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Microgliosis plays a critical role in diet-induced hypothalamic inflammation. A few hours after a high-fat diet (HFD), hypothalamic microglia shift to an inflammatory phenotype, and prolonged fat consumption leads to the recruitment of bone marrow-derived cells to the hypothalamus. However, the transcriptional signatures and functions of these cells remain unclear. Using dual-reporter mice, this study reveals that CX3CR1-positive microglia exhibit minimal changes in response to a HFD, while significant transcriptional differences emerge between microglia and CCR2-positive recruited myeloid cells, particularly affecting chemotaxis. These recruited cells also show sex-specific transcriptional differences impacting neurodegeneration and thermogenesis. The chemokine receptor CXCR3 is emphasized for its role in chemotaxis, displaying notable differences between recruited cells and resident microglia, requiring further investigation. Central immunoneutralization of CXCL10, a ligand for CXCR3, resulted in increased body mass and decreased energy expenditure, especially in females. Systemic chemical inhibition of CXCR3 led to significant metabolic changes, including increased body mass, reduced energy expenditure, elevated blood leptin, glucose intolerance, and decreased insulin levels. This study elucidates the transcriptional differences between hypothalamic microglia and CCR2-positive recruited myeloid cells in diet-induced inflammation and identifies CXCR3-expressing recruited immune cells as protective in metabolic outcomes linked to HFD consumption, establishing a new concept in obesity-related hypothalamic inflammation.

Interactions between top-down attention and bottom-up visceral inputs are assumed to produce conscious perceptions of interoceptive states, and while each process has been independently associated with aberrant interoceptive symptomatology in psychiatric disorders, the neural substrates of this interface are unknown. We conducted a preregistered functional neuroimaging study of 46 individuals with anxiety, depression, and/or eating disorders (ADE) and 46 propensity-matched healthy comparisons (HC), comparing their neural activity across two interoceptive tasks differentially recruiting top-down or bottom-up processing within the same scan session. During an interoceptive attention task, top-down attention was voluntarily directed towards cardiorespiratory or visual signals. In contrast, during an interoceptive perturbation task, intravenous infusions of isoproterenol (a peripherally-acting beta-adrenergic receptor agonist) were administered in a double-blinded and placebo-controlled fashion to drive bottom-up cardiorespiratory sensations. Across both tasks, neural activation converged upon the insular cortex, localizing within the granular and ventral dysgranular subregions bilaterally. However, contrasting hemispheric differences emerged, with the ADE group exhibiting (relative to HCs) an asymmetric pattern of overlap in the left insula, with increased or decreased proportions of co-activated voxels within the left or right dysgranular insula, respectively. The ADE group also showed less agranular anterior insula activation during periods of bodily uncertainty (i.e. when anticipating possible isoproterenol-induced changes that never arrived). Finally, post-task changes in insula functional connectivity were associated with anxiety and depression severity. These findings confirm the dysgranular mid-insula as a key cortical interface where attention and prediction meet real-time bodily inputs, especially during heightened awareness of interoceptive states. Furthermore, the dysgranular mid-insula may indeed be a 'locus of disruption' for psychiatric disorders.

Mammalian sperm delve into the female reproductive tract to fertilize the female gamete. The available information about how sperm regulate their motility during the final journey to the fertilization site is extremely limited. In this work, we investigated the structural and functional changes in the sperm flagellum after acrosomal exocytosis (AE) and during the interaction with the eggs. The evidence demonstrates that the double helix actin network surrounding the mitochondrial sheath of the midpiece undergoes structural changes prior to the motility cessation. This structural modification is accompanied by a decrease in diameter of the midpiece and is driven by intracellular calcium changes that occur concomitant with a reorganization of the actin helicoidal cortex. Midpiece contraction occurs in a subset of cells that undergo AE, and live-cell imaging during in vitro fertilization showed that the midpiece contraction is required for motility cessation after fusion is initiated. These findings provide the first evidence of the F-actin network's role in regulating sperm motility, adapting its function to meet specific cellular requirements during fertilization, and highlighting the broader significance of understanding sperm motility.

O-GlcNAcylation is an essential intracellular protein modification mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Recently, missense mutations in OGT have been linked to intellectual disability, indicating that this modification is important for the development and functioning of the nervous system. However, the processes that are most sensitive to perturbations in O-GlcNAcylation remain to be identified. Here, we uncover quantifiable phenotypes in the fruit fly Drosophila melanogaster carrying a patient-derived OGT mutation in the catalytic domain. Hypo-O-GlcNAcylation leads to defects in synaptogenesis and reduced sleep stability. Both these phenotypes can be partially rescued by genetically or chemically targeting OGA, suggesting that a balance of OGT/OGA activity is required for normal neuronal development and function.

Enhancers and promoters are classically considered to be bound by a small set of transcription factors (TFs) in a sequence-specific manner. This assumption has come under increasing skepticism as the datasets of ChIP-seq assays of TFs have expanded. In particular, high-occupancy target (HOT) loci attract hundreds of TFs with often no detectable correlation between ChIP-seq peaks and DNA-binding motif presence. Here, we used a set of 1003 TF ChIP-seq datasets (HepG2, K562, H1) to analyze the patterns of ChIP-seq peak co-occurrence in combination with functional genomics datasets. We identified 43,891 HOT loci forming at the promoter (53%) and enhancer (47%) regions. HOT promoters regulate housekeeping genes, whereas HOT enhancers are involved in tissue-specific process regulation. HOT loci form the foundation of human super-enhancers and evolve under strong negative selection, with some of these loci being located in ultraconserved regions. Sequence-based classification analysis of HOT loci suggested that their formation is driven by the sequence features, and the density of mapped ChIP-seq peaks across TF-bound loci correlates with sequence features and the expression level of flanking genes. Based on the affinities to bind to promoters and enhancers we detected five distinct clusters of TFs that form the core of the HOT loci. We report an abundance of HOT loci in the human genome and a commitment of 51% of all TF ChIP-seq binding events to HOT locus formation thus challenging the classical model of enhancer activity and propose a model of HOT locus formation based on the existence of large transcriptional condensates.

Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here, we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2 knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single-cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested hepatocyte nuclear factor 4 alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9, a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.

Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.

The low-complexity domain of hnRNPA1 (A1-LCD) phase separates in a salt-dependent manner. Unlike many intrinsically disordered proteins (IDPs) whose phase separation is suppressed by increasing salt concentrations, the phase separation of A1-LCD is promoted by >100 mM NaCl. To investigate the atypical salt effect on A1-LCD phase separation, we carried out all-atom molecular dynamics simulations of systems comprising multiple A1-LCD chains at NaCl concentrations from 50 to 1000 mM NaCl. The ions occupy first shell as well as more distant sites around the IDP chains, with Arg sidechains and backbone carbonyls the favored partners of Cl^- and Na⁺, respectively. They play two direct roles in driving A1-LCD condensation. The first is to neutralize the high net charge of the protein (+9) by an excess of bound Cl^- over Na⁺; the second is to bridge between A1-LCD chains, thereby fortifying the intermolecular interaction networks in the dense phase. At high concentrations, NaCl also indirectly strengthens π-π, cation-π, and amino-π interactions, by drawing water away from the interaction partners. Therefore, at low salt, A1-LCD is prevented from phase separation by net charge repulsion; at intermediate concentrations, NaCl neutralizes enough of the net charge while also bridging IDP chains to drive phase separation. This drive becomes even stronger at high salt due to strengthened π-type interactions. Based on this understanding, four classes of salt dependence of IDP phase separation can be predicted from amino-acid composition.

Many voltage-gated potassium (Kv) channels display a time-dependent phenomenon called C-type inactivation, whereby prolonged activation by voltage leads to the inhibition of ionic conduction, a process that involves a conformational change at the selectivity filter toward a non-conductive state. Recently, a high-resolution structure of a strongly inactivated triple-mutant channel kv1.2-kv2.1-3m revealed a novel conformation of the selectivity filter that is dilated at its outer end, distinct from the well-characterized conductive state. While the experimental structure was interpreted as the elusive non-conductive state, our molecular dynamics simulations and electrophysiological measurements show that the dilated filter of kv1.2-kv2.1-3m is conductive and, as such, cannot completely account for the inactivation of the channel observed in the structural experiments. The simulation shows that an additional conformational change, implicating isoleucine residues at position 398 along the pore lining segment S6, is required to effectively block ion conduction. The I398 residues from the four subunits act as a state-dependent hydrophobic gate located immediately beneath the selectivity filter. By mutating I398 to Asparagine, ion permeation can be resumed in the kv1.2-kv2.1-3m channel, which was not a reversion C-type inactivation, since AgTxII fails to block the ionic permeation of kv1.2-kv2.1-3m_I398N. As a critical piece of the C-type inactivation machinery, this structural feature is the potential target of a broad class of QA blockers and negatively charged activators thus opening new research directions towards the development of drugs that specifically modulate gating-states of Kv channels.

In congenital stationary night blindness, type 2 (CSNB2)-a disorder involving the Ca_v1.4 (L-type) Ca²⁺ channel-visual impairment is mild considering that Ca_v1.4 mediates synaptic release from rod and cone photoreceptors. Here, we addressed this conundrum using a Ca_v1.4 knockout (KO) mouse and a knock-in (G369i KI) mouse expressing a non-conducting Ca_v1.4. Surprisingly, Ca_v3 (T-type) Ca²⁺ currents were detected in cones of G369i KI mice and Ca_v1.4 KO mice but not in cones of wild-type mouse, ground squirrels, and macaque retina. Whereas Ca_v1.4 KO mice are blind, G369i KI mice exhibit normal photopic (i.e. cone-mediated) visual behavior. Cone synapses, which fail to form in Ca_v1.4 KO mice, are present, albeit enlarged, and with some errors in postsynaptic wiring in G369i KI mice. While Ca_v1.4 KO mice lack evidence of cone synaptic responses, electrophysiological recordings in G369i KI mice revealed nominal transmission from cones to horizontal cells and bipolar cells. In CSNB2, we propose that Ca_v3 channels maintain cone synaptic output provided that the nonconducting role of Ca_v1.4 in cone synaptogenesis remains intact. Our findings reveal an unexpected form of homeostatic plasticity that relies on a non-canonical role of an ion channel.