Cell Type-specific Roles Research Articles

Inflammasome-mediated pyroptosis in defense against pathogenic bacteria.

Macrophages, neutrophils, and epithelial cells are pivotal components of the host's immune response against bacterial infections. These cells employ inflammasomes to detect various microbial stimuli during infection, triggering an inflammatory response aimed at eradicating the pathogens. Among these inflammatory responses, pyroptosis, a lytic form of cell death, plays a crucial role in eliminating replicating bacteria and recruiting immune cells to combat the invading pathogen. The immunological function of pyroptosis varies across macrophages, neutrophils, and epithelial cells, aligning with their specific roles within the innate immune system. This review centers on elucidating the role of pyroptosis in resisting gram-negative bacterial infections, with a particular focus on the mechanisms at play in macrophages, neutrophils, and intestinal epithelial cells. Additionally, we underscore the cell type-specific roles of pyroptosis invivo in these contexts during defense.

Abstract P127: microRNA-204 protects against Angiotensin II-induced apoptosis in podocytes via Sox4

Background: We showed previously that global knockout (KO) of microRNA miR-204 attenuated hypertension but exacerbated renal injury including albuminuria in 14 days in C57BL/6J mice treated with uninephrectomy, angiotensin II, and a high-salt diet (Unx/AngII/salt). This suggests cell type-specific roles of miR-204. We hypothesize that miR-204 in podocytes protects against renal injury, specifically albuminuria via podocyte damage, in hypertension. Methods: We generated podocyte-specific miR-204 KO mice. Blood pressure and albuminuria were measured. RNA-seq in isolated miR-204 wild-type (WT) and KO mouse glomeruli, approximately 20% of which are podocytes, was performed. Human induced pluripotent stem cell-derived podocytes (iPSC-podocytes) were utilized to model and characterize podocyte injury. Results: Podocyte-specific miR-204 KO mice and WT littermates developed hypertension similarly following Unx/AngII/salt, but albuminuria was substantially exacerbated in miR-204 KO male mice (n=12, 8, 10 for WT, miR-204 +/- and miR-204 -/- , respectively; P<0.05, one-way ANOVA followed by Holm–Šidák test). RNA-seq analysis showed significant upregulation of Sox4, a target of miR-204 and a transcription factor promoting apoptotic p53, in the glomeruli of podocyte-specific miR-204 KO mice (P<0.05). A miR-204 KO iPSC line was generated by CRISPR/Cas9 and clonal expansion. Podocytes differentiated from miR-204 WT and KO iPSCs displayed arborized cell body, foot process-like structures, and significantly higher levels of podocyte markers Nphs1 and Nphs2. When apoptosis was induced via 24-hour AngII treatment, miR-204 KO iPSC-podocytes displayed upregulation of Sox4 (0.67±0.32 and 1.55±0.87, fold for WT and KO iPSC-podocytes compared to their control, n = 4, 4) and p53 (1.56±0.15 and 3.70±1.56, fold for WT and KO iPSC-podocytes compared to their control, n = 4, 4), and exacerbated AngII-induced apoptosis. Sox4 knockdown in miR-204 KO iPSC-podocytes mitigated the p53 upregulation (1.49±0.29 and 1.53±0.16, fold for WT and KO iPSC-podocytes compared to their control, n = 4, 4) and apoptosis. Conclusions: Our findings suggested that miR-204 in podocytes protect against AngII-induced apoptosis by suppressing Sox4, which in turn suppresses p53. Targeting the miR-204/Sox4 axis may hold therapeutic potential in attenuating podocyte injury and preserving renal function in hypertension patients.

LYSMD proteins promote activation of Rab32-family GTPases for lysosome-related organelle biogenesis.

Lysosome-related organelles (LROs) are specialized lysosomes with cell type-specific roles in organismal homeostasis. Dysregulation of LROs leads to many human disorders, but the mechanisms underlying their biogenesis are not fully understood. Here, we identify a group of LYSMD proteins as evolutionarily conserved regulators of LROs. In Caenorhabditis elegans, mutations of LMD-2, a LysM domain-containing protein, reduce the levels of the Rab32 GTPase ortholog GLO-1 on intestine-specific LROs, the gut granules, leading to their abnormal enlargement and defective biogenesis. LMD-2 interacts with GLO-3, a subunit of GLO-1 guanine nucleotide exchange factor (GEF), thereby promoting GLO-1 activation. Mammalian homologs of LMD-2, LYSMD1, and LYSMD2 can functionally replace LMD-2 in C. elegans. In mammals, LYSMD1/2 physically interact with the HPS1 subunit of BLOC-3, the GEF of Rab32/38, thus promoting Rab32 activation. Inactivation of both LYSMD1 and LYSMD2 reduces Rab32 activation, causing melanosome enlargement and decreased melanin production in mouse melanoma cells. These findings provide important mechanistic insights into LRO biogenesis and functions.

Lateral inhibition in V1 controls neural & perceptual contrast sensitivity.

Lateral inhibition is a central principle for sensory system function. It is thought to operate by the activation of inhibitory neurons that restrict the spatial spread of sensory excitation. Much work on the role of inhibition in sensory systems has focused on visual cortex; however, the neurons, computations, and mechanisms underlying cortical lateral inhibition remain debated, and its importance for visual perception remains unknown. Here, we tested how lateral inhibition from PV or SST neurons in mouse primary visual cortex (V1) modulates neural and perceptual sensitivity to stimulus contrast. Lateral inhibition from PV neurons reduced neural and perceptual sensitivity to visual contrast in a uniform subtractive manner, whereas lateral inhibition from SST neurons more effectively changed the slope (or gain) of neural and perceptual contrast sensitivity. A neural circuit model identified spatially extensive lateral projections from SST neurons as the key factor, and we confirmed this with anatomy and direct subthreshold measurements of a larger spatial footprint for SST versus PV lateral inhibition. Together, these results define cell-type specific computational roles for lateral inhibition in V1, and establish their unique consequences on sensitivity to contrast, a fundamental aspect of the visual world.

Cell type specific roles of FOXP1 during early neocortical murine development.

Cortical development is a tightly controlled process and any deviation during development may increase the susceptibility to neurodevelopmental disorders, such as autism spectrum disorders (ASD). Numerous studies identified mutations in FOXP1, a transcription factor enriched in the neocortex, as causal for ASD and FOXP1 syndrome. Our group has shown that Foxp1 deletion in the mouse cortex leads to overall reduced cortex thickness, alterations in cortical lamination, and changes in the relative thickness of cortical layers. However, the developmental and cell type-specific mechanisms underlying these changes remained unclear. This work characterizes the developmental requirement of neocortical Foxp1 at key embryonic and perinatal ages using a conditional knock-out of Foxp1. We find that Foxp1 deletion results in accelerated pseudo-age during early neurogenesis, increased cell cycle exit during late neurogenesis, altered gene expression and chromatin accessibility, and selective migration deficits in a subset of upper-layer neurons. These data explain the postnatal differences observed in cortical layers and relative cortical thickness. We also highlight genes regulated by FOXP1 and their enrichment with high-confidence ASD or synaptic genes. Together, these results underscore a network of neurodevelopmental disorder-related genes that may serve as potential modulatory targets for postnatal modification relevant to ASD and FOXP1 syndrome.

Impaired GABAergic regulation and developmental immaturity in interneurons derived from the medial ganglionic eminence in the tuberous sclerosis complex

GABAergic interneurons play a critical role in maintaining neural circuit balance, excitation–inhibition regulation, and cognitive function modulation. In tuberous sclerosis complex (TSC), GABAergic neuron dysfunction contributes to disrupted network activity and associated neurological symptoms, assumingly in a cell type-specific manner. This GABAergic centric study focuses on identifying specific interneuron subpopulations within TSC, emphasizing the unique characteristics of medial ganglionic eminence (MGE)- and caudal ganglionic eminence (CGE)-derived interneurons. Using single-nuclei RNA sequencing in TSC patient material, we identify somatostatin-expressing (SST+) interneurons as a unique and immature subpopulation in TSC. The disrupted maturation of SST+ interneurons may undergo an incomplete switch from excitatory to inhibitory GABAergic signaling during development, resulting in reduced inhibitory properties. Notably, this study reveals markers of immaturity specifically in SST+ interneurons, including an abnormal NKCC1/KCC2 ratio, indicating an imbalance in chloride homeostasis crucial for the postsynaptic consequences of GABAergic signaling as well as the downregulation of GABAA receptor subunits, GABRA1, and upregulation of GABRA2. Further exploration of SST+ interneurons revealed altered localization patterns of SST+ interneurons in TSC brain tissue, concentrated in deeper cortical layers, possibly linked to cortical dyslamination. In the epilepsy context, our research underscores the diverse cell type-specific roles of GABAergic interneurons in shaping seizures, advocating for precise therapeutic considerations. Moreover, this study illuminates the potential contribution of SST+ interneurons to TSC pathophysiology, offering insights for targeted therapeutic interventions.

Simultaneous deletion of ORMDL1 and ORMDL3 proteins disrupts immune cell homeostasis.

ORMDL3 is a prominent member of a family of highly conserved endoplasmic reticulum resident proteins, ORMs (ORM1 and ORM2) in yeast, dORMDL in Drosophila and ORMDLs (ORMDL1, ORMDL2, and ORMDL3) in mammals. ORMDL3 mediates feedback inhibition of de novo sphingolipid synthesis. Expression levels of ORMDL3 are associated with the development of inflammatory and autoimmune diseases including asthma, systemic lupus erythematosus, type 1 diabetes mellitus and others. It has been shown that simultaneous deletions of other ORMDL family members could potentiate ORMDL3-induced phenotypes. To understand the complex function of ORMDL proteins in immunity in vivo, we analyzed mice with single or double deletions of Ormdl genes. In contrast to other single and double knockouts, simultaneous deletion of ORMDL1 and ORMDL3 proteins disrupted blood homeostasis and reduced immune cell content in peripheral blood and spleens of mice. The reduced number of splenocytes was not caused by aberrant immune cell homing. A competitive bone marrow transplantation assay showed that the development of Ormdl1-/-/Ormdl3-/- B cells was dependent on lymphocyte intrinsic factors. Highly increased sphingolipid production was observed in the spleens and bone marrow of Ormdl1-/-/Ormdl3-/- mice. Slight, yet significant, increase in some sphingolipid species was also observed in the spleens of Ormdl3-/- mice and in the bone marrow of both, Ormdl1-/- and Ormdl3-/- single knockout mice. Taken together, our results demonstrate that the physiological expression of ORMDL proteins is critical for the proper development and circulation of lymphocytes. We also show cell-type specific roles of individual ORMDL family members in the production of different sphingolipid species.

The miR-183/96/182 cluster regulates sensory innervation, resident myeloid cells and functions of the cornea through cell type-specific target genes

The conserved miR-183/96/182 cluster (miR-183C) is expressed in both corneal resident myeloid cells (CRMCs) and sensory nerves (CSN) and modulates corneal immune/inflammatory responses. To uncover cell type-specific roles of miR-183C in CRMC and CSN and their contributions to corneal physiology, myeloid-specific miR-183C conditional knockout (MS-CKO), and sensory nerve-specific CKO (SNS-CKO) mice were produced and characterized in comparison to the conventional miR-183C KO. Immunofluorescence and confocal microscopy of flatmount corneas, corneal sensitivity, and tear volume assays were performed in young adult naïve mice; 3′ RNA sequencing (Seq) and proteomics in the trigeminal ganglion (TG), cornea and CRMCs. Our results showed that, similar to conventional KO mice, the numbers of CRMCs were increased in both MS-CKO and SNS-CKO vs age- and sex-matched WT control littermates, suggesting intrinsic and extrinsic regulations of miR-183C on CRMCs. The number of CRMCs was increased in male vs female MS-CKO mice, suggesting sex-dependent regulation of miR-183C on CRMCs. In the miR-183C KO and SNS-CKO, but not the MS-CKO mice, CSN density was decreased in the epithelial layer of the cornea, but not the stromal layer. Functionally, corneal sensitivity and basal tear volume were reduced in the KO and SNS-CKO, but not the MS-CKO mice. Tear volume in males is consistently higher than female WT mice. Bioinformatic analyses of the transcriptomes revealed a series of cell-type specific target genes of miR-183C in TG sensory neurons and CRMCs. Our data elucidate that miR-183C imposes intrinsic and extrinsic regulation on the establishment and function of CSN and CRMCs by cell-specific target genes. miR-183C modulates corneal sensitivity and tear production through its regulation of corneal sensory innervation.

Epithelial MAPK signaling directs endothelial NRF2 signaling and IL-8 secretion in a tri-culture model of the alveolar-microvascular interface following diesel exhaust particulate (DEP) exposure

BackgroundParticulate matter 2.5 (PM2.5) deposition in the lung’s alveolar capillary region (ACR) is significantly associated with respiratory disease development, yet the molecular mechanisms are not completely understood. Adverse responses that promote respiratory disease development involve orchestrated, intercellular signaling between multiple cell types within the ACR. We investigated the molecular mechanisms elicited in response to PM2.5 deposition in the ACR, in an in vitro model that enables intercellular communication between multiple resident cell types of the ACR.MethodsAn in vitro, tri-culture model of the ACR, incorporating alveolar-like epithelial cells (NCI-H441), pulmonary fibroblasts (IMR90), and pulmonary microvascular endothelial cells (HULEC) was developed to investigate cell type-specific molecular responses to a PM2.5 exposure in an in-vivo-like model. This tri-culture in vitro model was termed the alveolar capillary region exposure (ACRE) model. Alveolar epithelial cells in the ACRE model were exposed to a suspension of diesel exhaust particulates (DEP) (20 µg/cm2) with an average diameter of 2.5 µm. Alveolar epithelial barrier formation, and transcriptional and protein expression alterations in the directly exposed alveolar epithelial and the underlying endothelial cells were investigated over a 24 h DEP exposure.ResultsAlveolar epithelial barrier formation was not perturbed by the 24 h DEP exposure. Despite no alteration in barrier formation, we demonstrate that alveolar epithelial DEP exposure induces transcriptional and protein changes in both the alveolar epithelial cells and the underlying microvascular endothelial cells. Specifically, we show that the underlying microvascular endothelial cells develop redox dysfunction and increase proinflammatory cytokine secretion. Furthermore, we demonstrate that alveolar epithelial MAPK signaling modulates the activation of NRF2 and IL-8 secretion in the underlying microvascular endothelial cells.ConclusionsEndothelial redox dysfunction and increased proinflammatory cytokine secretion are two common events in respiratory disease development. These findings highlight new, cell-type specific roles of the alveolar epithelium and microvascular endothelium in the ACR in respiratory disease development following PM2.5 exposure. Ultimately, these data expand our current understanding of respiratory disease development following particle exposures and illustrate the utility of multicellular in vitro systems for investigating respiratory tract health.

Cell type-specific roles of APOE4 in Alzheimer disease.

The ɛ4 allele of the apolipoprotein E gene (APOE), which translates to the APOE4 isoform, is the strongest genetic risk factor for late-onset Alzheimer disease (AD). Within the CNS, APOE is produced by a variety of cell types under different conditions, posing a challenge for studying its roles in AD pathogenesis. However, through powerful advances in research tools and the use of novel cell culture and animal models, researchers have recently begun to study the roles of APOE4 in AD in a cell type-specific manner and at a deeper and more mechanistic level than ever before. In particular, cutting-edge omics studies have enabled APOE4 to be studied at the single-cell level and have allowed the identification of critical APOE4 effects in AD-vulnerable cellular subtypes. Through these studies, it has become evident that APOE4 produced in various types of CNS cell - including astrocytes, neurons, microglia, oligodendrocytes and vascular cells - has diverse roles in AD pathogenesis. Here, we review these scientific advances and propose a cell type-specific APOE4 cascade model of AD. In this model, neuronal APOE4 emerges as a crucial pathological initiator and driver of AD pathogenesis, instigating glial responses and, ultimately, neurodegeneration. In addition, we provide perspectives on future directions for APOE4 research and related therapeutic developments in the context of AD.

Genome-Wide Mapping of R-Loops in Single Cells Reveals Cell-Type Specific Roles for R-Loops in Hematopoietic Cell Identity

R-loops are three-stranded structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops play critical roles in gene regulation and pathologic roles in DNA damage response. Current methods to map R-loops require at least 500,000 cells. Consequently, R-loop characterization in primary cells is limited. Furthermore, there are no genomic tools to identify R-loops in single cells. We therefore developed here methods to map genome-wide R-loops at single cell resolution. R-loop mapping in primary stem and mature hematopoietic cell populations, and individual single cells revealed striking cell-type specific R-loops. Current methods to map R-loops utilize immunoprecipitation with a monoclonal antibody (clone “S9.6”) or catalytically inactive, but R-loop binding competent, RNAseH1. Motivated by success mapping chromatin modifications in single cells through fusion of Tn5 transposase to a nanobody as well as insertion of T7 promoters to amplify DNA at sites of transposition, we combined both techniques to identify R-loops in limited cell numbers, down to the single cell level. We used a nanobody-Tn5 fusion to identify S9.6 and developed an orthogonal recombinant fusion protein of enzymatic dead RNAseH1 fused to Tn5 (Figure A). We first applied our nanobody-Tn5 method to identify R-loops bound by S9.6 in mouse lineage-negative c-Kit + bone marrow cells in biological duplicates of increasingly reduced cell numbers (64,000, 32,000, 16,000, and 8,000 cells). This revealed remarkable reproducibility of R-loop detection and characteristics. R-loops identified were sensitive to RNAseH1 and enriched at sites with G/C skew and mitochondrial DNA (a site with abundant physiologic R-loops), suggesting authentic, bona fide R-loops mapped in as few 8,000 cells. We next evaluated R-loops across FACS-isolated hematopoietic stem and mature cells from mouse and human bone marrow. Mapping of R-loops in 10,000 cells in duplicate revealed reproducible R-loops which were characteristic of each cell population. Hierarchical clustering of R-loop profiles delineated mature from stem and progenitor cells and discovered increased abundance of genic R-loops in hematopoietic stem and progenitors relative to mature cells (Figure B). Application of our enzymatic dead RNaseH1/Tn5 fusion protein to CD34 + cells revealed a similar genome-wide distribution of R-loops as with the S9.6/nanobody-Tn5 method. There was high correlation of long- and short-term HSC-specific R-loops with sites characteristic of active transcription (based on H3K4me1/2/3 as well as H3K27Ac). Transcription factor motifs activated in unique hematopoietic cell types were enriched at R-loops within the same cell types. For example, GATA motif usage was enriched at R-loops in CMP, MEP, and erythroid precursors. We also detected massive increases in R-loop abundance at the immunoglobulin heavy chain locus in primary B-cells stimulated to undergo class switch recombination. This provides further proof of our low input method to identify R-loops, as physiologic R-loops are known to occur at this locus to promote class switch recombination. Despite recent advances in single cell genomics there are no single cell maps of R-loops. We therefore profiled R-loops using our nanobody-Tn5, low-input S9.6 CUT&Tag approach in individual human CD34 + and mouse c-Kit + cells. We obtained 1,579 single-cell profiles from human and 1,716 from mouse. Uniform manifold approximation and projection (UMAP) identified 12 clusters in human and 9 mouse hematopoietic clusters. Aggregated single-cell R-loop profiles closely resembled bulk profiles indicating that single-cell data captured features of multi-cell maps. Pseudotime analyses of R-loops inferred LT-HSCs as the shared root of hematopoiesis in both mouse and human and identified seven terminal states without prior knowledge of the number of branches in the hematopoietic development. These data provide the first maps of R-loops in primary normal hematopoietic cells and identify scheduled, physiologic cell-type specific R-loops which have not previously been uncovered. The atlas of R-loops across hematopoiesis generated here will motivate future studies to understand the roles of R-loops in hematopoietic differentiation. Moreover, the methods presented map R-loop abundance at nucleotide resolution in primary and limited cell types which can be used for wide-reaching applications.

Deciphering a critical role of uterine epithelial SHP2 in parturition initiation at single cell resolution

The timely onset of female parturition is a critical determinant for pregnancy success. The highly heterogenous maternal decidua has been increasingly recognized as a vital factor in setting the timing of labor. Despite the cell type specific roles in parturition, the role of the uterine epithelium in the decidua remains poorly understood. This study uncovers the critical role of epithelial SHP2 in parturition initiation via COX1 and COX2 derived PGF2α leveraging epithelial specific Shp2 knockout mice, whose disruption contributes to delayed parturition initiation, dystocia and fetal deaths. Additionally, we also show that there are distinct types of epithelium in the decidua approaching parturition at single cell resolution accompanied with profound epithelium reformation via proliferation. Meanwhile, the epithelium maintains the microenvironment by communicating with stromal cells and macrophages. The epithelial microenvironment is maintained by a close interaction among epithelial, stromal and macrophage cells of uterine stromal cells. In brief, this study provides a previously unappreciated role of the epithelium in parturition preparation and sheds lights on the prevention of preterm birth.

Identification of cell-type specific alternative transcripts in the multicellular alga Volvox carteri

BackgroundCell type specialization is a hallmark of complex multicellular organisms and is usually established through implementation of cell-type-specific gene expression programs. The multicellular green alga Volvox carteri has just two cell types, germ and soma, that have previously been shown to have very different transcriptome compositions which match their specialized roles. Here we interrogated another potential mechanism for differentiation in V. carteri, cell type specific alternative transcript isoforms (CTSAI).MethodsWe used pre-existing predictions of alternative transcripts and de novo transcript assembly with HISAT2 and Ballgown software to compile a list of loci with two or more transcript isoforms, identified a small subset that were candidates for CTSAI, and manually curated this subset of genes to remove false positives. We experimentally verified three candidates using semi-quantitative RT-PCR to assess relative isoform abundance in each cell type.ResultsOf the 1978 loci with two or more predicted transcript isoforms 67 of these also showed cell type isoform expression biases. After curation 15 strong candidates for CTSAI were identified, three of which were experimentally verified, and their predicted gene product functions were evaluated in light of potential cell type specific roles. A comparison of genes with predicted alternative splicing from Chlamydomonas reinhardtii, a unicellular relative of V. carteri, identified little overlap between ortholog pairs with alternative splicing in both species. Finally, we interrogated cell type expression patterns of 126 V. carteri predicted RNA binding protein (RBP) encoding genes and found 40 that showed either somatic or germ cell expression bias. These RBPs are potential mediators of CTSAI in V. carteri and suggest possible pre-adaptation for cell type specific RNA processing and a potential path for generating CTSAI in the early ancestors of metazoans and plants.ConclusionsWe predicted numerous instances of alternative transcript isoforms in Volvox, only a small subset of which showed cell type specific isoform expression bias. However, the validated examples of CTSAI supported existing hypotheses about cell type specialization in V. carteri, and also suggested new hypotheses about mechanisms of functional specialization for their gene products. Our data imply that CTSAI operates as a minor but important component of V. carteri cellular differentiation and could be used as a model for how alternative isoforms emerge and co-evolve with cell type specialization.

Local autocrine plasticity signaling in single dendritic spines by insulin-like growth factors.

The insulin superfamily of peptides is essential for homeostasis as well as neuronal plasticity, learning, and memory. Here, we show that insulin-like growth factors 1 and 2 (IGF1 and IGF2) are differentially expressed in hippocampal neurons and released in an activity-dependent manner. Using a new fluorescence resonance energy transfer sensor for IGF1 receptor (IGF1R) with two-photon fluorescence lifetime imaging, we find that the release of IGF1 triggers rapid local autocrine IGF1R activation on the same spine and more than several micrometers along the stimulated dendrite, regulating the plasticity of the activated spine in CA1 pyramidal neurons. In CA3 neurons, IGF2, instead of IGF1, is responsible for IGF1R autocrine activation and synaptic plasticity. Thus, our study demonstrates the cell type-specific roles of IGF1 and IGF2 in hippocampal plasticity and a plasticity mechanism mediated by the synthesis and autocrine signaling of IGF peptides in pyramidal neurons.

FOXP1 orchestrates neurogenesis in human cortical basal radial glial cells.

During cortical development, human basal radial glial cells (bRGCs) are highly capable of sustained self-renewal and neurogenesis. Selective pressures on this cell type may have contributed to the evolution of the human neocortex, leading to an increase in cortical size. bRGCs have enriched expression for Forkhead Box P1 (FOXP1), a transcription factor implicated in neurodevelopmental disorders (NDDs) such as autism spectrum disorder. However, the cell type-specific roles of FOXP1 in bRGCs during cortical development remain unexplored. Here, we examine the requirement for FOXP1 gene expression regulation underlying the production of bRGCs using human brain organoids. We examine a developmental time point when FOXP1 expression is highest in the cortical progenitors, and the bRGCs, in particular, begin to actively produce neurons. With the loss of FOXP1, we show a reduction in the number of bRGCs, as well as reduced proliferation and differentiation of the remaining bRGCs, all of which lead to reduced numbers of excitatory cortical neurons over time. Using single-nuclei RNA sequencing and cell trajectory analysis, we uncover a role for FOXP1 in directing cortical progenitor proliferation and differentiation by regulating key signaling pathways related to neurogenesis and NDDs. Together, these results demonstrate that FOXP1 regulates human-specific features in early cortical development.

Cell type-specific roles of NLRP3, inflammasome-dependent and -independent, in host defense, sterile necroinflammation, tissue repair, and fibrosis.

The NLRP3 inflammasome transforms a wide variety of infectious and non-infectious danger signals that activate pro-inflammatory caspases, which promote the secretion of IL-1β and IL-18, and pyroptosis, a pro-inflammatory form of cell necrosis. Most published evidence documents the presence and importance of the NLRP3 inflammasome in monocytes, macrophages, and neutrophils during host defense and sterile forms of inflammation. In contrast, in numerous unbiased data sets, NLRP3 inflammasome-related transcripts are absent in non-immune cells. However, an increasing number of studies report the presence and functionality of the NLRP3 inflammasome in almost every cell type. Here, we take a closer look at the reported cell type-specific expression of the NLRP3 inflammasome components, review the reported inflammasome-dependent and -independent functions, and discuss possible explanations for this discrepancy.

The World according to IL-9.

Among the cytokines regulating immune cells, IL-9 has gained considerable attention for its ability to act on multiple cell types as a regulator of beneficial and pathologic immune responses. Yet, it is still not clearly defined how IL-9 impacts immune responses. IL-9 demonstrates a remarkable degree of tissue-specific functionality and has cellular sources that vary by tissue site and the context of the inflammatory milieu. Here, we provide perspective to summarize the biological activities of IL-9 and highlight cell type-specific roles in the immune pathogenesis of diseases. This perspective will be important in defining the diseases where targeting IL-9 as a therapeutic strategy would be beneficial and where it has the potential to complicate clinical outcomes.

Mitofusin-2 in nucleus accumbens D2-MSNs regulates social dominance and neuronal function

The nucleus accumbens (NAc) is a brain hub regulating motivated behaviors, including social competitiveness. Mitochondrial function in the NAc links anxiety with social competitiveness, and the mitochondrial fusion protein mitofusin 2 (Mfn2) in NAc neurons regulates anxiety-related behaviors. However, it remains unexplored whether accumbal Mfn2 levels also affect social behavior and whether Mfn2 actions in the emotional and social domain are driven by distinct cell types. Here, we found that subordinate-prone highly anxious rats show decreased accumbal Mfn2 levels and that Mfn2 overexpression promotes dominant behavior. In mice, selective Mfn2 downregulation in NAc dopamine D2 receptor-expressing medium spiny neurons (D2-MSNs) induced social subordination, accompanied by decreased accumbal mitochondrial functions and decreased excitability in D2-MSNs. Instead, D1-MSN-targeted Mfn2 downregulation affected competitive ability only transiently and likely because of an increase in anxiety-like behaviors. Our results assign dissociable cell-type specific roles to Mfn2 in the NAc in modulating social dominance and anxiety.

Protective and Adverse Roles of DDX3X in Different Cell Types in Nonalcoholic Steatohepatitis Progression

Persistent hepatic cellular metabolic stress and liver inflammatory stimuli are key signatures of nonalcoholic steatohepatitis (NASH). DDX3X is a vital molecule involved in cell fate decisions in both pro-survival stress granule (SG) and pro-death NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome assembly in response to stress signals. However, the role of DDX3X in NASH remains unclear. We characterized the cell type-specific roles of DDX3X in NASH. Human liver tissues from NASH patients and normal control subjects were collected to assess DDX3X expression and distribution. Nutritional steatohepatitis models were constructed by feeding macrophage-specific DDX3X knockout (DDX3X ΔMφ ), hepatocyte-specific DDX3X knockout (DDX3X Δhep ), and wild-type control (DDX3X fl/fl ) mice a high-fat and high-cholesterol (HFHC) diet, a methionine- and choline-deficient (MCD) diet, and a high-fat/high-iron/high-fructose/high-cholesterol, low-methionine, and choline-deficient (HFHIHFHC-MCD) diet. The study demonstrated that DDX3X was predominantly expressed in macrophages and hepatocytes in control liver tissues, and its expression was down-regulated in patients or mice with NASH. Compared to DDX3X fl/fl littermates, DDX3X ΔMφ mice showed improved liver histology in nutritional steatohepatitis models. Loss of macrophage DDX3X inhibited NLRP3 inflammasome-mediated pyroptosis, causing anti-inflammatory M2 polarization and alleviating hepatocyte steatohepatitic changes. DDX3X Δhep mice developed marked steatohepatitis in multiple nutritional steatohepatitis models compared to DDX3X fl/fl littermates. DDX3X-deleted hepatocytes showed impaired SG assembly, leading to increased sensitivity and intolerance to metabolic stimulation and resultant steatohepatitis. In conclusion, DDX3X plays opposite roles in different cell types during the progression of NASH. A better understanding of the cell-specific differences in the crosstalk between SG formation and NLRP3 activation is crucial for developing prospective targeted DDX3X inhibitors for the treatment of NASH.

Subtype-Specific Roles of Ellipsoid Body Ring Neurons in Sleep Regulation in Drosophila.

The ellipsoid body (EB) is a major structure of the central complex of the Drosophila melanogaster brain. Twenty-two subtypes of EB ring neurons have been identified based on anatomic and morphologic characteristics by light-level microscopy and EM connectomics. A few studies have associated ring neurons with the regulation of sleep homeostasis and structure. However, cell type-specific and population interactions in the regulation of sleep remain unclear. Using an unbiased thermogenetic screen of EB drivers using female flies, we found the following: (1) multiple ring neurons are involved in the modulation of amount of sleep and structure in a synergistic manner; (2) analysis of data for ΔP(doze)/ΔP(wake) using a mixed Gaussian model detected 5 clusters of GAL4 drivers which had similar effects on sleep pressure and/or depth: lines driving arousal contained R4m neurons, whereas lines that increased sleep pressure had R3m cells; (3) a GLM analysis correlating ring cell subtype and activity-dependent changes in sleep parameters across all lines identified several cell types significantly associated with specific sleep effects: R3p was daytime sleep-promoting, and R4m was nighttime wake-promoting; and (4) R3d cells present in 5HT7-GAL4 and in GAL4 lines, which exclusively affect sleep structure, were found to contribute to fragmentation of sleep during both day and night. Thus, multiple subtypes of ring neurons distinctively control sleep amount and/or structure. The unique highly interconnected structure of the EB suggests a local-network model worth future investigation; understanding EB subtype interactions may provide insight how sleep circuits in general are structured.SIGNIFICANCE STATEMENT How multiple brain regions, with many cell types, can coherently regulate sleep remains unclear, but identification of cell type-specific roles can generate opportunities for understanding the principles of integration and cooperation. The ellipsoid body (EB) of the fly brain exhibits a high level of connectivity and functional heterogeneity yet is able to tune multiple behaviors in real-time, including sleep. Leveraging the powerful genetic tools available in Drosophila and recent progress in the characterization of the morphology and connectivity of EB ring neurons, we identify several EB subtypes specifically associated with distinct aspects of sleep. Our findings will aid in revealing the rules of coding and integration in the brain.