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Peptidergic dense-core vesicles are involved in packaging and releasing neuropeptides and peptide hormones—critical processes underlying brain, endocrine and exocrine function. Yet, the heterogeneity within these organelles, even for morphologically defined vesicle types, is not well characterized because of their small volumes. We present image-guided, high-throughput mass spectrometry-based protocols to chemically profile large populations of both dense-core vesicles and lucent vesicles for their lipid and peptide contents, allowing observation of the chemical heterogeneity within and between these two vesicle populations. The proteolytic processing products of four prohormones are observed within the dense-core vesicles, and the mass spectral features corresponding to the specific peptide products suggest three distinct dense-core vesicle populations. Notable differences in the lipid mass range are observed between the dense-core and lucent vesicles. These single-organelle mass spectrometry approaches are adaptable to characterize a range of subcellular structures.This article presents a workflow for image-guided MALDI MS characterization of peptide and lipid content in single organelles.

Single-cell mass spectrometry (MS) empowers metabolomic investigations by decreasing analytical dimensions to the size of individual cells and subcellular structures. We describe a protocol for investigating and quantifying metabolites in individual isolated neurons using single-cell capillary electrophoresis (CE) coupled to electrospray ionization (ESI) time-of-flight (TOF) MS. The protocol requires ∼2 h for sample preparation, neuron isolation and metabolite extraction, and 1 h for metabolic measurement. We used the approach to detect more than 300 distinct compounds in the mass range of typical metabolites in various individual neurons (25–500 μm in diameter) isolated from the sea slug ( Aplysia californica ) central and rat ( Rattus norvegicus ) peripheral nervous systems. We found that a subset of identified compounds was sufficient to reveal metabolic differences among freshly isolated neurons of different types and changes in the metabolite profiles of cultured neurons. The protocol can be applied to the characterization of the metabolome in a variety of smaller cells and/or subcellular domains.

The progressive accumulation of amyloid beta (Aβ) plaques is a hallmark of Alzheimer’s disease (AD). However, the biochemical mechanisms of their formation and the consequences associated with plaque formation remain elusive. In female 5xFAD and APP NL-G-F mice, we map region-specific, plaque-associated lipids with large molecular coverage including isomers. We describe a multimodal framework that integrates matrix assisted laser desorption/ionization with laser-induced postionization (MALDI-2) mass spectrometry imaging, trapped ion mobility spectrometry, and fluorescence microscopy. Our approach improves detectability and spatial-chemical resolution. We couple these measurements with a computational pipeline for multimodal image coregistration and discovery of plaque-altered lipids. Here, we show the lipids in and around Aβ plaques are highly heterogeneous. Integration of our data with existing spatial transcriptomics data suggests that region-specific accumulation of simple gangliosides is likely driven by lysosomal degradation of complex species. Together, this work provides a generalizable framework to understand lipid alterations within the Aβ plaque microenvironment. Amyloid plaques are a hallmark of Alzheimer’s disease. Better understanding of their biochemistry can inspire new biomarkers and therapeutics. Using multimodal mass spectrometry imaging, this work reveals surprising lipid heterogeneity in plaque microenvironments across the brain.

Accumulating evidence has supported diverse regulatory functions of astrocytes in different neural circuits as well as various aspects of complex behaviors. However, little is known about how astrocytes regulate different neuronal subpopulations that are linked to specific behavioral aspects within a single brain region. Here, we show that astrocytes in the medial prefrontal cortex (mPFC) encode anxiogenic environmental cues in freely behaving mice. Silencing mPFC astrocyte Ca 2+ signaling heightens anxiety-like behavior and triggers opposing functional responses in excitatory and inhibitory neurons. Moreover, neuronal subpopulations tuned to anxiety-like behavior are differentially modulated by mPFC astrocytes at single cell and network levels. Using cell type-specific proximity biotinylation approaches, we identified significant intracellular and intercellular proteomic alterations in mPFC astrocytes and at the astrocyte-neuron interface associated with anxiety. Collectively, our findings uncover mechanisms underpinning the heterogenous astrocyte-neuron interaction that is behaviorally relevant and offer critical insights into the pathophysiology of emotional disorders. Whether and how prefrontal astrocyte Ca 2+ signaling modulates different neuronal populations in aiding or inhibiting anxiety-like behavior remains not fully understood. Here authors show that prefrontal astrocytes encode anxiogenic cues and modulate excitatory and inhibitory neurons differently. Silencing prefrontal astrocytes heightens anxiety-like behavior and induces proteomic changes in astrocytes and neurons.

Daily rhythms of mammalian physiology, metabolism, and behavior parallel the day-night cycle. They are orchestrated by a central circadian clock in the brain, the suprachiasmatic nucleus (SCN). Transcription of clock genes is sensitive to metabolic changes in reduction and oxidation (redox); however, circadian cycles in protein oxidation have been reported in anucleate cells, where no transcription occurs. We investigated whether the SCN also expresses redox cycles and how such metabolic oscillations might affect neuronal physiology. We detected self-sustained circadian rhythms of SCN redox state that required the molecular clockwork. The redox oscillation could determine the excitability of SCN neurons through nontranscriptional modulation of multiple potassium (K + ) channels. Thus, dynamic regulation of SCN excitability appears to be closely tied to metabolism that engages the clockwork machinery.

Pseudomonas aeruginosa commonly infects immunocompromised patients, including those with cystic fibrosis (CF). These infections are difficult to treat due to a variety of factors including the ability of Pseudomonas aeruginosa to resist to antibiotic treatment in part due to formation of biofilms. D-amino acids have known biofilm-disruption and antibacterial properties in some bacteria including P. aeruginosa . However, this treatment remains underexplored especially for inhibiting biofilm biomass production under CF environments. We explore the effects of six individual D-amino acids (alanine, aspartic acid, tyrosine, glutamic acid, serine, and proline) on the quorum sensing signaling and biofilm biomass production of two strains: PAO1 and the CF isolate FRD1. The D-amino acid causing the most significant decrease in biofilm mass and a decrease in quorum sensing molecules was D-aspartic acid. Meanwhile D-glutamic acid and D-serine had the opposite effects with an increase in biofilm mass and increase in quorum sensing molecule abundance. D-proline also showed a decrease in quorum sensing signaling with a decrease in biofilm biomass. P. aeruginosa had a lower or delayed quorum sensing response in the presence of D-aspartic acid and the absence of its L- counterpart at 48 h, a potential therapeutic route to explore.

Bioactive peptides (i.e., neuropeptides or peptide hormones) represent the largest class of cell-cell signaling molecules in metazoans and are potent regulators of neural and physiological function. In vertebrates, peptide hormones play an integral role in endocrine signaling between the brain and the gonads that controls reproductive development, yet few of these molecules have been shown to influence reproductive development in invertebrates. Here, we define a role for peptide hormones in controlling reproductive physiology of the model flatworm, the planarian Schmidtea mediterranea. Based on our observation that defective neuropeptide processing results in defects in reproductive system development, we employed peptidomic and functional genomic approaches to characterize the planarian peptide hormone complement, identifying 51 prohormone genes and validating 142 peptides biochemically. Comprehensive in situ hybridization analyses of prohormone gene expression revealed the unanticipated complexity of the flatworm nervous system and identified a prohormone specifically expressed in the nervous system of sexually reproducing planarians. We show that this member of the neuropeptide Y superfamily is required for the maintenance of mature reproductive organs and differentiated germ cells in the testes. Additionally, comparative analyses of our biochemically validated prohormones with the genomes of the parasitic flatworms Schistosoma mansoni and Schistosoma japonicum identified new schistosome prohormones and validated half of all predicted peptide-encoding genes in these parasites. These studies describe the peptide hormone complement of a flatworm on a genome-wide scale and reveal a previously uncharacterized role for peptide hormones in flatworm reproduction. Furthermore, they suggest new opportunities for using planarians as free-living models for understanding the reproductive biology of flatworm parasites.

Schistosomes are parasitic flatworms that infect over 200 million people, causing the neglected tropical disease, schistosomiasis. A single drug, praziquantel, is used to treat schistosome infection. Limitations in mass drug administration programs and the emergence of schistosomiasis in nontropical areas indicate the need for new strategies to prevent infection. It has been known for several decades that rotifers colonizing the schistosome's snail intermediate host produce a water-soluble factor that paralyzes cercariae, the life cycle stage infecting humans. In spite of its potential for preventing infection, the nature of this factor has remained obscure. Here, we report the purification and chemical characterization of Schistosome Paralysis Factor (SPF), a novel tetracyclic alkaloid produced by the rotifer Rotaria rotatoria. We show that this compound paralyzes schistosome cercariae and prevents infection and does so more effectively than analogous compounds. This molecule provides new directions for understanding cercariae motility and new strategies for preventing schistosome infection.

While we now appreciate that many infections are polymicrobial, we understand little of the specific actions between a given set of microbes to enable combinatorial survival and pathogenesis. The bacteria Pseudomonas aeruginosa and Enterococcus faecalis are both prevalent pathogens in wound, urinary tract, and bacteremic infections. While P. aeruginosa often kills other species in standard laboratory culture conditions, we present here that E. faecalis can be reliably co-cultured with P. aeruginosa . We specifically detail that ornithine produced by E. faecalis reduces the Pseudomonas quinolone signal response of P. aeruginosa . This reduction of the Pseudomonas quinolone signal response aids E. faecalis growth.

Pathway docking ( in silico docking of metabolites to several enzymes and binding proteins in a metabolic pathway) enables the discovery of a catabolic pathway for the osmolyte trans -4-hydroxy- l -proline betaine. Structural key to predicting enzyme function Overprediction and database annotation errors in genome-sequencing projects have caused much confusion because of the difficulty of assigning valid functions to the proteins identified. These authors use structure-guided approaches for predicting the substrate specificities of several enzymes encoded by a bacterial gene cluster to correctly predict the in vitro activity of an enzyme of unknown function and identify the catabolic pathway in which it participates in cells. The substrate-liganded pose predicted by virtual library screening was confirmed experimentally, enzyme activities in the predicted pathway were confirmed by in vitro assays and genetic analyses, the intermediates were identified by metabolomics, and repression of the genes encoding the pathway by high salt concentrations was established by transcriptomics. This study establishes the utility of structure-guided functional predictions for the discovery of new metabolic pathways. Assigning valid functions to proteins identified in genome projects is challenging: overprediction and database annotation errors are the principal concerns 1 . We and others 2 are developing computation-guided strategies for functional discovery with ‘metabolite docking’ to experimentally derived 3 or homology-based 4 three-dimensional structures. Bacterial metabolic pathways often are encoded by ‘genome neighbourhoods’ (gene clusters and/or operons), which can provide important clues for functional assignment. We recently demonstrated the synergy of docking and pathway context by ‘predicting’ the intermediates in the glycolytic pathway in Escherichia coli 5 . Metabolite docking to multiple binding proteins and enzymes in the same pathway increases the reliability of in silico predictions of substrate specificities because the pathway intermediates are structurally similar. Here we report that structure-guided approaches for predicting the substrate specificities of several enzymes encoded by a bacterial gene cluster allowed the correct prediction of the in vitro activity of a structurally characterized enzyme of unknown function (PDB 2PMQ), 2-epimerization of trans -4-hydroxy- l -proline betaine (tHyp-B) and cis -4-hydroxy- d -proline betaine (cHyp-B), and also the correct identification of the catabolic pathway in which Hyp-B 2-epimerase participates. The substrate-liganded pose predicted by virtual library screening (docking) was confirmed experimentally. The enzymatic activities in the predicted pathway were confirmed by in vitro assays and genetic analyses; the intermediates were identified by metabolomics; and repression of the genes encoding the pathway by high salt concentrations was established by transcriptomics, confirming the osmolyte role of tHyp-B. This study establishes the utility of structure-guided functional predictions to enable the discovery of new metabolic pathways.