hypoxia induced stress pathways. "Transport" of materials to and from the site of rebuilding is also important and we identified several molecules that regulate trafficking of proteins along the axon. Synthesis of new membrane and proteins must be part of any regeneration program. A regenerating axon may increase its total size several hundred fold during regrowth to a distant target. "ER and golgi" proteins are involved in regulating new protein and lipid synthesis. The ER proteins we discovered are also regulated by known stress activated pathways. A surprising finding was a number of "RNA binding factors" that normally act to inhibit axon regeneration. Our interpretation of these stored RNAs is that they represent ready repair kits that can be rapidly activated locally in case of emergency. They would not require the cell to wait for the injury signal to be transported to the cell nucleus and reprogram the cell for regrowth. Cellular damage, if not rapidly repaired, can lead to catastrophic consequences and ultimately cell death. The candidate genes identified in our genetic screen of the small roundworm C. elegans are conserved among animals, including humans. They should present several new targets to pharmaceutical and biotechnology companies for the treatment of injuries and diseases of the nervous system. Genes inhibiting regeneration are of particular interest because these pathways may be easier to eliminate via targeted drugs rather than by up regulating growth-promoting pathways. An important approach will be to determine whether elimination of particular pathways in C. elegans can also enhance regeneration in older animals or induce regeneration in neurons that do not normally regenerate.
