Cellular targeting techniques can isolate neuronal subpopulations and link them to behavioral states

As genetic sequencing technologies become more powerful, our understanding of cellular diversity simultaneously improves. In response, scientists at St. Jude Children's Research Hospital have developed tools that improve the ease and accuracy with which researchers can study specific cell subpopulations.

The tool, named “Conditional Viral Expression by Ribozyme-Induced Degradation (ConVERGD),” allows researchers to specifically access subgroups of these cells and precisely manipulate them based on multiple cellular characteristics.

ConVERGD offers many advantages over existing cross-expression platforms by accommodating more complex gene payloads and providing greater adaptability. The researchers demonstrated the utility of ConVERGD by studying a previously unidentified subpopulation of norepinephrine neurons.

The study, published today (May 27), demonstrates the profound impact that investigating cell subpopulations can have on basic research and medicine. Nature Neuroscience.

Same Cell Type, Different Functions

Dr. Lindsay Schwartz of St. Jude's Department of Developmental Neurobiology came up with the invention out of necessity while studying nerve cells, specifically the nerve cells that produce norepinephrine.

“Norepinephrine neurons have long been thought of as just one type of neuron, but when activated in the brain they can trigger a variety of behaviors, including increasing attention and memory formation, as well as triggering stress and fight-or-flight responses,” Schwartz said. “But how can it trigger a variety of behaviors if it's just one type of neuron that's releasing this molecule?”

Exploring such questions requires the ability to selectively interrogate cellular subpopulations in highly unbiased ways. To this end, Schwartz found that all attempts with current methods fell short. “We didn't set out on this project with the idea of ​​building a new tool, but it seemed like there was a need in the community.”

Improving current cell subpopulation targeting technologies

To target subpopulations of cells, they must be passed through multiple genetic filters. These cross filters examine which genes cells express and what pathways and connections they form, parsing different subpopulations and allowing researchers to focus on specific, isolated groups of cells.

The use of adeno-associated virus (AAV)-based reporter tools, which can deliver genetic material to specific cells with high precision, is an ideal way to apply these cross-filters. These reporter tools are used to label or monitor gene expression and protein localization within specific cells or regions. However, they can be complex to design and have limited space within the tool.

“One of our main goals was to design a tool that would only express a gene of interest if it was transferred with multiple functions, and that could be easily modified by the end user to insert any gene of their choice,” Schwarz explains.

Potent ribozymes provide next-generation specificity

Schwartz and lead author Alex Hughes, PhD, a St. Jude School of Biomedical Sciences alumnus and now at the Allen Institute for Brain Science, used inspiration from two different technologies to design ConVERGD: AAV-based reporter technology and the world of ribozymes, strands of RNA that act like enzymes by catalyzing biochemical reactions.

Importantly, ribozymes can be designed to control the on/off switch of gene expression with great precision. “We first heard about ribozymes from a journal club that was thinking about how to use AAVs therapeutically,” Schwartz says. “Alex came back and thought we could figure out how to leverage this into a neuroscience tool.”

This is exciting for the neuroscience community and beyond.

As proof of concept, Schwartz and Hughes used ConVERGD to study a subpopulation of norepinephrine neurons. “As a whole, norepinephrine neurons do a variety of things,” Schwartz explains. “The subset we were targeting produces norepinephrine, but they also produce another opioid peptide called dynorphin, which had not previously been characterized in these neurons. With ConVERGD, we found that activating only these dynorphin-expressing neurons was enough to cause an anxiety response.”

Schwartz hopes that parsing out function and assigning it to subgroups of cells will enable targeted therapies. “We treat anxiety and depression with drugs that target norepinephrine signaling, but they target it globally,” Schwartz says. “We'll also see negative effects on other important functions of norepinephrine that we don't want. Targeting these neurons more specifically could help improve that.”

The research will have ripple effects beyond St. Jude's. “We're really excited about this research for the community,” Schwartz said. “ConVERGD should be adaptable to any organization. It may be useful beyond neuroscience.”