Different Bioactive Neuropeptides are Expressed in Two Sub-Classes of GABAergic RME Nerve Ring Motorneurons in Ascaris suum

Abstract

Neuropeptides can have significant effects on neurons and synapses, but among the ∼250 predicted peptides in nematodes, few have been characterized functionally. Here, we report new neuropeptides in the 4 RME nerve ring motorneurons of the nematode Ascaris suum. These GABAergic neurons are involved in three-dimensional head movement. Mass spectrometry (MS) of single dissected RMEs detected a total of 12 neuropeptides (encoded by five genes), nine of which are novel. None of these are expressed in the DI/VI inhibitory GABAergic motorneurons that synapse onto body wall muscle. Using peptide sequences obtained by tandem MS, we cloned the peptide-encoding transcripts and synthesized riboprobes for in situ hybridization (ISH). This complementary technique corroborated the results from single-cell MS, showing that the dissections were not contaminated with adhering tissue from other cells. We also synthesized a multiple antigenic peptide to raise a highly specific antibody against one of the endogenous peptides, which labeled the same cells detected by MS and ISH. Our results show that the RMEs can be divided into two subsets: RMED/V (expressing afp-2, afp-15, Asu-nlp-58, and high levels of afp-16) and RMEL/R (expressing afp-15 and low levels of afp-4 and afp-16). Almost all of these peptides are bioactive in A. suum.

Sydney Brenner. 13 January 1927—5 April 2019

“…In addition, a small group started studying the parasitic nematode Ascaris with a view to undertaking electrophysiological experiments on this much larger organism. However, Ascaris research was not continued at the LMB, although Stretton took the project to the University of Wisconsin where he used this organism to good effect in studies of electrophysiology (Davis & Stretton 1996) and of neuropeptides (Knickelbine et al 2018). The main thrust of Sydney's early work with C. elegans was the isolation and mapping of behavioural and morphological mutants.…”

A Potential Golden Age to Come—Current Tools, Recent Use Cases, and Future Avenues for De Novo Sequencing in Proteomics

“…De novo sequencing is an important tool here, too, as accurate sequence references may be lacking and the peptides are often chemically modified. In their study Knickelbine et al. were able to obtain 12 bioactive neuropeptides that are expressed in the nematode Ascaris suum using de novo sequencing.…”

My life with Sydney, 1961–1971

“…But 3 years after I went back to Cambridge, my appointment ran out, and I was on the job market. I have been in the Zoology Department (now Department of Integrative Biology) of the University of Wisconsin-Madison ever since, and was lucky to have fantastic students and postdocs, and we did the electrophysiology, biochemistry, electron microscopy, and mass spectrometry (to characterize the multiplicity of neuropeptides that make the functional circuitry much more complicated than we ever imagined) (Davis & Stretton, 1996;Konop et al, 2015a,b;Knickelbine et al, 2018).…”

“…Kari Andersen discovered how to dissect identified neuronal cell bodies, and Jessie Jarecki performed single cell MS-the cells are big enough that neuropeptides can be characterized and sequenced by MS, all from a single cell (Jarecki et al, 2010), and with no constraint imposed by relatedness to known peptides. This work was continued by Molly Sygulla and Chris Konop (Konop, Knickelbine, Sygulla, Vestling, & Stretton, 2015a,b), and by Jenny Knickelbine, my last graduate student (Knickelbine et al, 2018). We were building up a robust map of the neurons that synthesize different neuropeptides, robust because it relies on 3 independent techniques, antibody staining, in situ hybridization, and single cell MS, all carried out directly on normal unmodified cells.…”

“…We also continued to explore bioactivity, either by observing peptide-induced effects on locomotory behavior (Reinitz et al, 2000(Reinitz et al, , 2011 or effects on muscle contraction (Knickelbine et al, 2018). We were surprised to find that almost all the peptides we isolated from A. suum affect locomotion and/or muscle contraction.…”