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Microglia sculpt developing neural circuits by eliminating excess synapses in a process called synaptic pruning, by removing apoptotic neurons, and by promoting neuronal survival. To elucidate the role of microglia during embryonic and postnatal brain development, we used a mouse model deficient in microglia throughout life by deletion of the fms-intronic regulatory element (FIRE) in the Csf1r locus. Surprisingly, young adult Csf1r ΔFIRE/ΔFIRE mice display no changes in excitatory and inhibitory synapse number and spine density of CA1 hippocampal neurons compared with Csf1r + /+ littermates. However, CA1 neurons are less excitable, receive less CA3 excitatory input and show altered synaptic properties, but this does not affect novel object recognition. Cytokine profiling indicates an anti-inflammatory state along with increases in ApoE levels and reactive astrocytes containing synaptic markers in Csf1r ΔFIRE/ΔFIRE mice. Notably, these changes in Csf1r ΔFIRE/ΔFIRE mice closely resemble the effects of acute microglial depletion in adult mice after normal development. Our findings suggest that microglia are not mandatory for synaptic pruning, and that in their absence pruning can be achieved by other mechanisms., (© 2024. The Author(s).)

Neuroinflammation is highly influenced by microglia, particularly through activation of the NLRP3 inflammasome and subsequent release of IL-1β. Extracellular ATP is a strong activator of NLRP3 by inducing K + efflux as a key signaling event, suggesting that K + -permeable ion channels could have high therapeutic potential. In microglia, these include ATP-gated THIK-1 K + channels and P2X7 receptors, but their interactions and potential therapeutic role in the human brain are unknown. Using a novel specific inhibitor of THIK-1 in combination with patch-clamp electrophysiology in slices of human neocortex, we found that THIK-1 generated the main tonic K + conductance in microglia that sets the resting membrane potential. Extracellular ATP stimulated K + efflux in a concentration-dependent manner only via P2X7 and metabotropic potentiation of THIK-1. We further demonstrated that activation of P2X7 was mandatory for ATP-evoked IL-1β release, which was strongly suppressed by blocking THIK-1. Surprisingly, THIK-1 contributed only marginally to the total K + conductance in the presence of ATP, which was dominated by P2X7. This suggests a previously unknown, K + -independent mechanism of THIK-1 for NLRP3 activation. Nuclear sequencing revealed almost selective expression of THIK-1 in human brain microglia, while P2X7 had a much broader expression. Thus, inhibition of THIK-1 could be an effective and, in contrast to P2X7, microglia-specific therapeutic strategy to contain neuroinflammation., (© 2024. The Author(s).)

In microglia, changes in intracellular calcium concentration ([Ca 2+ ] i ) may regulate process motility, inflammasome activation, and phagocytosis. However, while neurons and astrocytes exhibit frequent spontaneous Ca 2+ activity, microglial Ca 2+ signals are much rarer and poorly understood. Here, we studied [Ca 2+ ] i changes of microglia in acute brain slices using Fluo-4-loaded cells and mice expressing GCaMP5g in microglia. Spontaneous Ca 2+ transients occurred ~ 5 times more frequently in individual microglial processes than in their somata. We assessed whether microglial Ca 2+ responses change in Alzheimer's disease (AD) using App NL-G-F knock-in mice. Proximity to Aβ plaques strongly affected microglial Ca 2+ activity. Although spontaneous Ca 2+ transients were unaffected in microglial processes, they were fivefold more frequent in microglial somata near Aβ plaques than in wild-type microglia. Microglia away from Aβ plaques in AD mice showed intermediate properties for morphology and Ca 2+ responses, partly resembling those of wild-type microglia. By contrast, somatic Ca 2+ responses evoked by tissue damage were less intense in microglia near Aβ plaques than in wild-type microglia, suggesting different mechanisms underlying spontaneous vs. damage-evoked Ca 2+ signals. Finally, as similar processes occur in neurodegeneration and old age, we studied whether ageing affected microglial [Ca 2+ ] i . Somatic damage-evoked Ca 2+ responses were greatly reduced in microglia from old mice, as in the AD mice. In contrast to AD, however, old age did not alter the occurrence of spontaneous Ca 2+ signals in microglial somata but reduced the rate of events in processes. Thus, we demonstrate distinct compartmentalised Ca 2+ activity in microglia from healthy, aged and AD-like brains., (© 2023. The Author(s).)

More than 30% of all human cancers are driven by RAS mutations and activating KRAS mutations are present in 40% of colorectal cancer (CRC) in the two main CRC subgroups, MSS (Microsatellite Stable) and MSI (Microsatellite Instable). Studies in RAS-driven tumors have shown essential roles of the RAS effectors RAF and specifically of RAF1, which can be dependent or independent of RAF's ability to activate the MEK/ERK module. In this study, we demonstrate that RAF1, but not its kinase activity, plays a crucial role in the proliferation of both MSI and MSS CRC cell line-derived spheroids and patient-derived organoids, and independently of KRAS mutation status. Moreover, we could define a RAF1 transcriptomic signature which includes genes that contribute to STAT3 activation, and could demonstrate that RAF1 ablation decreases STAT3 phosphorylation in all CRC spheroids tested. The genes involved in STAT3 activation as well as STAT3 targets promoting angiogenesis were also downregulated in human primary tumors expressing low levels of RAF1. These results indicate that RAF1 could be an attractive therapeutic target in both MSI and MSS CRC regardless of their KRAS status and support the development of selective RAF1 degraders rather than RAF1 inhibitors for clinical use in combination therapies., (© 2023. The Author(s).)

Neuroinflammation, specifically the NLRP3 inflammasome cascade, is a common underlying pathological feature of many neurodegenerative diseases. Evidence suggests that NLRP3 activation involves changes in intracellular K + . Nuclear Enriched Transcript Sort Sequencing (NETSseq), which allows for deep sequencing of purified cell types from human post-mortem brain tissue, demonstrated a highly specific expression of the tandem pore domain halothane-inhibited K + channel 1 (THIK-1) in microglia compared to other glial and neuronal cell types in the human brain. NETSseq also showed a significant increase of THIK-1 in microglia isolated from cortical regions of brains with Alzheimer's disease (AD) relative to control donors. Herein, we report the discovery and pharmacological characterisation of C101248, the first selective small-molecule inhibitor of THIK-1. C101248 showed a concentration-dependent inhibition of both mouse and human THIK-1 (IC50: ∼50 nM) and was inactive against K2P family members TREK-1 and TWIK-2, and Kv2.1. Whole-cell patch-clamp recordings of microglia from mouse hippocampal slices showed that C101248 potently blocked both tonic and ATP-evoked THIK-1 K + currents. Notably, C101248 had no effect on other constitutively active resting conductance in slices from THIK-1-depleted mice. In isolated microglia, C101248 prevented NLRP3-dependent release of IL-1β, an effect not seen in THIK-1-depleted microglia. In conclusion, we demonstrated that inhibiting THIK-1 (a microglia specific gene that is upregulated in brains from donors with AD) using a novel selective modulator attenuates the NLRP3-dependent release of IL-1β from microglia, which suggests that this channel may be a potential therapeutic target for the modulation of neuroinflammation in AD., Competing Interests: Declaration of competing interest All authors except for Ali R, SD, CB, Michael H, DB, CM were employers of Cerevance Ltd at the time their contribution to this work. Ali R and CM were supported by Cerevance Ltd for this work. CB, Michael H, and DB have no interest to declare., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

ABCB4 (ATP-binding cassette subfamily B member 4) is a hepatocanalicular floppase involved in biliary phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene give rise to several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), an autosomal recessive disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ten ABCB4 missense variations in NBD1:NBD2 homologous positions (Y403H/Y1043H, K435M/K1075M, E558K/E1200A, D564G/D1206G and H589Y/H1231Y) all localized at the conserved and functionally critical motifs of ABC transporters, six of which are mutated in patients. By combining structure analysis and in vitro studies, we found that all ten mutants were normally processed and localized at the canalicular membrane of HepG2 cells, but showed dramatically impaired PC transport activity that was significantly rescued by treatment with the clinically approved CFTR potentiator ivacaftor. Our results provide evidence that functional ABCB4 mutations are rescued by ivacaftor, paving the way for the repositioning of this potentiator for the treatment of selected patients with PFIC3 caused by mutations in the ATP-binding sites of ABCB4.

Aims: Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to alveolar hypoxia that diverts blood flow from poorly ventilated to better aerated lung areas to optimize ventilation-perfusion matching. Yet, the exact sensory and signalling mechanisms by which hypoxia triggers pulmonary vasoconstriction remain incompletely understood. Recently, ATP release via pannexin 1 (Panx1) and subsequent signalling via purinergic P2Y receptors has been identified as regulator of vasoconstriction in systemic arterioles. Here, we probed for the role of Panx1-mediated ATP release in HPV and chronic hypoxic pulmonary hypertension (PH)., Methods and Results: Pharmacological inhibition of Panx1 by probenecid, spironolactone, the Panx1 specific inhibitory peptide (10Panx1), and genetic deletion of Panx1 specifically in smooth muscle attenuated HPV in isolated perfused mouse lungs. In pulmonary artery smooth muscle cells (PASMCs), both spironolactone and 10Panx1 attenuated the increase in intracellular Ca2+ concentration ([Ca2+]i) in response to hypoxia. Yet, genetic deletion of Panx1 in either endothelial or smooth muscle cells did not prevent the development of PH in mice. Unexpectedly, ATP release in response to hypoxia was not detectable in PASMC, and inhibition of purinergic receptors or ATP degradation by ATPase failed to attenuate HPV. Rather, transient receptor potential vanilloid 4 (TRPV4) antagonism and Panx1 inhibition inhibited the hypoxia-induced [Ca2+]i increase in PASMC in an additive manner, suggesting that Panx1 regulates [Ca2+]i independently of the ATP-P2Y-TRPV4 pathway. In line with this notion, Panx1 overexpression increased the [Ca2+]i response to hypoxia in HeLa cells., Conclusion: In the present study, we identify Panx1 as novel regulator of HPV. Yet, the role of Panx1 in HPV was not attributable to ATP release and downstream signalling via P2Y receptors or TRPV4 activation, but relates to a role of Panx1 as direct or indirect modulator of the PASMC Ca2+ response to hypoxia. Panx1 did not affect the development of chronic hypoxic PH., Competing Interests: Conflict of interest: none declared., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)