Summary: Researchers have developed ConVERGD, a tool for the precise manipulation of specific cell subpopulations that improves the study of cell diversity. The study demonstrated the utility of ConVERGD by identifying a subpopulation of norepinephrine neurons associated with anxiety. This innovative approach could influence research and treatment in various fields.
Key facts:
- Precision tool: ConVERGD enables precise targeting and manipulation of cell subpopulations.
- Neuroscience Applications: Used to identify a subpopulation of norepinephrine neurons influencing anxiety.
- Broad impact: Potential applications outside of neuroscience that benefit various fields of research.
Source: Children’s Research Hospital St. Jude
As gene sequencing technologies become more powerful, our understanding of cellular diversity grows in parallel. This led scientists from the Children’s Research Hospital St. Jude to create a tool to improve the ease and precision with which investigators can study specific subpopulations of cells.
A tool called Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD) allows researchers to specifically access these subsets of cells and precisely manipulate them based on different cell properties.
ConVERGD offers numerous advantages over existing intersectional expression platforms by accommodating more complex genetic payloads and increased adaptability.
The researchers demonstrated the utility of ConVERGD by studying a previously unidentified subpopulation of norepinephrine neurons. The work demonstrates the substantial impact that cell subpopulation research could have on basic research and healthcare.
The findings were published today in Nature Neuroscience.
Same cell type, different functions
For Lindsay Schwarz, PhD, St. Jude Department of Developmental Neurobiology, necessity drove the invention as she explored the landscape of neuronal cells, and in particular the neuronal cells that produce norepinephrine.
“Norepinephrine neurons were thought to be one type of neuron.” But when they’re activated in the brain, they can cause many different types of behavior, such as enhancing attention and forming memory or triggering a stress response or a fight-or-flight response,” Schwarz said.
“But if it’s just one type of neuron releasing this one molecule, how does that make you do different things?”
Probing such questions requires the ability to selectively interrogate cell subpopulations with extreme biases. To this end, Schwarz found that all attempts using current procedures were unsuccessful.
“We didn’t go into this project thinking we were going to create a new tool, but it felt like a need in the community.”
Improving current cell subpopulation targeting technology
Targeting subpopulations of cells requires passing them through several genetic filters. These intersection filters detect what genes cells express and what pathways and connections they make, analyzing different subpopulations to allow researchers to focus on a select group of isolated cells.
The use of adeno-associated virus (AAV)-based reporter tools that can deliver genetic material to specific cells with high fidelity is an ideal approach for the application of these intersection filters.
These reporter tools are used to label or monitor gene expression and protein localization in specific cells or regions. However, their design can be complex and offer a limited amount of space.
“One of our main goals was to design a tool where your desired gene will only be expressed when it’s mediated with a lot of features, but it’s really easy for end users to modify it and put in whatever genes they want,” Schwarz explained.
Robust ribozymes offer next-generation specificity
Schwarz and first author Alex Hughes, PhD, a graduate of St. Jude Graduate School of Biomedical Sciences, now the Allen Institute for Brain Science, used two separate technologies to design ConVERGD, namely an AAV-based reporter technology and inspiration from the world of ribozymes, chains of RNA that can act like enzymes by catalyzing biochemical reactions .
Importantly, ribozymes can be engineered to control the on/off switch for gene expression with extreme precision.
“We initially heard about ribozymes from a journal that was thinking more therapeutically about how to use AAV,” Schwarz said. “Alex came back and figured he could figure out a way to use them in neuroscience tools.
Exciting for the neuroscience community and beyond
As a proof of concept, Schwarz and Hughes used ConVERGD to interrogate a subpopulation of noradrenaline neurons.
“Together, norepinephrine neurons do a lot of different things,” Schwarz explained.
“The subset we focused on makes norepinephrine, but it also makes this other opioid peptide called dynorphin, which hasn’t been characterized in these neurons yet.” With ConVERGD, we found that activating just these dynorphin-expressing neurons was enough to trigger an anxiety response.”
By analyzing the functions and assigning them to subpopulations of cells, Schwarz hopes that targeted therapy is a possibility.
“We treat anxiety and depression with drugs that target norepinephrine signaling, but they target it globally,” Schwarz said.
“You’ll also see damage to other important functions of norepinephrine that you don’t want to see.” Targeting these neurons more specifically could help improve this.”
The work will have a ripple effect beyond St. Jude. “We’re really excited about this for the community,” Schwarz said. “ConVERGD should be accessible to any tissue.” It could be useful beyond neuroscience.”
Authors and funding
Additional study authors include Brittany Pittman, Beisi Xu, Jesse Gammons, Charis Webb, Hunter Nolen, Phillip Chapman and Jay Bikoff, St. Jude.
The study was supported by grants from the Brain & Behavior Research Foundation (NARSAD Young Investigator Grant), the National Institutes of Health (1DP2NS115764, P30 CA021765), and ALSAC, a fundraising and awareness organization for St. Jude.
About this news from neurotechnological research
Author: Chelsea Bryant
Source: Children’s Research Hospital St. Jude
Contact: Chelsea Bryant – Children’s Research Hospital St. Jude
Picture: Image is credited to Neuroscience News
Original Research: Closed access.
“A single-vector intersectional AAV strategy for probing cellular diversity and brain function” by Lindsay Schwarz et al. Nature Neuroscience
Abstract
A single-vector intersectional AAV strategy for interrogating brain cellular diversity and function
As the discovery of cellular diversity in the brain accelerates, so does the need for tools that target cells based on multiple functions.
Here, we developed Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), a single-construct adeno-associated viral intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches to deliver molecular cargo to specific neuronal subtypes.
ConVERGD offers advantages over existing intersectional expression platforms, such as expanded intersectional targeting with up to five recombinase-based functions, accommodating larger and more complex payloads, and a vector that can be easily modified for rapid toolbox expansion.
In this report, we used ConVERGD to characterize an unexplored subpopulation of norepinephrine (NE)-producing neurons in the locus coeruleus of rodents that coexpress the endogenous opioid gene prodynorphin (Pdyn).
These studies show ConVERGD as a versatile tool for targeting different cell types and detection Pdyn-expressing NO+ locus coeruleus neurons as a small neuronal subpopulation capable of driving anxiogenic behavioral responses in rodents.