Van Bael, Sven et al. (2017) International Worm Meeting "MS-based neuropeptidomics in C. elegans."

Boonen, Kurt, De Haes, Wouter, Temmerman, Liesbet, Van Bael, Sven, Edwards, Samantha Louise, Schoofs, Liliane

[International Worm Meeting, 2017]

Neuropeptides constitute a functionally diverse family of neurochemical signaling molecules. C. elegans is well-suited for the study of neuropeptide biochemistry and function, since numerous peptides are known to contribute to many behavioral regimens, yet deleting peptides - or their processing enzymes altogether - does not affect C. elegans viability. We have developed an optimized protocol that enables the detection of about 120 individual peptides per sample (under reference conditions; treatment may increase or decrease this number), as such vastly increasing the detection potential of C. elegans peptidomics. As a proof of concept, we applied this protocol to characterize the C. elegans enzymes required for the last step in the production of many bioactive peptides - the carboxyterminal amidation reaction - via mutant analysis, and faulty carboxyterminal amidation indeed results in a severely altered neuropeptide profile. Overall, about half of the more than 250 predicted peptides have been detected with our current method under standard conditions, and we are working on expanding the potential even further. We estimate the number of currently detectable peptides to be somewhat larger, as it can be expected that several peptides would be produced under specific conditions only, yet some low-abundant peptides that are expressed in only a few cells still escape detection in whole-mount peptidomics. This problem should be solved by our efforts in establishing single-cell peptidomics approaches, which will increase the signal-to-noise ratio drastically.

Van Bael, Sven et al. (2021) International Worm Meeting "Quantitative peptidomics in C. elegans via targeted mass spectrometry of neuropeptides"

Van Bael, Sven, Ludwig, Christina, Boonen, Kurt, Temmerman, Liesbet, Abele, Miriam

[International Worm Meeting, 2021]

Neuropeptides are a class of bioactive peptides that obtain their biological activity after cleavage from larger precursor proteins. Once released, these peptides are able to regulate standard physiological functions such as digestion and reproduction but also exert long-term effects in more complex processes, including behavioral adaptation, memory processes and aging. Hence, neuropeptides are often an interesting entry point in an effort to study and better understand these types of behavior. Current strategies for studying relative differences in the neuropeptidome often rely on RNA sequencing, which remains completely blind to the real abundancies of actual neuropeptides, as these result from extensive post-translational processing. On the other hand, current mass spectrometric methods for neuropeptide identification are discovery-driven and lack robustness and reproducibility, hampering quantitative studies. We here present a mass spectrometry-based approach for the relative quantification of the neuropeptidome in C. elegans, using a parallel reaction monitoring method. Our current method can detect and quantify 288 mature neuropeptides, this corresponds to 67% of the (known and predicted) neuropeptidome of C. elegans. When applied to wild type controls, our method identified 178 neuropeptides with a 96% overlap between samples. Insofar as we are aware, we hereby are able to provide the most extensive method to map the peptidome of an animal. We are validating the method for differential studies, delivering a promising avenue to finally enable the detection and differential analysis of neuropeptidomic variations over different conditions.

Rafi, Ibnul et al. (2019) International Worm Meeting "The neuropeptidergic connectome of Caenorhabditis elegans."

Temmerman, Liesbet, Vertes, Petra, Hobert, Oliver, YANG, Xinyi, Vandewyer, Elke, Schafer, William, Beets, Isabel, Sanchez, Lidia, Chen, Chi, Rafi, Ibnul, Bael, Sven

[International Worm Meeting, 2019]

A "connectome" describes the complete synaptic wiring diagram of a brain. Past and current connectomic efforts are focused on determining the anatomical, i.e. chemical and electrical synaptic connections between neurons in a brain, thereby completely ignoring aspects of neuronal communication that are likely of equal importance but are not captured by anatomical connections: Neuromodulatory communication by neuropeptides and their cognate receptors. Neuropeptidergic communication is usually non-synaptic, i.e. neuropeptides are often released from non-synaptic sites and cognate neuropeptide receptors are often located distal from the source of the cognate neuropeptide. While the importance of a number of neuropeptides and their receptors in controlling behavior are well appreciated, the extent of usage of neuropeptidergic signaling is only beginning to be fully appreciated. Every neuron in an animal nervous system is now thought to express at least one neuropeptide, but the pathways of communication of these neuropeptidergic signals have not been comprehensibly mapped and, hence, our understanding of information flow in the nervous system remains incomplete. A consortium of four laboratories (Hobert, Schafer, Beets, Temmerman Labs) has received NIH funds to establish the first comprehensive neuropeptidergic connectome. We build such a connectome through (1) comprehensively defining ligand/receptor pairs through in vitro receptor activation assays, (2) defining the expression patterns of all neuropeptide and neuropeptide receptor encoding genes, (3) synthesizing these data into a neuropeptidergic network and computationally comparing the topology of this network to the synaptic connectivity network and (4) undertaking a preliminary functional validation of specific nodes and edges of this network. Comparing a neuropeptidergic connectome to that of the completely established synaptic connectome, we expect to describe a "multilayer connectome" with substantially distinct pathways of information flow, as well as distinct and similar topological features.

Cockx, Bram et al. (2021) International Worm Meeting "Peptidergic modulation of dispersal behavior in pathogenic and free-living nematodes"

Beets, Isabel, Dalzell, Johnathan, Lee, Junho, Yang, Heeseung, Van Bael, Sven, Vandewyer, Elke, Kieswetter, Amanda, Boelen, Rose, Cockx, Bram, Temmerman, Liesbet

[International Worm Meeting, 2021]

Emerging evidence suggests that behavioral changes associated with host finding of pathogenic nematodes may be regulated by neuropeptides. Hence, we started from peptidomic discovery in such a species, Steinernema carpocapsae, to direct functional discovery. We observe numerous similarities between the well-studied C. elegans peptidome and the S. carpocapsae peptidome, which we further exploit to understand neuropeptidergic contributions to regulating nictation, an evolutionary conserved behavior for foraging in (these) nematodes. An in-house method based on acidified methanol was used to extract endogenous neuropeptides of Steinernema carpocapsae infective juveniles, a life stage similar to the C. elegans dauer stage. Neuropeptide identification was done by state-of-the-art UHPLC-MS/MS. We detected 30% (139) of the predicted peptidome in these infective juveniles, which provides a resource for comparison with the C. elegans dauer peptidome (in house). We hypothesized that nictation-relevant peptides will be abundant in infective juveniles and/or dauers, and prioritized these for functional assays. Out of several tested target genes, we found at least one neuropeptidergic signaling system that is involved in modulation of nictation behavior, which we assayed using microdirt arenas. In addition, using phylogenetic analyses, we aim to get a better global understanding of conserved peptidergic signaling systems in parasitic and free living nematodes. Steinernema spp. are used as eco-friendly alternative for chemicals to combat pest insects. Knowledge on neuropeptidergic regulation of host-finding strategies will help understand how entomopathogenic nematodes regulate their behavior. This should contribute to improving their applicability and host specificity in the field.