| NSF Org: |
ECCS Division of Electrical, Communications and Cyber Systems |
| Recipient: |
|
| Initial Amendment Date: | August 27, 2019 |
| Latest Amendment Date: | August 27, 2019 |
| Award Number: | 1926793 |
| Award Instrument: | Standard Grant |
| Program Manager: |
Svetlana Tatic-Lucic
ECCS Division of Electrical, Communications and Cyber Systems ENG Directorate for Engineering |
| Start Date: | September 15, 2019 |
| End Date: | August 31, 2023 (Estimated) |
| Total Intended Award Amount: | $1,000,000.00 |
| Total Awarded Amount to Date: | $1,000,000.00 |
| Funds Obligated to Date: |
|
| History of Investigator: |
|
| Recipient Sponsored Research Office: |
3112 LEE BUILDING COLLEGE PARK MD US 20742-5100 (301)405-6269 |
| Sponsor Congressional District: |
|
| Primary Place of Performance: |
2236 Jeong H. Kim Engineering Bu College Park MD US 20742-3285 |
| Primary Place of
Performance Congressional District: |
|
| Unique Entity Identifier (UEI): |
|
| Parent UEI: |
|
| NSF Program(s): | IntgStrat Undst Neurl&Cogn Sys |
| Primary Program Source: |
|
| Program Reference Code(s): |
|
| Program Element Code(s): |
|
| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.041 |
ABSTRACT
The goal of this project is to create an engineering solution to measure and predict the molecular communication across the gut-microbiome-brain axis. This platform has the potential to facilitate the fundamental understanding of gut microbiome communication with the nervous system. The project will quantify release patterns of key molecules involved in this cross-talk and identify their influence on neural activation and behavior. The gut-microbiome-brain axis, comprising a vast network of nerves innervating the gut and propagating signals to the brain, is a major influencer of behavior and cognition. The neurotransmitter serotonin is a key molecule in this pathway; gut epithelial cells sense luminal conditions and release serotonin to stimulate nearby neurons. The gut microbiome has been shown to mediate this serotonin release, a process that is also linked to the co-occurrence of gastric and neural disorders. The technical underpinnings of this work involve designing and constructing a device that enables researchers to assemble the essential components of the gut-microbiome-neuron tissue interface. The device is fabricated with sensors to obtain information that is currently inaccessible - collecting molecular information at the length and time scales of the cells and tissues under investigation. The data extracted from this platform will enable temporal correlation and prediction of microbial, gut, and neural signaling patterns. This work provides opportunities to bring together researchers and stakeholders from various disciplines including electrical and computer engineering, bioengineering, molecular biology, neuroscience, and data science to develop a system-oriented approach. Further, this project promotes the participation of women, historically underrepresented in engineering, and undergraduates through programs such as Women in Engineering Research Fellowship and First-Year Innovation and Research Experience (FIRE).
Multidisciplinary engineering methods are essential to building an in vitro discovery platform capable of directly monitoring chemical transduction patterns along the gut-neuron axis. In TASK 1, electrochemical sensors will be directly fabricated on a porous cell culture substrate, allowing direct access to cellular and molecular mechanisms of an in vitro model gut epithelium. Impedance monitoring of the cell layer will detect physical changes over time (e.g., barrier integrity). Potentiometric monitoring will detect real-time serotonin released from gut cells due to bacterial metabolite stimulation. In TASK 2, the neural effect of gut serotonin signaling will be studied by exposing this cell-released serotonin to an isolated ex vivo crayfish nerve cord with connected and innervated hindgut. Neurobehavioral activation patterns will be recorded during hindgut peristalsis in motor and sensory neurons that bidirectionally connect the central and enteric nervous systems. Machine learning approaches will identify key variables to quantify discrete serotonin release and neuronal activation patterns. In TASK 3, the mucosal layer of the gut epithelium will be colonized with specific gut microbes to assess bacterial influence on barrier integrity, serotonin release patterns, and resulting neuromuscular activation. Classification via machine learning will quantify the wholistic and synergistic effects of different microbial combinations on time-dependent serotonin release profiles and downstream effects. There are multiple novel aspects of this work. First, this is a new platform implementing extensive integrated cell-interfacial sensors for direct access to real-time cell and molecular data. Second, the use of this technology to investigate the interplay between gut and nervous system can give unprecedented insight into the vast and relatively inaccessible gut-brain transduction pathways. Third, machine learning analysis can identify meaningful patterns of serotonergic communication and predict the expected impact of gut bacteria on neural behavior.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH
Note:
When clicking on a Digital Object Identifier (DOI) number, you will be taken to an external
site maintained by the publisher. Some full text articles may not yet be available without a
charge during the embargo (administrative interval).
Some links on this page may take you to non-federal websites. Their policies may differ from
this site.
PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
Recent research has unveiled the intricacies of the gut-microbiome-brain-axis (GMBA), an extensive network of enteric nerves that innervate the gut and transmit signals to the brain. This axis profoundly influences behavior and cognition. Notably, the neurotransmitter serotonin (5-HT) plays a pivotal role in this pathway, with gut epithelial cells releasing 5-HT to activate nearby neurons and the gut microbiome concurrently stimulating 5-HT release. Additionally, the GMBA is associated with the co-occurrence of gastric and neural disorders. However, a scarcity of methodologies capable of studying cellular and molecular interactions between these cells and tissues at their natural time and length scales persists. Such methods are vital for comprehending how the gut microbiome impacts both the enteric nervous system (ENS) and the central nervous system (CNS), as well as the interactions between these two systems.
The highly complex and challenging nature of this interface in animal models has left significant gaps in our knowledge regarding specific molecular interactions within the GMBA. These interactions encompass three primary components: (1) the interplay between the microbiome and the gut, (2) the biosynthesis and release of 5-HT from the gut epithelium, and (3) the subsequent activation of the nervous systems. To address these gaps, we developed an interdisciplinary discovery platform?a 3D printed transwell housing with impedimetric, potentiometric, and electrophysiological interfacial sensors. This platform, in an unprecedented manner, enables real-time monitoring of signal transduction between the microbiome, gut epithelium, and an ex vivo invertebrate CNS-ENS-hindgut preparation. Our developed system investigates serotonin-induced neurobehavioral effects, utilizing various tasks to unravel multi-tissue signaling mechanisms.
Our specific objectives can be summarized into three tasks: (1) developing an in vitro multimodal sensor-integrated discovery platform?a system equipped with electrochemical sensors for real-time potentiometric sensing of released 5-HT (2) eliciting patterns of stimulated 5-HT release from cultured gut cells and examine their corresponding effects on the crayfish preparation, and (3) engineering bacteria to produce γ-aminobutyric (GABA), a common metabolite of commensal gut microorganisms, to investigate the influence of gut microbes on 5-HT dynamics.
Our project resulted in unique modules capable of growing an in vitro model of the gut epithelium on a substrate integrated with an electrochemical 5-HT sensor. This achievement was made possible through a straightforward yet robust hybrid fabrication protocol that combines cleanroom technologies with additive manufacturing. This approach allowed us to create electrode-integrated porous member-based and glass slide-based in vitro modules. These modules feature robust and biocompatible materials for cell growth and the detection of cell-released 5-HT. Additionally, we designed and fabricated a 3D printed villi structure to mimic intestinal epithelial physiology, providing a larger surface area to host more 5-HT-producing cells and potentially yielding more chemical signals for downstream sensors or compartments. Furthermore, an electrophysiological module was developed to house a dissected crayfish nerve cord, allowing for electrode accessibility to assess nerve responses to 5-HT.
For the first time, our system integrates these platforms, enabling the study of molecular signaling between gut and nerve cells, and facilitating real-time monitoring of both tissues (gut epithelium and nerve tissues) within the GMBA. The in vitro gut cell culture module offers a controlled and accessible platform for the study of gut physiology, while the electrophysiology module supports the investigation of nerve signaling in the ex vivo crayfish abdominal nerve cord.
Furthermore, we engineered E. coli to express glutamate decarboxylase (GAD), the enzyme responsible for producing GABA, and optimized the fermentation conditions to produce biologically relevant levels of GABA from engineered E. coli when provided with glutamate. We selected GAD from archaeon P. horikoshii (GadB) and fused it with the engineered AIDA-I autotransporter to enable surface expression on bacterial cells. The engineering bacteria showed a successful GABA production, which can be used to study the inhibited 5-HT release and suppressed inflammatory response from the EC cells.
Overall, identifying the relevant mechanisms underlying GMBA function is crucial for advancing future clinical outcomes. Our multi-disciplinary research represents the first direct examination of model cell systems with interfacial biosensor technology to comprehend the impact of gut microbes on interconnected neural pathways. This approach offers cellular and molecular precision that is not achievable with existing systems. We anticipate that the new knowledge and clinical health advancements generated by this discovery platform will benefit researchers and the public by advancing our understanding of signaling events within the GMBA, ultimately aiding in the diagnosis and treatment of related disorders.
Last Modified: 09/16/2023
Modified by: Reza Ghodssi
Please report errors in award information by writing to: awardsearch@nsf.gov.
