Researchers from The University of Texas at Dallas' Center for Advanced Pain Studies (CAPS) and their colleagues have made a fundamental discovery about a key mechanism that enables nervous system connections to strengthen.
The findings have direct implications for better understanding the underlying biochemical mechanisms involved in learning and memory, as well as pain, said Dr. Ted Price BS'97, Ashbel Smith Professor of neuroscience in the School of Behavioral and Brain Sciences , CAPS director and a co-corresponding author of the study published Nov. 20 in the journal Science.
"This study gets to the core of how synaptic plasticity works — how connections between neurons evolve," he said. "It has very broad implications for neuroscience."
The discovery, based on studies in mice and on human tissue, revolves around phosphorylation, a biochemical process in which an enzyme called a kinase modifies another protein's function by adding a phosphate molecule to it. This process is considered fundamental for functions within cells, such as metabolism, structural processes and subcellular signaling.
The role of extracellular phosphorylation, which occurs outside of cells, has been less clear, however, especially in synapses, the spaces between nerve cells. It is into this space that a presynaptic neuron releases neurotransmitters, peptides and proteins that then bind to, activate and regulate receptors on postsynaptic neurons. This information transfer forms the basis of neuronal plasticity, a process that can enhance or diminish the strength of connections between nerve cells involved in learning, memory and pain.
The researchers focused on the role that phosphorylating kinases secreted by neurons might play in regulating synaptic signaling.
"Extracellular phosphorylation occurs via ectokinases — kinases secreted outside of cells. It has been known to occur for almost 150 years, but almost nothing has been learned about it in the nervous system," Price said. "In this study we found that kinases within the synaptic cleft itself play an important role in synaptic plasticity. These results alter our textbook-level understanding of how synapses work."
Researchers knew that phosphorylation via ectokinases was linked to pain but did now know which specific kinase was responsible.
An ectokinase called vertebrate lonesome kinase (VLK) was recently shown to have a role in platelet function and bone development. The new research by teams led by Price and Dr. Matthew Dalva, director of the Tulane Brain Institute and professor of cell and molecular biology at Tulane University, suggests VLK is also needed for a key interaction between neurons that mediates injury-induced pain.
They discovered that, as the result of an injury, presynaptic neurons secrete VLK, which then phosphorylates the extracellular side of ephrin type-B receptor 2 (EphB2) extruding from the membranes of postsynaptic nerve cells. This unique process attracts N-methyl-D-aspartate (NMDA) receptor proteins, which then cluster in the membrane with the EphB2 receptors. NMDA receptors play a key role in learning and memory formation by regulating the electrical potential of neurons to strengthen synaptic connections.
"Increased concentrations of NMDA receptors at the synapse allows for high levels of neuronal activation, leading to greater postsynaptic potentials — a fundamental mechanism of synaptic plasticity," said Hajira Elahi BS'17, MS'19, PhD'23, a co-first author of the study who completed much of the work as part of her dissertation.
The researchers found that mice genetically engineered to lack VLK in sensory neurons involved in pain did not develop acute hypersensitivity to pain after surgery. Conversely, administering VLK to normal mice induced robust pain hypersensitivity that was mediated by NMDA receptor activation.
"Complementing the mouse studies, we found that human sensory neurons also express and secrete VLK, and that VLK induces the EphB2 and NMDA receptor interaction in human tissue too," Elahi said. "This really highlights the translational impact of our work — the ectokinase role of VLK is likely important in human synaptic signaling as well."
"We started this project 10 years ago based on our long-standing collaboration with the Dalva lab, initially synthesizing kinases here at UT Dallas. It took us years to get around to testing VLK," Price said. "When we did, it became very clear that VLK is the kinase that phosphorylates EphB1 and EphB2 receptors, and that VLK activity is sufficient to cause NMDA receptor clustering."
"The finding that neurons release a protein kinase to modify synaptic function is broadly important and suggests many new and unexpected targets," said Dalva, the co-corresponding author of the paper. "Our work is a great example of collaborative science. The project and our findings would not have been possible without the shared expertise of the different teams participating."
Although NMDA receptors have long been a potential pain-relief drug target, direct approaches to modulate them are fraught with side effects.
"NMDA receptors are involved in almost every aspect of how the nervous system works," Price said. "Our findings suggest a new way to manipulate NMDA receptors through VLK targeting potentially without huge side effects. In cortical neurons from the brain, VLK seems to be released in an activity-dependent fashion. Because of that, we can envision a model with many implications within the nervous system."
A potential therapeutic approach to pain relief might include local injections to block VLK in the spine, he said, although more research is needed to determine how widespread the synaptic VLK mechanism is in the nervous system.
"We're most excited about having discovered that kinases act within the synapse, not just inside neurons. It's a huge update to our understanding of the basic mechanisms that regulate receptors involved in synaptic plasticity," Price said. "And I think we're just scratching the surface. Showing that kinase activity within the synaptic cleft is important for how synapses work will have a big impact on how we think about synaptic plasticity."
UT Dallas-affiliated authors of the paper include neuroscience research scientists Moeno Kume BS'17 PhD'23, Ishwarya Sankaranarayanan PhD'22 and Stephanie Shiers PhD'19; cognition and neuroscience doctoral students Lucy He and Khadijah Mazhar; and Juliet Mwirigi BS'17, PhD'23, Jessica Loucks BS'22 and Rohita Arjarapu BS'24.
Additional authors are from Tulane, the UT San Antonio Health Science Center, UT MD Anderson Cancer Center, the University of Houston Tilman J. Fertitta Family College of Medicine, Princeton University, the University of Wisconsin-Madison, NYU Grossman School of Medicine and Thomas Jefferson University.
This research was supported by grants from the National Institute of Neurological Disorders and Stroke ( R01NS111976 , R01NS115441 , U19NS130608 ), National Institute on Drug Abuse ( R01DA022727 ) and the National Center for Research Resources ( S10RR027990 ), all of which are components of the National Institutes of Health.
