isolation-active-caenorhabditis-elegans-nuclear-extract

Isolation of Active <em>Caenorhabditis elegans</em> Nuclear Extract and Reconstitution for <em>In Vitro</em> Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

Washington State University

Upon pathogen infection, the nervous system regulates innate immunity to confer coordinated protection to the host. However, the precise mechanisms of such regulation remain unclear. Previous studies have demonstrated that OCTR-1, a putative G protein-coupled receptor for catecholamine, functions in the sensory neurons designated "ASH" to suppress innate immune responses in  It is unknown what molecules act as OCTR-1 ligands in the neural immune regulatory circuit. Here we identify neurotransmitter octopamine (OA) as an endogenous ligand for OCTR-1 in immune regulation and show that the OA-producing RIC neurons function in the OCTR-1 neural circuit to suppress innate immunity. RIC neurons are deactivated in the presence of pathogens but transiently activated by nonpathogenic bacteria. Our data support a model whereby an octopaminergic immunoinhibitory pathway is tonically active under normal conditions to maintain immunological homeostasis or suppress unwanted innate immune responses but downregulated upon pathogen infection to allow enhanced innate immunity. As excessive innate immune responses have been linked to a myriad of human health concerns, our study could potentially benefit the development of more-effective treatments for innate immune disorders. Insufficient or excessive immune responses to pathogen infection are major causes of disease. Increasing evidence indicates that the nervous system regulates the immune system to help maintain immunological homeostasis. However, the precise mechanisms of this regulation are largely unknown. Here we show the existence of an octopaminergic immunoinhibitory pathway in  Our study results indicate that this pathway is tonically active under normal conditions to maintain immunological homeostasis or suppress unwanted innate immune responses but downregulated upon pathogen infection to allow enhanced innate immunity. As excessive innate immune responses have been linked to human health conditions such as Crohn's disease, rheumatoid arthritis, atherosclerosis, diabetes, and Alzheimer's disease, elucidating octopaminergic neural regulation of innate immunity could be helpful in the development of new treatments for innate immune diseases.

Octopaminergic Signaling Mediates Neural Regulation of Innate Immunity in Caenorhabditis elegans.

Increasing evidence indicates that infection-triggered host defenses are regulated by the nervous system. However, the precise mechanisms of this regulation are not well understood. Here, we demonstrate that neuronal G protein-coupled receptor NPR-8 negatively regulates  defense against pathogen infection by suppressing cuticular collagen expression. NPR-8 controls the dynamics of cuticle structure in response to infection, likely through its regulation of cuticular collagen genes which, in turn, affects the nematode's defense. We further show that the defense activity of NPR-8 is confined to amphid sensory neurons AWB, ASJ, and AWC. It is generally believed that physical barrier defenses are not a response to infections but are part of the body's basic innate defense against pathogens. Our results challenge this view by showing not only that  cuticle structure dynamically changes in response to infection but also that the cuticle barrier defense is regulated by the nervous system.

Neuronal GPCR NPR-8 regulates  defense against pathogen infection.

Caenorhabditis elegans is an excellent model organism for biological research, but its contributions to biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest obstacles for C. elegans biochemical studies is the high difficulty of obtaining functionally active nuclear extract due to its thick surrounding cuticle. A C. elegans in vitro transcription system was once developed by Lichtsteiner and Tjian in the 1990s, but it has not become widely used, most likely because the transcription reactions were re-constituted with nuclear extract from embryos, not from larval or adult worms, and the method of Dounce homogenization used to prepare the nuclear extract could lead to protein instability. Besides Dounce homogenization, several other techniques were developed to break worms, but no transcription reactions were re-constituted following worm disruption using these approaches. A C. elegans transcription system with effective preparation of functionally active nuclear extract from larval or adult worms has yet to be established. Additionally, non-radioactive methods for detecting transcription as alternatives to the conventional radioactive detection also need to be adapted into such an in vitro system.

A novel in vitro Caenorhabditis elegans transcription system.

The innate immune system is a complex collection of physical barriers and physiological defense responses to internal and external environmental assaults. Recent studies in the model organism  have highlighted how the nervous system interacts with the innate immune system to generate coordinated protective responses. Indeed, studies on neuro-immune interaction pathways have provided mechanistic insights into the roles of neuro-immune communication in modulating both immune and behavioral responses to pathogen attacks. The nervous system releases a variety of neurotransmitters, peptides, and hormones that regulate the innate immune response, while the innate immune system also relays information to the nervous system to affect learning and behavioral responses. Although these interactions still need further investigation, the knowledge that we have gained thus far has improved our understanding of how separate biological systems can act collectively for the survival and well-being of an organism. Here, we review recent studies on neuro-immune communication related to the survival and defense of  against pathogens.

Phillip Wibisono

Department of Translational Medicine and Physiology,
Elson S. Floyd College of Medicine,
Department of Translational Medicine and Physiology, Elson S. Floyd College of Medicine

Washington State University

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Phillip Wibisono

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Translational Medicine and Physiology,Elson S. Floyd College of Medicine,Department of Translational Medicine and Physiology, Elson S. Floyd College of Medicine

Washington State University

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Department of Translational Medicine and Physiology,
Elson S. Floyd College of Medicine,
Department of Translational Medicine and Physiology, Elson S. Floyd College of Medicine