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Specialized proresolving mediators (SPMs) and their cognate G protein-coupled receptors are implicated in autoimmune disorders, including chronic inflammation, rheumatoid arthritis, systemic scleroderma, and lupus erythematosus. To date, six G protein-coupled receptors (GPCRs) have been paired with numerous endogenous and synthetic ligands. However, the function and downstream signaling of these receptors remains unclear. To address this knowledge gap, we systematically expressed each receptor in a human embryonic kindney 293 (HEK293)-Flp-In-CD8a-FLAG cell system. Each receptor was pharmacologically characterized with both synthetic and putative endogenous ligands across different signaling assays, covering both G protein-dependent (G

Dual agonists that can activate both the glucagon-like peptide-1 receptor (GLP-1R) and the gastric inhibitory polypeptide receptor (GIPR) have demonstrated high efficacy for the treatment of metabolic disease. Peptide-19 is a prototypical dual agonist that has high potency at both GLP-1R and GIPR but has a distinct signalling profile relative to the native peptides at the cognate receptors. In this study, we solved the structure of peptide-19 bound to the GLP-1R in complex with Gs protein, and compared the structure and dynamics of this complex to that of published structures of GLP-1R:Gs in complex with other receptor agonists. Unlike other peptide-bound receptor complexes, peptide-19:GLP-1R:Gs demonstrated a more open binding pocket where transmembrane domain (TM) 6, TM7 and the interconnecting extracellular loop 3 (ECL3) were located away from the peptide, with no interactions between peptide-19 and TM6/ECL3. Analysis of conformational variance of the complex revealed that peptide-19 was highly dynamic and underwent binding and unbinding motions facilitated by the more open TM binding pocket. Both the consensus structure of the GLP-1R complex with peptide-19 and the dynamics of this complex were distinct from previously described GLP-1R structures providing unique insights into the mode of GLP-1R activation by this dual agonist.

Summary Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies., Highlights • Integrated molecular characterization reveals differences in four iMGL protocols • A dual tdTomato and nanoluciferase reporter tool was generated to track iMGLs via CX3CR1 • Kinetics of surface marker expression during iMGL differentiation • Co-culture with glia/neurons restores ex vivo microglial transcription factors, In this article, Jose Polo and colleagues molecularly compare existing microglia differentiation methods from stem cells, generate a new dual fluorescent and enzymatic reporter tool to study microglia, and show that human glia/neuron co-culture pushes the regulatory landscape of in vitro microglia-like cells to more closely resemble bona fide microglia.

The role of microglia cells in Alzheimer’s disease (AD) is well recognized, however their molecular and functional diversity remain unclear. Here, we isolated amyloid plaque-containing (using labelling with methoxy-XO4, XO4+) and non-containing (XO4−) microglia from an AD mouse model. Transcriptomics analysis identified different transcriptional trajectories in ageing and AD mice. XO4+ microglial transcriptomes demonstrated dysregulated expression of genes associated with late onset AD. We further showed that the transcriptional program associated with XO4+ microglia from mice is present in a subset of human microglia isolated from brains of individuals with AD. XO4− microglia displayed transcriptional signatures associated with accelerated ageing and contained more intracellular post-synaptic material than XO4+ microglia, despite reduced active synaptosome phagocytosis. We identified HIF1α as potentially regulating synaptosome phagocytosis in vitro using primary human microglia, and BV2 mouse microglial cells. Together, these findings provide insight into molecular mechanisms underpinning the functional diversity of microglia in AD., Microglia associated with Aβ plaques may have a distinct transcriptional signature compared to those in plaque-free areas of the brain in Alzheimer’s disease (AD) models. Here the authors show that amyloid plaque phagocytosis is associated with a specific microglia transcriptional signature in a mouse model of AD.

[Image: see text] There are no effective therapeutics for cognitive impairments associated with schizophrenia (CIAS), which includes deficits in executive functions (working memory and cognitive flexibility) and episodic memory. Compounds that have entered clinical trials are inadequate in terms of efficacy and/or tolerability, highlighting a clear translational bottleneck and a need for a cohesive preclinical drug development strategy. In this review we propose hippocampal–prefrontal-cortical (HPC–PFC) circuitry underlying CIAS-relevant cognitive processes across mammalian species as a target source to guide the translation-focused discovery and development of novel, procognitive agents. We highlight several G protein-coupled receptors (GPCRs) enriched within HPC–PFC circuitry as therapeutic targets of interest, including noncanonical approaches (biased agonism and allosteric modulation) to conventional clinical targets, such as dopamine and muscarinic acetylcholine receptors, along with prospective novel targets, including the orphan receptors GPR52 and GPR139. We also describe the translational limitations of popular preclinical cognition tests and suggest touchscreen-based assays that probe cognitive functions reliant on HPC–PFC circuitry and reflect tests used in the clinic, as tests of greater translational relevance. Combining pharmacological and behavioral testing strategies based in HPC–PFC circuit function creates a cohesive, translation-focused approach to preclinical drug development that may improve the translational bottleneck currently hindering the development of treatments for CIAS.

Renal podocyte survival depends upon the dynamic regulation of a complex cell architecture that links the glomerular basement membrane to integrins, ion channels, and receptors. Alport syndrome is a heritable chronic kidney disease where mutations in α3, α4, or α5 collagen genes promote podocyte death. In rodent models of renal failure, activation of the calcium-sensing receptor (CaSR) can protect podocytes from stress-related death. In this study, we assessed CaSR function in podocyte-like cells derived from induced-pluripotent stem cells from two patients with Alport Syndrome (AS1 & AS2) and a renal disease free individual [normal human mesangial cell (NHMC)], as well as a human immortalized podocyte-like (HIP) cell line. Extracellular calcium elicited concentration-dependent elevations of intracellular calcium in all podocyte-like cells. NHMC and HIP, but not AS1 or AS2 podocyte-like cells, also showed acute reductions in intracellular calcium prior to elevation. In NHMC podocyte-like cells this acute reduction was blocked by the large-conductance potassium channel (KCNMA1) inhibitors iberiotoxin (10 nM) and tetraethylammonium (5 mM), as well as the focal adhesion kinase inhibitor PF562271 (N-methyl-N-(3-((2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino)-methyl)-pyridin-2-yl)-methanesulfonamide, 10 nM). Quantitative polymerase chain reaction (qPCR) and immunolabeling showed the presence of KCNMA1 transcript and protein in all podocyte-like cells tested. Cultivation of AS1 podocytes on decellularized plates of NHMC podocyte-like cells partially restored acute reductions in intracellular calcium in response to extracellular calcium. We conclude that the AS patient-derived podocyte-like cells used in this study showed dysfunctional integrin signaling and potassium channel function, which may contribute to podocyte death seen in Alport syndrome.

The important role of microglia, the brain’s resident immune cells, in Alzheimer’s disease (AD) is now well recognized, however their molecular and functional diversity and underlying mechanisms still remain controversial. To transcriptionally and functionally characterize the diversity of microglia in AD and aging, we isolated the amyloid plaque-containing (XO4+) and non-containing (XO4−) microglia from an AD mouse model. Transcriptomics analysis unveiled independent transcriptional trajectories in ageing and AD. XO4+ microglial transcriptomes linked plaque phagocytosis to altered expression of bona fide late onset AD genetic risk factors. We further revealed that the XO4+ transcriptional program is present in a subset of human microglia from AD patients and is a direct and reversible consequence of Aβ plaque phagocytosis. Conversely, XO4− microglia in AD displayed an accelerated ageing signature and contained more intracellular post synaptic material than plaque-containing microglia, despite reduced active synaptosome phagocytosis. Mechanistically, we predicted HIF1α as a core regulator of the XO4−/XO4+ axis, and further validated the mechanism in vitro using human stem cell-derived microglia like cells and primary human microglia. Together these findings unveiled the molecular mechanism underpinning the functional diversity of microglia in AD, providing opportunities to develop treatments targeted at subset specific manipulation of the microglial niche.