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New analytical methods focusing on polar metabolite analysis in mass spectrometry and NMR-based metabolomics.
Following in the footsteps of genomics and proteomics, metabolomics has revolutionised the way we investigate and understand biological systems. Rapid development in the last 25 years has been driven largely by technical innovations in mass spectrometry and nuclear magnetic resonance spectroscopy. However, despite the modest size of metabolomes relative to proteomes and genomes, methodological capabilities for robust, comprehensive metabolite analysis remain a major challenge. Therefore, development of new methods and techniques remains vital for progress in the field. Here, we review developments in LC-MS, GC-MS and NMR methods in the last few years that have enhanced quantitative and comprehensive metabolome coverage, highlighting the techniques involved, their technical capabilities, relative performance, and potential impact.
Development of an inhibitor of the mutagenic SOS response that suppresses the evolution of quinolone antibiotic resistance
Bacterial DNA-damage repair activates the SOS response, which promotes hypermutation and antibiotic resistance. SAR investigation identified OXF-077, a small molecule DNA-repair and SOS-response inhibitor that suppresses resistance evolution.
Optogenetic Determination of Dynamic and Cell-Type-Specific Inhibitory Reversal Potentials.
The reversal potential refers to the membrane potential at which the net current flow through a channel reverses direction. The reversal potential is determined by transmembrane ion gradients and, in turn, determines how the channel's activity will affect the membrane potential. Traditional investigation into the reversal potential of inhibitory ligand-gated ion channels (EInh) has relied upon the activation of endogenous receptors, such as the GABA-A receptor (GABAAR). There are, however, challenges associated with activating endogenous receptors, including agonist delivery, isolating channel responses, and the effects of receptor saturation and desensitization. Here, we demonstrate the utility of using a light-gated anion channel, stGtACR2, to probe EInh in the rodent brain. Using mice of both sexes, we demonstrate that the properties of this optically activated channel make it a suitable proxy for studying GABAAR receptor-mediated inhibition. We validate this agonist-independent optogenetic strategy in vitro and in vivo and further show how it can accurately capture differences in EInh dynamics following manipulations of endogenous ion fluxes. This allows us to explore distinct resting EInh differences across genetically defined neuronal subpopulations. Using this approach to challenge ion homeostasis mechanisms in neurons, we uncover cell-specific EInh dynamics that are supported by the differential expression of endogenous ion handling mechanisms. Our findings therefore establish an effective optical strategy for revealing novel aspects of inhibitory reversal potentials and thereby expand the repertoire of optogenetics.
Two-pore channels: going with the flows.
In recent years, our understanding of the structure, mechanisms and functions of the endo-lysosomal TPC (two-pore channel) family have grown apace. Gated by the second messengers, NAADP and PI(3,5)P2, TPCs are an integral part of fundamental signal-transduction pathways, but their array and plasticity of cation conductances (Na+, Ca2+, H+) allow them to variously signal electrically, osmotically or chemically. Their relative tissue- and organelle-selective distribution, together with agonist-selective ion permeabilities provides a rich palette from which extracellular stimuli can choose. TPCs are emerging as mediators of immunity, cancer, metabolism, viral infectivity and neurodegeneration as this short review attests.
Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion.
Oxytocin (OT) is a prominent regulator of many aspects of mammalian social behavior and stored in large dense-cored vesicles (LDCVs) in hypothalamic neurons. It is released in response to activity-dependent Ca2+ influx, but is also dependent on Ca2+ release from intracellular stores, which primes LDCVs for exocytosis. Despite its importance, critical aspects of the Ca2+-dependent mechanisms of its secretion remain to be identified. Here we show that lysosomes surround dendritic LDCVs, and that the direct activation of endolysosomal two-pore channels (TPCs) provides the critical Ca2+ signals to prime OT release by increasing the releasable LDCV pool without directly stimulating exocytosis. We observed a dramatic reduction in plasma OT levels in TPC knockout mice, and impaired secretion of OT from the hypothalamus demonstrating the importance of priming of neuropeptide vesicles for activity-dependent release. Furthermore, we show that activation of type 1 metabotropic glutamate receptors sustains somatodendritic OT release by recruiting TPCs. The priming effect could be mimicked by a direct application of nicotinic acid adenine dinucleotide phosphate, the endogenous messenger regulating TPCs, or a selective TPC2 agonist, TPC2-A1-N, or blocked by the antagonist Ned-19. Mice lacking TPCs exhibit impaired maternal and social behavior, which is restored by direct OT administration. This study demonstrates an unexpected role for lysosomes and TPCs in controlling neuropeptide secretion, and in regulating social behavior.
Neurophysiological functions and pharmacological tools of acidic and non-acidic Ca2+ stores.
Ca2+ signalling is of prime importance in controlling numerous cell functions in the brain. Endolysosomes are acidic organelles currently emerging as important Ca2+ stores in astrocytes, microglia, endothelial cells, and neurons. In neurons, these acidic Ca2+ stores are found in axons, soma, dendrites, and axon endings and could provide local sources of Ca2+ to control synaptic transmission, neuronal plasticity, and autophagy to name a few. This review will address how acidic Ca2+ stores are recruited in response to cell stimulation. We will focus on the role of the endolysosomal two-pore channels (TPCs) and their physiological agonist nicotinic acid adenine dinucleotide phosphate (NAADP) and how they interact with cyclic ADP-ribose and ryanodine receptors from the endoplasmic reticulum. Finally, this review will describe new pharmacological tools and animal mutant models now available to explore acidic Ca2+ stores as key elements in brain function and dysfunction.
Low Temperature Delays the Effects of Ischemia in Bergmann Glia and in Cerebellar Tissue Swelling.
Cerebral ischemia results in oxygen and glucose deprivation that most commonly occurs after a reduction or interruption in the blood supply to the brain. The consequences of cerebral ischemia are complex and involve the loss of metabolic ATP, excessive K+ and glutamate accumulation in the extracellular space, electrolyte imbalance, and brain edema formation. So far, several treatments have been proposed to alleviate ischemic damage, yet few are effective. Here, we focused on the neuroprotective role of lowering the temperature in ischemia mimicked by an episode of oxygen and glucose deprivation (OGD) in mouse cerebellar slices. Our results suggest that lowering the temperature of the extracellular 'milieu' delays both the increases in [K+]e and tissue swelling, two dreaded consequences of cerebellar ischemia. Moreover, radial glial cells (Bergmann glia) display morphological changes and membrane depolarizations that are markedly impeded by lowering the temperature. Overall, in this model of cerebellar ischemia, hypothermia reduces the deleterious homeostatic changes regulated by Bergmann glia.
A multiscale analysis in CD38-/- mice unveils major prefrontal cortex dysfunctions.
Autism spectrum disorder (ASD) is characterized by early onset of behavioral and cognitive alterations. Low plasma levels of oxytocin (OT) have also been found in ASD patients; recently, a critical role for the enzyme CD38 in the regulation of OT release was demonstrated. CD38 is important in regulating several Ca2+-dependent pathways, but beyond its role in regulating OT secretion, it is not known whether a deficit in CD38 expression leads to functional modifications of the prefrontal cortex (PFC), a structure involved in social behavior. Here, we report that CD38-/- male mice show an abnormal cortex development, an excitation-inhibition balance shifted toward a higher excitation, and impaired synaptic plasticity in the PFC such as those observed in various mouse models of ASD. We also show that a lack of CD38 alters social behavior and emotional responses. Finally, examining neuromodulators known to control behavioral flexibility, we found elevated monoamine levels in the PFC of CD38-/- adult mice. Overall, our study unveiled major changes in PFC physiologic mechanisms and provides new evidence that the CD38-/- mouse could be a relevant model to study pathophysiological brain mechanisms of mental disorders such as ASD.-Martucci, L. L., Amar, M., Chaussenot, R., Benet, G., Bauer, O., de Zélicourt, A., Nosjean, A., Launay, J.-M., Callebert, J., Sebrié, C., Galione, A., Edeline, J.-M., de la Porte, S., Fossier, P., Granon, S., Vaillend, C., Cancela, J.-M., A multiscale analysis in CD38-/- mice unveils major prefrontal cortex dysfunctions.
NAADP-Mediated Ca2+ Signalling.
The discovery of NAADP-evoked Ca2+ release in sea urchin eggs and then as a ubiquitous Ca2+ mobilizing messenger has introduced several novel paradigms to our understanding of Ca2+ signalling, not least in providing a link between cell stimulation and Ca2+ release from lysosomes and other acidic Ca2+ storage organelles. In addition, the hallmark concentration-response relationship of NAADP-mediated Ca2+ release, shaped by striking activation/desensitization mechanisms, influences its actions as an intracellular messenger. There has been recent progress in our understanding of the molecular mechanisms underlying NAADP-evoked Ca2+ release, such as the identification of the endo-lysosomal two-pore channel family of cation channels (TPCs) as their principal target and the identity of NAADP-binding proteins that complex with them. The NAADP/TPC signalling axis has gained recent prominence in pathophysiology for their roles in such disease processes as neurodegeneration, tumorigenesis and cellular viral entry.
Higher-order thalamocortical circuits are specified by embryonic cortical progenitor types in the mouse brain.
The sensory cortex receives synaptic inputs from both first-order and higher-order thalamic nuclei. First-order inputs relay simple stimulus properties from the periphery, whereas higher-order inputs relay more complex response properties, provide contextual feedback, and modulate plasticity. Here, we reveal that a cortical neuron's higher-order input is determined by the type of progenitor from which it is derived during embryonic development. Within layer 4 (L4) of the mouse primary somatosensory cortex, neurons derived from intermediate progenitors receive stronger higher-order thalamic input and exhibit greater higher-order sensory responses. These effects result from differences in dendritic morphology and levels of the transcription factor Lhx2, which are specified by the L4 neuron's progenitor type. When this mechanism is disrupted, cortical circuits exhibit altered higher-order responses and sensory-evoked plasticity. Therefore, by following distinct trajectories, progenitor types generate diversity in thalamocortical circuitry and may provide a general mechanism for differentially routing information through the cortex.
A multinational report on SARS-COV-2 infection outcomes in people with CF and Aspergillus infection or ABPA.
BACKGROUND: Aspergillus infection is known to be associated with worse respiratory outcomes in people with CF (pwCF) and is a well-recognised complication of severe SARS-CoV-2 infection. The aim of this observational cross-sectional study was to examine the association of pre-existing Aspergillus infection and/or allergic bronchopulmonary aspergillosis (ABPA) in pwCF and severity of COVID-19. METHODS: Data on SARS-CoV-2 infections in pwCF from January 2020 to June 2021 were collected by the European Cystic Fibrosis Society Patient Registry. The primary outcome was COVID-19 severity measured by hospitalisation comparing those with Aspergillus infection and/or ABPA in the 12 months preceding COVID-19and those without. RESULTS: In total, 1095 pwCF were diagnosed with SARS-CoV-2 and information on pre-existing Aspergillus/ABPA status was available from 807. PwCF and SARS-CoV-2 in the Aspergillus/ABPA group (n = 153), in comparison to the non-Aspergillus/ABPA group (n = 654), were more likely to be hospitalised (adjusted OR 1.79 (1.19 to 2.85); p = 0.005) and their disease course was more likely to be complicated by sepsis (adjusted OR 7.78 (1.78 to 49.43); p = 0.008). The association with hospital admission was no longer significant after excluding patients with ABPA. Secondary analysis comparing pwCF who received antifungal treatment (n = 18), versus those who did not (n = 474) during COVID-19, showed a higher rate of hospitalisation (p
Correction: Modified minimal-size fragments of heparan sulfate as inhibitors of endosulfatase-2 (Sulf-2).
Correction for 'Modified minimal-size fragments of heparan sulfate as inhibitors of endosulfatase-2 (Sulf-2)' by Alice Kennett et al., Chem. Commun., 2024, 60, 436-439, https://doi.org/10.1039/D3CC02565A.
Rebound activation of 5-HT neurons following SSRI discontinuation.
Cessation of therapy with a selective serotonin (5-HT) reuptake inhibitor (SSRI) is often associated with an early onset and disabling discontinuation syndrome, the mechanism of which is surprisingly little investigated. Here we determined the effect on 5-HT neurochemistry of discontinuation from the SSRI paroxetine. Paroxetine was administered repeatedly to mice (once daily, 12 days versus saline controls) and then either continued or discontinued for up to 5 days. Whereas brain tissue levels of 5-HT and/or its metabolite 5-HIAA tended to decrease during continuous paroxetine, levels increased above controls after discontinuation, notably in hippocampus. In microdialysis experiments continuous paroxetine elevated hippocampal extracellular 5-HT and this effect fell to saline control levels on discontinuation. However, depolarisation (high potassium)-evoked 5-HT release was reduced by continuous paroxetine but increased above controls post-discontinuation. Extracellular hippocampal 5-HIAA also decreased during continuous paroxetine and increased above controls post-discontinuation. Next, immunohistochemistry experiments found that paroxetine discontinuation increased c-Fos expression in midbrain 5-HT (TPH2 positive) neurons, adding further evidence for a hyperexcitable 5-HT system. The latter effect was recapitulated by 5-HT1A receptor antagonist administration although gene expression analysis could not confirm altered expression of 5-HT1A autoreceptors following paroxetine discontinuation. Finally, in behavioural experiments paroxetine discontinuation increased anxiety-like behaviour, which partially correlated in time with the measures of increased 5-HT function. In summary, this study reports evidence that, across a range of experiments, SSRI discontinuation triggers a rebound activation of 5-HT neurons. This effect is reminiscent of neural changes associated with various psychotropic drug withdrawal states, suggesting a common unifying mechanism.