Cell reports最新文献

The function of the mammalian neocortex relies on the timing of axon extension and establishment of cell-type-biased patterns of excitatory synaptic connections. A subtype of excitatory neurons, layer 6 corticothalamic neurons (L6CThNs), ultimately exhibits a marked preference for synapsing onto parvalbumin-positive (PV) inhibitory interneurons over more common excitatory cells in layers 6 and 4 (L6, L4). We show that the intracortical axons of L6CThNs develop in phases, first elongating within L6, then pausing before extending translaminar branches into L4. Decreasing L6CThN excitability selectively enhances axon growth in L6 but not the later elaboration in L4. For both layers, we test whether preferential synaptogenesis onto rarer PV interneurons, or non-selective synapse formation followed by selective pruning, generates adult connectivity. We find that L6CThNs form functional AMPA-receptor-containing synapses preferentially onto PV interneurons. Silent L6CThN synapses are not detected. Our findings show that cell-type-biased synaptogenesis underlies the formation of functional cell-type-specific excitatory connections in the neocortex.

A major obstacle to identifying effective therapies for the aggressive brain tumor glioblastoma is the lack of human-specific, immunocompetent models that reflect the human tumor microenvironment. To address this, we developed the immune-human organoid tumor transplantation (iHOTT) model, an autologous co-culture platform that integrates patient-derived tumor cells and matched peripheral blood mononuclear cells within human cortical organoids to enable the study of patient-specific immune responses and tumor-immune interactions. This platform preserves tumor and immune populations, immune signaling, and cell-cell interactions observed in patient tumors. Treatment of iHOTT with pembrolizumab, a checkpoint inhibitor, mirrors cell-type shifts and cell-cell interactions observed in patients. T cell receptor (TCR) sequencing further reveals pembrolizumab-driven expansion of stem-like CD4 T cell clonotypes exhibiting patient-specific repertoires. These findings establish iHOTT as a physiologically relevant platform for exploring autologous tumor-immune interactions and underscore the need for antigen-targeted strategies to enhance immunotherapy in glioblastoma.

Human liver ductal epithelium is morphologically, functionally, and transcriptionally heterogeneous. Understanding the impact of this heterogeneity has been challenging due to the absence of systems that recapitulate this heterogeneity in vitro. Here, we found that human liver cholangiocyte organoids do not retain the complex cellular heterogeneity of the native ductal epithelium. Inspired by the knowledge of the cellular niche, we refined our previous organoid medium to fully capture the in vivo cellular heterogeneity. We employed this refined system to analyze the relationships between human biliary epithelial cell states. In our refined model, cholangiocytes transition toward hepatocyte-like states through a bipotent state. Additionally, inhibiting WNT signaling enhances the differentiation capacity of the cells toward hepatocyte-like states. By capturing the in vivo cholangiocyte heterogeneity, our improved organoid model represents a platform to investigate the impact of the different liver ductal cell states in cell plasticity, regeneration, and disease.

Formation of the immune synapse (IS) following T cell antigen recognition includes recruitment of the linker for activation of T cells (LAT). Once at the IS, LAT tyrosines are phosphorylated, allowing it to serve as a scaffold for the formation of the "signalosome," a multiprotein complex that drives T cell receptor signaling. Here, we show that upon T cell activation, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) interacts with LAT and localizes to the IS. Inhibition of DNA-PKcs diminishes LAT localization at the IS. We identified two LAT serines phosphorylated by DNA-PKcs, S224 and S241, that impact LAT tyrosine phosphorylation, protein binding, and cytokine production. Using our mouse model designed to delete DNA-PKcs expression in mature CD4⁺ or CD8⁺ T cells, we show that loss of DNA-PKcs results in T cells that are unable to control tumor growth or induce allogeneic graft rejection. These data highlight DNA-PKcs as a pivotal protein in T cell function.

Chimeric brain models generated by transplanting human pluripotent stem cell (hPSC)-derived neural cells are valuable for studying the development and function of human neural cells in vivo. To explore glial-neuronal and glial-glial interactions, we co-engraft hPSC-derived primitive neural progenitor cells and primitive macrophage progenitors into neonatal mouse brains, generating chimeric brains containing human microglia, macroglia, and neurons. Using super-resolution imaging and 3D reconstruction, we observe human microglia pruning synapses and engulfing neurons. Single-cell RNA sequencing reveals human glial progenitor populations and dynamic stages of astroglial development resembling those in the human brain. Cell-cell communication analysis identifies strong human neural interactions, including NRXN-NLGN3 signaling between neurons and astrocytes and SPP1- and PTN-MK-mediated communication between microglia and astroglia. This co-transplantation model provides a powerful approach to study complex human glial-neuronal interactions and mechanisms underlying neurological diseases.

ADP-ribosylation (ADPr) is a reversible modification of macromolecules critical for the regulation of genome stability, stress responses, and proteostasis. While the roles of ADPr transferases such as PARP1/2 and TNKS1/2 are well established, the functions and regulatory mechanisms of ADPr hydrolases are still poorly understood. Here, we identify a function of the poly(ADP-ribose) glycohydrolase PARG in regulating protein degradation. Using quantitative proteomics, we show that PARG inhibition depletes protein levels of the mono-ADPr hydrolase TARG1. We demonstrate that this TARG1 depletion is both PAR and proteasome dependent and identify the E3 ubiquitin ligases HUWE1 and TRIP12 as mediators of this process. Our findings establish TARG1 as a substrate of PAR-dependent protein degradation and uncover a PARG-dependent mechanism controlling its stability. This work highlights an interplay between the two ADP-ribosyl hydrolases, with implications for the refinement of PARG-targeted therapeutic strategies.

The five pseudo-response regulators (PRRs) in Arabidopsis are key transcriptional repressors in regulating the circadian clock, yet the mechanisms by which they cooperate to maintain circadian homeostasis and adapt to environmental changes remains unclear. We show that PRR9, PRR5, and TOC1 have interchangeable roles in determining circadian pace through promoter swapping and chromatin immunoprecipitation assays. PRRs form homodimers and heterodimers with sequential rhythms lasting approximately 12 h, with the pseudo-receiver (PR), ethylene-responsive element-binding factor-associated amphiphilic repression motif (EAR), and C-terminal CONSTANS, CONSTANS-like, and TOC1 motif (CCT) domains being essential for dimer formation. These interactions coincide with extended periods of high protein abundance and are essential for sustaining the 24-h circadian oscillation. Based on these dynamics, we propose a "PRR escapement" model that explains the diverse circadian periods of prr mutants. Additionally, PRRs rapidly respond to environmental cues, such as light and temperature, adjusting transcription and protein-complex assembly to align growth with environmental changes.

Alpha-ketoglutarate (α-KG), a key intermediate in the tricarboxylic acid (TCA) cycle, was found to be significantly decreased in nucleus pulposus (NP) tissues of patients with intervertebral disc degeneration (IVDD). Supplementation with α-KG restored nucleus pulposus cell (NPC) proliferation, reduced apoptosis, and reestablished extracellular matrix (ECM) metabolic homeostasis. Mechanistically, α-KG enhanced mitophagy and suppressed reactive oxygen species (ROS) accumulation, effects that were abolished by the mitophagy inhibitor Mdivi-1. Further investigation identified isocitrate dehydrogenase 1 (IDH1) as essential for α-KG production and mitochondrial maintenance, with its expression controlled by the METTL3/MALAT1/miR-23c axis. Specifically, METTL3-mediated m⁶A modification destabilized MALAT1, attenuating its sponging of miR-23c and ultimately leading to IDH1 suppression. These findings reveal a novel regulatory pathway governing mitophagy and oxidative stress in NPCs, highlighting potential therapeutic targets for IVDD.

An altered gut microbiota, particularly the expansion of Prevotellaceae members, is implicated in the pathogenesis of rheumatoid arthritis (RA), yet the mechanisms behind this phenomenon remain unclear. Here, we demonstrate that Palleniella intestinalis, a member of the Prevotellaceae family, induces 100% of arthritis incidence in genetically resistant C57BL/6 mice. Inoculation with P. intestinalis modifies gut microbiota ecology, increases intestinal permeability, and selectively activates colonic CD11b⁺CD11c⁺ myeloid cells, facilitating T helper 17 (Th17) differentiation and driving joint inflammation. In vitro, outer membrane vesicles (OMVs) from P. intestinalis and Segatella copri (formerly known as Prevotella copri) prime bone marrow-derived dendritic cells (BMDCs) to drive Th17 differentiation in an interleukin-6 (IL-6)-dependent manner. Similar innate immune activation with elevated IL-6 levels was observed in gut biopsies from new-onset patients with RA. Transfer of Prevotellaceae-derived OMVs or Prevotellaeae-primed BMDCs replicates the heightened arthritis incidence in resistant mice, highlighting the critical role of intestinal immune activation in RA.

Increasing investigations indicate that neurotransmitters shape immune cell function; however, current results about glycine (Gly) in inflammatory macrophage responses are conflicting. Here, we found that Gly transporters support interleukin-1β (IL-1β) production in inflammatory macrophages, while Gly receptors inhibit it. Inflammatory macrophages have higher expression of Gly transporter 1 (GlyT1; also known as SLC6A9). Notably, SLC6A9 inhibition leads to extracellular accumulation of Gly and limits IL-1β production in inflammatory macrophages. Mechanically, extracellular Gly suppresses phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT1)/mammalian target of rapamycin (mTOR) signaling through the Gly receptor alpha-4 (Glrα4), thereby inhibiting activation of the NOD-like receptor 3 (NLRP3) inflammasome and IL-1β production. Furthermore, Gly supplementation or myeloid-specific SLC6A9 depletion alleviates the lipopolysaccharide (LPS)-induced inflammatory response in vivo. Collectively, our findings reveal a previously uncharacterized mechanism for the Gly-ergic system in regulating inflammatory macrophage function, providing a potential alleviating target for macrophage-associated diseases.