Gauri Kulkarni, PhD (center), and Haichang Li, PhD (right), postdoctoral scientists, with mentor William Wadsworth, PhD, professor, Department of Pathology, UMDNJ-Robert Wood Johnson Medical School

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urrently, there is no cure available for the disability caused by spinal cord injuries. Thousands of people suffer from these injuries, which are commonly caused by motor vehicle accidents, acts of violence, falls, sports and recreation injuries or diseases. The spinal cord is made up of motor and sensory cells called neurons that are connected to each other and muscles by thread-like structures called axons. The injuries cause damage to these axons, resulting in varying degrees of paralysis or loss of senses. Stimulating the regeneration of broken axons to increase recovery is important for spinal cord repair. However, the ability to grow new axons is limited and, therefore, new treatments are needed to target the regeneration of damaged axons following injury and to help restore the lost sensory or motor functions. Current research advances towards understanding the basic mechanism of axon growth and their guidance to target cells have a strong potential to create breakthrough therapies to treat people with disability caused by spinal cord injuries.

In the Wadsworth laboratory, we are looking for the proteins that drive axon growth and help them to make functional connections. We use a powerful genetics and cell biology approach employing a simple, nematode model system, C. elegans, which shares with humans several key components involved in the development of the nervous system. A better understanding of the molecular mechanism of axon guidance and circuit formation is likely to be beneficial in the development of new therapies to help repair a damaged spinal cord.

Spinal cord injuries result in varying degrees of paralysis or loss of senses. Though rehabilitation may result in some improvement, there is no therapy that can repair the broken connections to regain fully functional recovery. Following spinal cord injury, new axons can grow and form new connections that help compensate for the regions lost due to injury. However, this response is quite limited. For effective therapies, we need innovative treatments and medications that will promote the regeneration of axons and reconnect them to their target cells (synapse formation) to get functional recovery. This process is important both during the development of the nervous system and following injury. Therefore, we must understand which molecules/proteins are involved in axon growth and synapse formation during development. This could help us find ways to manipulate such molecules to restore proper connections following injury.

In the Wadsworth laboratory, our research is focused on understanding the molecular mechanism that regulates the axon growth and guidance mediated by secreted UNC-6/Netrin protein. We use C. elegans, a free–living soil roundworm, as a model system. Compared to the complexity of the human nervous system, which is comprised of billions of neurons that make trillions of connections, C. elegans offers a perfect model because its nervous system is composed of just 302 neurons and their connections are well described. Further, there are several fluorescent-tagged proteins available that can be expressed in specific motor or sensory neurons and we can observe growing axons and their connections directly due to transparent worm skin. The C. elegans genome encodes about 20,000 genes and a high percentage of these have related genes encoded in the human genome. Several key molecules that are involved in axon guidance during the development of the human nervous system were first discovered in C. elegans.

Figure 1. Motor neurons (green glow), which control the movement of C.elegans, are visualized with a Green Fluorescent Protein. (A) Wild type animals show the motor neuron cell bodies (green round bodies) situated on the ventral cord (vc) and their axons (green thread-like structures) are directed towards the dorsal cord (dc) by UNC-6/Netrin via receptors, UNC-40/DCC and UNC-5. After reaching dorsal cord, they connect to muscles and thus contribute movement. (B) In the absence of UNC-6 protein, the mutant unc-6 animals, show misdirected axons that meet the dorsal sublateral cord (dsl) without connecting the muscles. This results in uncoordinated/paralyzed movement (unc).

During development, axons need to grow in different directions to reach the target cells. They express multiple receptors that recognize specific extracellular proteins, which act as guidance cues. The secreted UNC-6/netrin proteins function in axon guidance in both invertebrates and vertebrates. The UNC-40/DCC family of receptors mediate attraction of the axon to UNC-6/netrin, whereas, the UNC-5 family of receptors, alone or in combination with UNC-40/DCC, mediate repulsion from UNC-6/netrin. Netrins and netrin receptors are found to be constitutively expressed in adult rats, and during the injury, netrin receptors are down-regulated, suggesting that receptor down-regulation could contribute to the limited capacity of regenerating axons to reach the targets. However, very little is known about how these receptors are modulated.

By genetic screen, we have identified two proteins that control the UNC-6/netrin receptor mediated axon growth as well synapse formation. Our research provides an important link between the coordination of axon growth and synapse formation. When the UNC-6/netrin gene is knocked out in C. elegans, the axons grow in aberrant directions and fail to connect with target cells. This results in paralysis and loss of touch sense in the worms. We used a green fluorescent protein (GFP) that glows in the motor neurons of the transparent worms. These motor neurons in normal animals circumferentially migrate away from the ventral cord and make connections to the dorsal muscles to form a dorsal cord (Figure 1A). The mutant animals without UNC-6 protein show defects in these axons that do not connect with the dorsal muscles (Figure 1B), resulting in paralyzed movement.

We uncovered two mutations that could improve the motor axon defects and movement in the unc-6 mutant strain having partial UNC-6 activity. These mutations were identified in two different genes by genetic and single nucleotide polymorphism (SNP) mapping. One gene was found to encode a novel protein encoding a transmembrane receptor with conserved C-type lectin-like domains and the second gene encodes a highly conserved RPM-1/highwire/PAM, encoding a putative RING E3-ubiquitin ligase. Further, we found that both the genes are expressed within the motor neurons we studied. By extensive genetic analysis, we found that CLEC-38 negatively regulates the activity of the UNC-40/DCC receptor (Kulkarni et al; J Neurosci. 2008 Apr 23; 28(17):4541-50.) and RPM-1 negatively regulates the activity of the UNC-5 receptor (Li et al; J Neurosci. 2008 Apr 2; 28(14):3595-603.). In the absence of CLEC-38 function, UNC-40/DCC levels are up-regulated in the neurons. On the other hand, UNC-5 levels are up-regulated in the absence of RPM-1 function.

For the first time, these studies uncovered the role of a previously uncharacterized protein, CLEC-38, in the regulation of UNC-40/DCC mediated axon growth. We also found that CLEC-38 protein promotes the formation of connections (synapses) between motor axons and muscles. RPM-1, a protein already known to play a role in building synapses, was identified as playing a new role in the regulation of the UNC-5 receptor. Thus, these two proteins regulate two processes, axon growth and synapse formation. In the absence of either of these two proteins, the axon fails to recognize the target cell and keeps on growing further without making functional synapses. This sheds light on the coordination between axon growth and synapse formation, processes that were previously considered to be independent. When an axon encounters the target cell, the guidance receptors might be down-regulated by CLEC-38 or RPM-1, stopping its growth and promoting the synapse formation. Currently, we are investigating the mechanism by which CLEC-38 and RPM-1 function and trying to understand which signals trigger these proteins to down-regulate the growth receptors while promoting synaptogenesis.

Our studies provide an important molecular link between two processes needed for correct wiring of the nervous system and for functional recovery from this type of injury. Understanding such molecular pathways could afford novel insights into developing targeted therapies to damaged neurons, and aiding the recovery of victims of spinal cord injuries.

This research was funded by the National Institute of Neurological Disorders and Stroke (NINDS) and by grants from the New Jersey Commission on Spinal Cord Research.

Gauri Kulkarni earned a PhD in molecular biology from Pune University in India in 2002 for her functional genomics studies of a gene involved in neuropeptide regulation in Drosophila. She joined Dr. Wadsworth’s laboratory for her postdoctoral work. She received a $100,000 postdoctoral fellowship grant from the New Jersey Commission on Spinal Cord Research to further her research into understanding the molecular mechanism of axon guidance and synaptogenesis that target spinal cord injury and related disorders.

Haichang Li received a PhD in developmental biology from Gifu University of Japan in 2001. He was a research scientist at the Riken-Brain Science Institute (BSI). In 2004, he joined the Wadsworth lab as a postdoctoral scientist and is currently working on neuronal development. His research is focused on understanding the molecular mechanism of axon outgrowth and guidance using C.elegans as a model system.