Summary: Researchers developed ConVERGD, a tool enabling precise manipulation of specific cell subpopulations, thereby improving studies of cellular diversity. The study demonstrated the utility of ConVERGD by identifying a subpopulation of norepinephrine neurons linked to anxiety. This innovative approach could impact research and treatment in various fields.
Highlights:
- Precision tool: ConVERGD allows precise targeting and manipulation of cell subpopulations.
- Application to neuroscience: Used to identify a subpopulation of norepinephrine neurons affecting anxiety.
- Broad impact: Potential applications beyond neuroscience, benefiting diverse research areas.
Source: St. Jude Children’s Research Hospital
As genetic sequencing technologies have become more powerful, our understanding of cellular diversity has grown in parallel. This led scientists at St. Jude Children’s Research Hospital to create a tool to improve the ease and precision with which investigators can study specific subpopulations of cells.
The tool, named Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), allows researchers to specifically access these subgroups of cells and precisely manipulate them based on multiple characteristics of the cell.
ConVERGD offers many advantages over existing intersectional expression platforms by supporting more complex genetic loads 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 research on cellular subpopulations could have on basic research and health care.
The results were published today in Natural neuroscience.
Same cell type, different functions
For Lindsay Schwarz, PhD, Department of Developmental Neurobiology at St. Jude, necessity led to invention as she explored the landscape of neuronal cells, and in particular neuronal cells that produce norepinephrine.
“Norepinephrine neurons were thought to be just one type of neuron. But when activated in the brain, they can cause many different types of behaviors, such as improving attention and memory formation or triggering a stress response or reaction. fight or flight,” Schwarz said.
“But if it’s only one type of neuron that releases this molecule, then how does that make you do different things?”
Exploring such questions requires the ability to selectively interrogate cellular subpopulations with extreme biases. To this end, Schwarz found that all attempts using current practices failed.
“We didn’t start this project thinking we were creating a new tool, but it seemed like a need in the community. »
Improving current technology for targeting cellular subpopulations
Targeting subpopulations of cells requires passing them through several genetic filters. These intersectional filters interrogate the genes expressed by cells and the pathways and connections they make, analyzing different subpopulations so that researchers can focus on a select group of isolated cells.
The use of adeno-associated virus (AAV)-based reporter tools, capable of delivering genetic material into specific cells with high precision, constitutes an ideal approach to apply these intersectional filters.
These reporter tools are used to mark or monitor gene expression and protein localization in specific cells or regions. However, they can be complex to design and offer limited space within them.
“One of our main goals was to design a tool in which your gene of interest was only expressed when transmitted with multiple features, but in which end users could very easily edit and insert the genes of their choice,” Schwarz explained.
Robust ribozymes deliver next-generation specificity
Schwarz and first author Alex Hughes, PhD, a graduate of the St. Jude Graduate School of Biomedical Sciences, currently at the Allen Institute for Brain Science, leveraged two distinct technologies in the design of ConVERGD, namely the reporter technology based on AAV and inspiration from the world of ribozymes, strands of RNA that can behave like enzymes by catalyzing biochemical reactions.
Importantly, ribozymes can be engineered to control the on/off switch of gene expression with extreme precision.
“We first learned about ribozymes from a journal club that was thinking more therapeutically about how to use AAVs,” Schwarz said. “Alex came back and thought he could find 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 norepinephrine neurons.
“Collectively, norepinephrine neurons do a lot of different things,” Schwarz explained.
“The subgroup we were targeting produces norepinephrine, but it also produces this other opioid peptide called dynorphin, which has not yet been characterized in these neurons. With ConVERGD, we found that activation of these dynorphin-expressing neurons was sufficient to elicit an anxiety response.
By analyzing functions and assigning them to a subpopulation 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 on a global scale,” Schwarz said.
“You’re also going to see harm 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 implications outside of St. Jude. “We’re really excited about this for the community,” Schwarz said. “ConVERGD should be compatible with any tissue. This could be useful beyond neuroscience.
Authors and funding
Other authors of the study are 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, the fundraising and awareness organization of St. Jude.
About this neurotechnology research news
Author: Chelsea Bryant
Source: St. Jude Children’s Research Hospital
Contact: Chelsea Bryant – St. Jude Children’s Research Hospital
Picture: Image is credited to Neuroscience News
Original research: Closed access.
“A single-vector intersectional AAV strategy for interrogating cellular diversity and brain function” by Lindsay Schwarz et al. Natural neuroscience
Abstract
A single-vector intersectional AAV strategy to interrogate cellular diversity and brain function
As the discovery of cellular diversity in the brain accelerates, so does the need for tools that target cells based on multiple characteristics.
Here, we developed conditional viral expression by guided ribozyme degradation (ConVERGD), a single-construct, adeno-associated virus-based intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches to deliver a 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 features, support for larger and more complex payloads, and easy vectoring. to modify for rapid expansion of the toolbox.
In the present report, we used ConVERGD to characterize an unexplored subpopulation of norepinephrine (NE)-producing neurons within the rodent locus coeruleus that co-express the endogenous opioid gene, prodynorphin (Pdyn).
These studies present ConVERGD as a versatile tool for targeting diverse cell types and reveal Pdyn-expressing NE+ locus coeruleus neurons as a small neuronal subpopulation capable of eliciting anxiety-like behavioral responses in rodents.