Ancient Molecules Guide New Synapse Growth

During the larval stage of development, Drosophila muscles grow hundreds of times in mass in just a few days. As they grow, the neurons that innervate them must stretch out to reach the muscle, find a proper target, and establish a synaptic connection. Research has shown that although these nerve endings essentially have a predetermined flight plan, they do not run entirely on autopilot. Because the terrain is dynamic, the nerve cells use external cues to guide them to suitable targets.

Photo by Graham Ramsay

Clockwise from left are Misao Higashi, April Duckworth, Aurnab Ghose, Thomas Schwarz, Alan Tenney, and David Van Vactor, who with John Flanagan and other colleagues found that versatile extracellular molecules help guide the formation of a functioning nervous system during development.

Recent studies have zeroed in on heparan sulfate proteoglycans (HSPGs), ancient molecules that appear evolutionarily at about the same time as the first nervous systems appear, as key cue-providers in the extracellular matrix. So far, the accepted model has described HSPGs as a scaffolding, quite literally like the scaffolding employed on construction sites. But new findings from a study published in the Feb. 16 Neuron, led by David Van Vactor, HMS associate professor of cell biology, suggest that HSPGs may do more than simply provide extracellular structure. Van Vactor and colleagues connect a protein tyrosine phosphatase receptor called LAR with two HSPGs, revealing that HSPGs influence a wide range of developmental nervous system changes and contribute to a rich and complex extracellular system beyond that of a scaffold.

Network Drivers
Previous investigations of nervous-system development have linked LAR to synapse formation in worms, flies, and mammals. The mechanism for nervous-system formation identified by Van Vactor and colleagues may therefore be found across organisms. Moreover, certain HSPG genetic defects have been associated with abnormal neural maturation in rat cell cultures. This evidence suggests that HSPGs and LAR may play roles in maintaining a healthy adult nervous system. “Understanding the basic science of how the brain develops and functions has great implications for neural disorders,” said co-author John Flanagan, HMS professor of cell biology.

The investigation found that through LAR, different HSPGs play distinct roles in driving synapse-forming machinery. “As the arbor of connections unfolds, both the form of the synapse and the orchestration of neurotransmitters work together to determine the functional capacity of the synapse,” said Van Vactor. “LAR is cleverly coupling that morphology with the assembly of active zones through two ligands that regulate distinct aspects of this growth.”

The researchers teased out the roles of these molecules by observing neuromuscular-junction growth in gain- and loss-of-function mutants. The team focused on the relationship between the LAR receptor and two Drosophila HSPGs, syndecan (Sdc) and the glycosylphosphatidylinositol-anchored glypican Dallylike (Dlp). Compared with wild types, mutant drosophila lacking Sdc developed significantly fewer boutons, the bulb-shaped heads of axons that form one side of a synaptic junction, suggesting that Sdc drives the normal development of boutons. Mutants lacking Dlp showed increased electrophysiological activity compared with wild types given similar nerve stimulation. The researchers found that the Dlp mutants formed smaller, more densely packed active zones, sites on the bouton membrane that determine the flow of neurotransmitters. This observation suggests that Dlp dictates the development of active zones and possibly the level of electrophysiological signaling.

Having shown that Sdc regulates bouton formation and Dlp regulates the functionality of the bouton, the next step was to determine the mechanism through which these HSPGs act. Cell surface binding assays showed that both Sdc and Dlp bind to LAR and revealed one other curious detail. “It seems that Dlp wins the binding competition and outcompetes Sdc for the attention of LAR,” said co–first author Alan Tenney, HMS research fellow in cell biology. Further genetic experiments using LAR-deficient mutants suggested that Sdc activates LAR and Dlp antagonizes the receptor. “This evidence suggests that LAR’s first priority may be active zone construction,” said Van Vactor.

Rules of Order
Taking these findings into account, Van Vactor proposes one possible model of synapse formation involving a time-dependent mechanism that makes physical sense the same way it makes physical sense to ice a cake after baking it. First, an axon forms a bouton, then it adds the neurotransmitter-signaling active zones, with LAR linking these two activities together. At the molecular level in the neuromuscular junction, the process begins when the LAR receptor appears on the nerve cell surface, and Sdc in the extracellular matrix stimulates bouton growth. “At this point, the clock starts ticking,” said Van Vactor. “Dlp and Sdc both vie for LAR’s attention, but the system is set up so that Dlp always wins. It’s just a matter of time.” This period may naturally correspond to the time it takes to form a bouton that is ready for active-zone development.

Image courtesy of David Van Vactor

Wiring diagram. During development, axons grow into the neuromuscular junction and send nerve endings out to form synaptic connections. When the nerve processes find a suitable target, specialized proteoglycans work to form bulb-shaped boutons and then construct active zones on their surface.

Any hypothetical model has limitations, of course. “You can construct a model that makes sense with three genes, but there are hundreds involved in the system, and many haven’t been studied yet,” said biologist Kai Zinn of the California Institute of Technology. In addition to exploring this and other competing models, Van Vactor’s lab is exploring the roles these LAR signaling pathways play in adult synapse plasticity. Their first experiment probes whether the activity-dependent plasticity observed at the fly neuromuscular junction requires the LAR receptor.

In addition to illuminating potential steps of synapse formation, the Neuron study brings researchers closer to understanding protein tyrosine phosphatase (PTP) receptors. PTPs have been relatively intractable when compared with their sister receptors, protein tyrosine kinases, which have been characterized in detail and exploited in targeted drug therapies. “The exciting thing about this study is that it puts most of the elements in place—binding, functionality, and some signaling,” said Flanagan. “After we understand how the biology [of PTPs] works, then we can try to understand how to tinker with it.”

—Elizabeth Dougherty

top