Final Report Summary - AXON RE-EXTENSION (The molecular mechanisms of axon re-extension following developmental axon pruning)
A summary description of the project objectives:
Lack of neuronal regeneration following injury such as spinal cord injury is the major cause for poor functional recovery. It has long been appreciated that young neurons can grow, undergo reorganization and synapse on appropriate targets during development while adult neurons cannot, however the molecular basis of these differences remain unclear. Studying the molecular mechanisms that regulate axon re-extension following remodeling during development holds the promise to uncover molecular rules underlying this phenomena and are therefore of great potential. A model system that allows a unique molecular exploration of axon re-extension is that of axon pruning in Drosophila. Pruning is a process in which neurons first extend excessive branches, later prune away inappropriate ones with precise spatial and temporal control and, at least in the some cases, re-extend their axons to form mature connections. Pruning was found to essential for sculpting the mature nervous systems of vertebrates and invertebrates. During my post-doctoral studies, I performed a mosaic screen and identified a mutant exhibiting normal pruning but lacking the stereotypical axon re-extension that follows. Remarkably, other neurons, belonging to the mutant clone but which do not undergo pruning, extend their axons normally at the same developmental time, indicating that the mutation selectively affected axon re-extension following pruning but not axon extension per se. This novel finding suggests that there is molecular switch dedicated to changing the growing status of a neuron. The causal gene was mapped to an uncharacterized steroid hormone receptor, HR51, also known as UNF. This project was focused on characterising the role of HR51/UNF in axon re-extension and initiate experiments to uncover its ligand and potential co-receptor.
Work performed and main results since the beginning of the project:
In our work so far, we have established that Hr51/UNF is specifically required for axon growth of mushroom body (MB) gamma neurons following pruning but not their initial axon outgrowth. Hr51/UNF is also not required for the initial axon outgrowth of other MB neurons that extend their axons at the same time as gamma neurons undergo re-extension. We also ruled out the possibility that Hr51/UNF is required for initial guidance or cell identity. Due to this distinction with initial axon growth, we coined the term "developmental axon regrowth".
Antibody staining and transgene expression confirmed that Hr51/UNF is expressed in MB neurons and is localized to nuclei. We performed a structure function analysis and found that all domains that were predicted to be required for the function of nuclear receptors, are indeed required in vivo for developmental axon regeneration.
Overexpression of full length Hr51/UNF rescued the mutant phenotype but in addition resulted in a gain of function defect of a partial axon pruning defect. We found that Hr51 can inhibit the expression of EcR-B1, thereby repressing pruning. This finding suggests that in order to activate an axon regrowth program, it may be required to inactivate a degeneration (pruning) program at the same time. These findings (attached as PDF) were published in Yaniv et al Curr. Biol, 2012. and Yaniv and Schuldiner, commentary in: Axons: Cell Biology, Molecular Dynamics and Roles in Neural Repair and Rehabilitation, Ed. Yamamoto and Oshiro, Nova publishing, 2013.
In our study we also tested whether another nuclear receptor, E75, is important for developmental axon pruning. The reason E75 was a candidate gene is that its mammalian ortholog (NR1D1) was shown to interact with the ortholog of Hr51 (NR2E3). Indeed, using mosaic loss of function experiments and rescue experiments, we found that E75 isoform C is also required for developmental axon regeneration suggesting that Hr51 and E75 function as co-receptors. We have also identified a putative ligand and are preparing these data for publication.
Expected final results and potential impact:
In our studies we discovered a new developmental program for axon growth that is distinct from initial axon outgrowth. These findings have initiated a new and growing avenue of research in the lab and were the foundation of an ERC consolidator proposal that was recently accepted for funding.
The failure of axon regeneration following injury of central nervous system (CNS) injury is extremely debilitating to the patients. One strategy to identify the molecular machinery that determines the intrinsic growth potential of neurons is to understand what allows young, developing neurons, to undergo neuronal plasticity. Nevertheless, several studies have shown that neurogenesis during development is different from axon regeneration following injury. Our study suggests that they have been comparing the wrong developmental program. We identify and provide molecular insights into a novel developmental program that is specifically required for switching the growth of axons from degeneration to regeneration and thus hold great promise to be more similar to axon regeneration. Understanding the intrinsic growth potential of axons during initial growth, developmental regrowth and regeneration following injury is the topic of our followup ERC grant.