There are billions of nerve cells and connections in the developed brain. How much is currently known about the way these neurons coordinate growth and form connections within the developing brain?
Firstly, I would like to clarify that there’s a big difference between understanding coordination and actually building connectivity.
In terms of building connectivity, several molecules have been identified that control this process and a lot has been learned from both genetic and biochemical research in a variety of different systems, particularly studies in the nematode C. elegans, the fruit fly Drosophila and mice. Much is also already known about how axons navigate over long distances through the brain. However, it is very poorly understood how the process of axon growth is coordinated with building connections.
There is clear evidence from certain types of systems that these events are coordinated and happen in a clear and precise temporal pattern, with an axon building connections while it is actively in the process of growth. It is definitely not the case that an axon grows to a target cell, stops, and then builds all its connectivity. It's clearly a much more active and integrated process than that.
In terms of understanding how coordination could be occurring, very little is known. To my knowledge, RPM-1, the protein that my lab has been studying recently, represents the best example that’s been described to date of a coordinating molecule that functions within neurons.
Could you please give an introduction to the intracellular signalling protein RPM-1 that your recent research looked at?
RPM-1, or Regulator of Presynaptic Morphology -1, was first described in a microscopic nematode called C. elegans and in Drosophila or fruit flies. It was identified based on its important role in building synaptic connections. Synapses are the structures that allow information relay in a neuronal circuit.
From there, RPM-1 was also found to play a role in regulating how an axon stops growing or how it “terminates.” Further examples of its roles in guiding axons to their targets also began to emerge.
So, RPM-1 plays a really core and important role in the basic mechanism of how synapses and therefore neuronal circuits are built.
RPM-1 is very large - almost five times the size of an average protein. It is made up of a series of different protein domains, which are smaller pieces of the protein that fold independently. In humans, almost all of those domains are heavily conserved and we only partly understand the functional role for some domains.
We know that RPM-1 controls several signalling mechanisms within the cell and it does so both positively and negatively. It therefore has the potential to act as an integrator of multiple signalling events,
What did your research show?
Together, our latest studies make two really striking points. The first is that we identified a phosphatase. It is called Protein Phosphatase Magnesium dependent 2 or PPM-2. We now know that RPM-1 binds to PPM-2 in order to inhibit or inactivate a target protein called DLK-1.
At the same time, it is known that RPM-1 has an intrinsic ability to modify the DLK-1 protein so that it degrades. Our finding that RPM-1 actually uses two independent mechanisms to control the activity of DLK1 was completely unexpected.
Potentially RPM-1 turns DLK-1 off within the short-term, while also making a targeted effort to destroy it in the long-term. Nobody was expecting that RPM-1 would function with that level of sophistication.
Were you surprised by the degree to which RPM-1 harnesses sophisticated mechanisms to regulate neuron development?
Absolutely. If you had asked me eight years ago if RPM-1 was simply functioning through a single mechanism, I would have told you that it's unlikely because of how large the protein is. Yet, there was only evidence for one molecular function downstream of RPM-1. I am still shocked that RPM-1 is now know to regulate at least five different pathways and possibility more.
I was very surprised to find that RPM-1 can control both the activation and degradation of a single target molecule. What also surprised me was the fact that, even though RPM-1 was controlling a lot of different pathways, none of the molecules or pathways that had been identified were receiving any input from outside of the neuron. They were all mechanisms that acted internally, within the cell.
Another of our latest findings shows, for the first time, that RPM-1 activity is converging in a unique way upon a target molecule called beta-catenin, as are extracellular signals. That was the level of sophistication I was hoping we might come across, but we had to look for a very long time to find a link to information coming from outside the cell.
Why is it extremely challenging to understand how the length of axons is determined at the molecular level?
Many labs around the world have made really great progress in understanding how axon length is regulated and how axons are guided to targets. However, understanding how neurons coordinate growth with the construction of connections is extremely difficult to assess.
You can't simply study one process or event; you have to integrate information on multiple events and understand how they're related. Understanding how things are coordinated is also difficult, because it requires getting a handle on the temporal control of a series of events and whenever you add a temporal element, things become much more challenging.
Finally, because we know that axon growth is coupled with building synaptic connectivity, it is difficult not to argue that if one event is impaired, then the other will automatically be affected.
One way to address this issue involves our recent observation that RPM-1 is located both at the mature axon tip where it forms a cap, as well as at the synapse.
This means RPM-1 meets critical criteria as a coordinating molecule, because not only is it regulating both axon termination and synapse formation, but it is in both of the relevant subcellular compartments of the neuron.
This finding, while not definitive, suggests that RPM-1 function in one location is not necessarily having a secondary effect at the other location.
What implications does your research have for neurodegenerative diseases?
These are very early days but there are definitely some important implications. Studies using mice conducted by Aaron Di Antonio's group at Washington University show that when axons are damaged, they go through an intrinsic degeneration process over the course of about 24 hours.
The axons essentially completely degrade following trauma and all synaptic connectivity is lost. However, in mice that lack RPM-1, this axon degeneration is greatly restricted for two weeks. This means RPM-1 has broad implications for a range of neurodegenerative diseases and trauma. If we can understand how RPM-1 functions, we may be able to understand how to halt the intrinsic degradation of axons.
If you consider Alzheimer's disease, for example, a lot of axon degradation occurs for a variety of reasons and we have a very poor understanding of the disease. However, presumably, once the axon is engaged in the degenerative process, there are native and internal mechanisms that take over and hasten the degradation process. If we could impair those mechanisms (and one of them may in fact be RPM-1), that might have some fairly broad implications.
Another implication is simply that the DLK-1 molecule that RPM-1 degrades, and that the PPM-2 phosphatase inhibits, is an essential molecule in axon regeneration. All the way from a small microscopic worm through a mouse, if DLK-1 is not present, axon regeneration is not possible.
So, we think that understanding the mechanisms that regulate DLK-1 activation could faciliate an understanding of how to improve regeneration. However, that requires a whole series of different types of experiments, and we are mainly focused on development at the moment.
I hope, however, that down the road, other groups will take an active interest in pursuing the mechanistic implications of our work regarding regeneration.
Does RPM-1 work in connection with extracellular signals?
During more than a decade of research, we have identified five pathways that are downstream of RPM-1, all of which are clearly required for building synaptic connectivity and regulating axon length, so they fit into the core mechanism of how RPM-1 works.
However, until our recent study, RPM-1 seemed to be acting almost in a vacuum within the cell. It was controlling a variety of signals, but all of these signals functioned within the neuron and there was no evidence of a link to signals originating outside of the cell.
Our study now shows that RPM-1 functions in combination with extracellular signals to regulate a single target molecule during axon synaptic development. In fact, RPM-1 is likely to function in combination with some of the most important extracellular regulators of development that have been described so far.
This was really critical because signals outside of the cell are clearly very important for an axon to grow and build synaptic connections. There is no doubt that information is coming from outside the cell, so to find that a molecule as important as RPM-1 does so many things within the cell, but without integrating with any activity from outside the cell was very puzzling.
In terms of a molecule coordinating different events during development, it cannot be acting in isolation within the cell. It would need to integrate information from outside the cell.
What are your further research plans?
We are actively trying to generate real-time in vivo imaging to provide further cell biological evidence of what I call the “coordinator hypothesis” of RPM function.
Gathering a body of evidence to support that RPM regulates coordination can be challenging and I think our observation that RPM-1 actually exist in two compartments in a single neuron at the same time is key.
To really understand coordination, we need to dissect in real-time a single neuron that is building connections and growing and actually examine what happens, including where RPM-1 is overtime.
Then, we need to look at whether coordination breaks down when RPM-1 is genetically removed from the neuron. We are currently trying to do these live imaging experiments on an active, live, growing axon inside a microscopic animal called C. elegans.
We're also trying to understand what is upstream of RPM-1. One of the really big mysteries right now is that numerous pathways have been found to function downstream of RPM-1, but we have no idea what is upstream of RPM-1.
It actually remains an open question whether anything even is upstream of RPM-1. I think there have to be regulators of RPM-1. It doesn't make sense that a molecule like RPM-1 would be regulating so many downstream pathways in different ways and controlling single targets with using multiple functions if RPM-1 wasn't somehow being regulated.
Where can readers find more information?
- Baker ST, Opperman KJ, Tulgren ED, Turgeon SM, Bienvenut W, et al. (2014) RPM-1 Uses Both Ubiquitin Ligase and Phosphatase-Based Mechanisms to Regulate DLK-1 during Neuronal Development. PLoS Genet 10(5): e1004297. doi:10.1371/journal.pgen.1004297
- Tulgren ED, Turgeon SM, Opperman KJ, Grill B (2014) The Nesprin Family Member ANC-1 Regulates Synapse Formation and Axon Termination by Functioning in a Pathway with RPM-1 and β-Catenin. PLoS Genet 10(7): e1004481. doi:10.1371/journal.pgen.1004481
- Opperman KJ, Grill B (2014) RPM-1 is localized to distinct subcellular compartments and regulates axon length in GABAergic motor neurons. Neural Dev. 2014; 9: 10. Published online 2014 May 10. doi: 10.1186/1749-8104-9-10
About Dr. Brock Grill
Brock Grill is an Assistant Professor in the Department of Neuroscience at The Scripps Research Institute - Florida.
Dr. Grill’s research team uses proteomic, genetic and transgenic approaches to understand the signaling networks that govern axon growth and synapse formation.
He has a particular interest in intracellular signaling molecules that coordinate synapse formation with termination of axon growth. Understanding the molecular underpinnings of neural development is critical for devising new therapeutic approaches to treat neurodegenerative diseases, and injury from stroke and trauma.
Dr. Grill received his B.Sc. in Microbiology from the University of Alberta, Canada; his doctorate in Experimental Medicine from the University of British Columbia, Canada; and received his postdoctoral training at UC Santa Cruz and Stanford University. Prior to joining Scripps – Florida, Dr. Grill was an Assistant Professor in the Department of Pharmacology at the University of Minnesota Medical School.
He has received several awards during his research career including: The Dean’s Silver Medal in Science (University of Alberta), an NSERC graduate fellowship, a CIHR graduate fellowship, and a CIHR postdoctoral fellowship.