Cell Type-specific Mechanisms Research Articles

SIV infection induces alterations in gene expression and loss of interneurons in Rhesus Macaque frontal cortex during early systemic infection.

Understanding the neurobiological mechanisms underlying HIV-associated neurocognitive decline in people living with HIV is frequently complicated by an inability to analyze changes across the course of the infection and frequent presence of comorbid psychiatric and substance use disorders. Preclinical non-human primate simian immunodeficiency virus (SIV) models help address these shortcomings. However, SIV studies frequently target protracted endpoints, limiting our understanding of the neuromolecular alterations during the early post-infection window. To begin to address this knowledge gap, we utilized single nuclei transcriptomics to examine frontal cortex samples of rhesus macaques 10- and 20-days post-SIV infection, compared to non-infected controls. We identify and validated a decrease in inhibitory neurons during the early post infection window, representing a potential substrate of longer-term injury and neurocognitive impairment in people living with HIV. Differential expression identified alterations in cellular subtype gene expression that persisted over the 20-day time course and short-lived differences only detected at 10-days post-SIV infection. In silico predicted regulatory mechanisms and dysregulated neural signaling pathways are presented. Analysis of cell-cell interaction networks identify altered signal pathways in the frontal cortex that may represent regional alterations in cell-cell communications. In total, these results identify cell type-specific molecular mechanisms putatively capable of underlying long-term neurocognitive alterations in persons living with HIV.

Cell-cell communications in the brain of hepatic encephalopathy: The neurovascular unit.

Many patients with liver diseases are exposed to the risk of hepatic encephalopathy (HE). The incidence of HE in liver patients is high, showing various symptoms ranging from mild symptoms to coma. Liver transplantation is one of the ways to overcome HE. However, not all patients can receive liver transplantation. Moreover, patients who have received liver transplantation have limitations in that they are vulnerable to hepatocellular carcinoma, allograft rejection, and infection. To find other therapeutic strategies, it is important to understand pathological factors and mechanisms that lead to HE after liver disease. Oxidative stress, inflammatory response, hyperammonaemia and metabolic disorders seen after liver diseases have been reported as risk factors of HE. These are known to affect the brain and cause HE. These peripheral pathological factors can impair the blood-brain barrier, cause it to collapse and damage the neurovascular unit component of multiple cells, including vascular endothelial cells, astrocytes, microglia, and neurons, leading to HE. Many previous studies on HE have suggested the impairment of neurovascular unit and cell-cell communication in the pathogenesis of HE. This review focuses on pathological factors that appear in HE, cell type-specific pathological mechanisms, miscommunication/incorrect relationships, and therapeutic candidates between brain cells in HE. This review suggests that regulating communications and interactions between cells may be important in overcoming HE.

Syndecan-1: a key player in health and disease.

Syndecan-1 (SDC-1) is a transmembrane protein localized on the basolateral surface of epithelial cells, encompassing a core protein with heparin sulfate and chondroitin sulfate glycosaminoglycan side chains. SDC-1 is involved in a panoply of cellular mechanismsincludingcell-to-cell adhesion, extracellular matrix interactions, cell cycle modulation, and lipid clearance. Alterations in the expression and function of SDC-1 are implicated in numerous disease entities, making it an attractive diagnostic and therapeutic target. However, despite its broad involvement in several disease processes, the underlying mechanism contributing to its diverse functions, pathogenesis, and therapeutic uses remains underexplored. Therefore, this review examines the role of SDC-1 in health and disease, focusing on liver pathologies, inflammatory diseases, infectious diseases, and cancer, and sheds light on SDC-1-based therapeutic approaches. Moreover, it delves into the mechanisms through which SDC-1 contributes to these diseases, emphasizing cell-type specific mechanisms. By comprehensively summarizing the significance of SDC-1, its association with several diseases, and its underlying mechanisms of action, the findings of this review could inform future research directions toward the development of targeted therapies and early diagnosis for a multitude of disease entities.

Abstract B018: Multifaceted pro-metastatic role of IL-17 signaling in modulating the tumor microenvironment of colorectal cancer

Abstract Colorectal cancer (CRC) ranks second in mortality and advanced CRCs are resistant to current immunotherapies and prone to metastasis, which necessitates research into enhancing innate and adaptive anti-cancer immune responses. Chronic inflammation mediated by cytokines like IL6 and notably, IL17 activates the oncogenic pathways in cancer cells, promoting sporadic and inflammatory CRC. What is unknown, is the ability of cytokines to control tumor microenvironment (TME) via cell type specific mechanisms to increase cancer progression, metastasis and/or therapy resistance. Increased levels of IL17A in CRC portends poor prognosis indicating its potential involvement into metastasis. However, the role of IL17 signaling in TME in the process of CRC progression and metastasis remains enigmatic. We utilized different models of CRC progression and metastasis, as well as variety of genetic mice models and therapeutic interventions. First, using a model of colitis-associated cancer we found that “late” IL17A neutralization decreases tumor burden, possibly acting through regulation of ferroptosis, NK cells mediated cytotoxicity, as well as myeloid cells and chemokine network essential for the control of immunosuppressive TME. Next, models of CRC metastasis, including those based on MC38 cells and APTAK cancerous organoids, showed that IL17A neutralization reduces metastasis growth. Also, FACS analysis revealed that IL17A promotes the recruitment of tumor- associated macrophages and other key myeloid subsets into the metastatic liver environment. Further research, using mice with conditional knockout of IL17RC receptor in myeloid cells (Il17rc f/f -LysMCre) or hepatocytes (Il17rc f/f -AlbCre) also showed significance of IL17 signaling activation in CRC metastasis through two distinct mechanisms. Employing different techniques, from FACS, immunohistochemistry, to RNAseq and scRNAseq data analysis, we observed that local hepatic activation of IL17RC signaling recruits neutrophils and reduces infiltration of cytotoxic CD8+T and NK cells to the metastatic niche supporting tumor growth, while myeloid IL17RC signaling reshapes TME, including macrophages and neutrophils, enhancing CRC metastasis. Our research indicates that IL17 signaling plays a multifaceted pathogenic role in CRC progression by operating in at least several cell types, providing new insights into the basic biology of CRC metastasis and potentially illuminating the design and improvement of CRC treatment strategies. Citation Format: Katarzyna Chojnacka, Katrina Mae Reyes, Hana Tomizawa, Sergei Grivennikov. Multifaceted pro-metastatic role of IL-17 signaling in modulating the tumor microenvironment of colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B018.

Single-Nucleus RNA Sequencing Reveals the Transcriptome Profiling of Ovarian Cells in Adolescent Cyprinus carpio.

The common carp (Cyprinus carpio) is a crucial freshwater species cultivated worldwide for food consumption. Female carp have better growth performance than males, which fascinates scholars to uncover the mechanism of gonadal differentiation and produce mono-sex populations. However, the mechanism of ovarian development at single-cell resolution is limited. Here, we conducted single-nucleus RNA sequencing in adolescent common carp ovaries. Our study obtained transcriptional profiles of 13,155 nuclei and revealed 13 distinct cell clusters in the ovaries, including three subtypes of germ cells and four subtypes of granulosa cells. We subsequently performed pseudotime trajectory analysis to delineate potential mechanisms underlying the development of germ cells and granulosa cells. We identified 1250 dynamic expression genes in germ cells and 1815 in granulosa cells (q-value < 0.01), including zp3, eif4a2 and aspm in germ cells and fshr and esr1 in granulosa cells. The functional annotation showed that dynamic expression genes in germ cells were involved in sperm-egg recognition and some terms related to meiosis, such as sister chromatid segregation and homologous recombination. Genes expressed dynamically in granulosa cells were related to the TGF-β signaling pathway, response to gonadotropin, and development of primary female sexual characteristics. In addition, the dynamic genes expressed in granulosa cells might relate to the complex communication between different cell types. In summary, our study provided a transcriptome profile of common carp ovaries at single-nucleus resolution, and we further revealed the potential cell type-specific mechanisms underlying oogenesis and the differentiation of granulosa cells, which will facilitate breeding all-female common carp populations.

Rare variant contribution to the heritability of coronary artery disease

Whole genome sequences (WGS) enable discovery of rare variants which may contribute to missing heritability of coronary artery disease (CAD). To measure their contribution, we apply the GREML-LDMS-I approach to WGS of 4949 cases and 17,494 controls of European ancestry from the NHLBI TOPMed program. We estimate CAD heritability at 34.3% assuming a prevalence of 8.2%. Ultra-rare (minor allele frequency ≤ 0.1%) variants with low linkage disequilibrium (LD) score contribute ~50% of the heritability. We also investigate CAD heritability enrichment using a diverse set of functional annotations: i) constraint; ii) predicted protein-altering impact; iii) cis-regulatory elements from a cell-specific chromatin atlas of the human coronary; and iv) annotation principal components representing a wide range of functional processes. We observe marked enrichment of CAD heritability for most functional annotations. These results reveal the predominant role of ultra-rare variants in low LD on the heritability of CAD. Moreover, they highlight several functional processes including cell type-specific regulatory mechanisms as key drivers of CAD genetic risk.

Alzheimer's disease risk gene CD2AP is a dose-sensitive determinant of synaptic structure and plasticity.

CD2-Associated protein (CD2AP) is a candidate susceptibility gene for Alzheimer's disease, but its role in the mammalian central nervous system remains largely unknown. We show that CD2AP protein is broadly expressed in the adult mouse brain, including within cortical and hippocampal neurons, where it is detected at pre-synaptic terminals. Deletion of Cd2ap altered dendritic branching and spine density, and impaired ubiquitin-proteasome system activity. Moreover, in mice harboring either one or two copies of a germline Cd2ap null allele, we noted increased paired-pulse facilitation at hippocampal Schaffer-collateral synapses, consistent with a haploinsufficient requirement for pre-synaptic release. Whereas conditional Cd2ap knockout in the brain revealed no gross behavioral deficits in either 3.5- or 12-month-old mice, Cd2ap heterozygous mice demonstrated subtle impairments in discrimination learning using a touchscreen task. Based on unbiased proteomics, partial or complete loss of Cd2ap triggered perturbation of proteins with roles in protein folding, lipid metabolism, proteostasis, and synaptic function. Overall, our results reveal conserved, dose-sensitive requirements for CD2AP in the maintenance of neuronal structure and function, including synaptic homeostasis and plasticity, and inform our understanding of possible cell-type specific mechanisms in Alzheimer's Disease.

Drug Metabolism and Transport Capacity of Endothelial Cells, Pericytes, and Astrocytes: Implications for CNS Drug Disposition.

Therapeutically targeting the brain requires interactions with endothelial cells, pericytes, and astrocytes at the blood brain barrier (BBB). We evaluated regional and cell-type specific drug metabolism and transport mechanisms using rhesus macaques and in vitro treatment of primary human cells. Here, we report heterogenous distribution of representative drugs, tenofovir (TFV), emtricitabine (FTC), and their active metabolites, which cerebrospinal fluid measures could not reflect. We found that all BBB cell types possessed functional drug metabolizing enzymes and transporters that promoted TFV and FTC uptake and pharmacologic activation. Pericytes and astrocytes emerged as pharmacologically dynamic cells that rivaled hepatocytes and were uniquely susceptible to modulation by disease and treatment. Together, our findings demonstrate the importance of considering the BBB as a unique pharmacologic entity, rather than viewing it as an extension of the liver, as each cell type possesses distinct drug metabolism and transport capacities that contribute to differential brain drug disposition.

The Musashi-1–type 2 deiodinase pathway regulates astrocyte proliferation

Thyroid hormone (TH) is a critical regulator of cellular function and cell fate. The circulating TH level is relatively stable, while tissue TH action fluctuates according to cell type-specific mechanisms. Here, we focused on identifying mechanisms that regulate TH action through the type 2 deiodinase (D2) in glial cells. Dio2 mRNA has an unusually long 3'UTR where we identified multiple putative MSI1 binding sites for Musashi-1 (MSI1), a highly conserved RNA-binding cell cycle regulator. Binding to these sites was confirmed through electrophoretic mobility shift assay. In H4 glioma cells, shRNA-mediated MSI1 knockdown increased endogenous D2 activity, whereas MSI1 overexpression in HEK293T cells decreased D2 expression. This latter effect could be prevented by the deletion of a 3.6 kb region of the 3'UTR of Dio2 mRNA containing MSI1 binding sites. MSI1 immunoreactivity was observed in 2mouse Dio2-expressing cell types, that is, cortical astrocytes and hypothalamic tanycytes, establishing the anatomical basis for a potential invivo interaction of Dio2 mRNA and MSl1. Indeed, increased D2 expression was observed in the cortex of mice lacking MSI1 protein. Furthermore, MSI1 knockdown-induced D2 expression slowed down cell proliferation by 56% in primary cultures of mouse cortical astrocytes, establishing the functionality of the MSI1-D2-T3 pathway. In summary, Dio2 mRNA is a target of MSI1 and the MSI1-D2-T3 pathway is a novel regulatory mechanism of astrocyte proliferation with the potential to regulate the pathogenesis of human glioblastoma.

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.

Single-Cell Transcriptomic Profiling Unveils Dynamic Immune Cell Responses during Haemonchus contortus Infection.

Haemonchus contortus is a parasite widely distributed in tropical, subtropical, and warm temperate regions, causing significant economic losses in the livestock industry worldwide. However, little is known about the genetics of H. contortus resistance in livestock. In this study, we monitor the dynamic immune cell responses in diverse peripheral blood mononuclear cells (PBMCs) during H. contortus infection in goats through single-cell RNA sequencing (scRNA-Seq) analysis. A total of four Boer goats, two goats with oral infection with the L3 larvae of H. contortus and two healthy goats as controls, were used in the animal test. The infection model in goats was established and validated by the fecal egg count (FEC) test and qPCR analysis of the gene expression of IL-5 and IL-6. Using scRNA-Seq, we identified seven cell types, including T cells, monocytes, natural killer cells, B cells, and dendritic cells with distinct gene expression signatures. After identifying cell subpopulations of differentially expressed genes (DEGs) in the case and control groups, we observed the upregulation of multiple inflammation-associated genes, including NFKBIA and NFKBID. Kyoto Encyclopedia of the Genome (KEGG) enrichment analysis revealed significant enrichment of NOD-like receptor pathways and Th1/Th2 cell differentiation signaling pathways in CD4 T cells DEGs. Furthermore, the analysis of ligand-receptor interaction networks showed a more active state of cellular communication in the PBMCs from the case group, and the inflammatory response associated MIF-(CD74 + CXCR4) ligand receptor complex was significantly more activated in the case group, suggesting a potential inflammatory response. Our study preliminarily revealed transcriptomic profiling characterizing the cell type specific mechanisms in host PBMCs at the single-cell level during H. contortus infection.

A single-cell profile reveals the transcriptional regulation responded for Abelmoschus manihot (L.) treatment in diabetic kidney disease

BackgroundHuangkui capsule (HKC), as an ethanol extract of Abelmoschus manihot (L.), has a significant efficacy in treatment of the patients with diabetic kidney disease (DKD). The bioactive ingredients of HKC mainly include the flavonoids such as rutin, hyperoside, hibifolin, isoquercetin, myricetin, quercetin and quercetin-3-O-robinobioside. PurposeTo explore the molecular mechanisms of A. manihot in treatment of DKD. Study designA single-cell RNA sequencing analysis of kidneys in db/db mice with and without HKC administration. MethodsUrinary biochemical and histopathological examination in C57BL/6 and db/db mice of DKD and HKC groups was done. Single-cell RNA sequencing pipeline was then performed. The regulatory mechanisms of seven flavonoids in HKC were revealed by cell communication, prediction of transcription factor regulatory network, and molecular docking. ResultsBy constructing ligand-receptor regulatory network and performing molecular docking between 75 receptors with different activities and seven flavonoids. 11 key receptors in 4 cell types (segment 3 proximal convoluted tubular cell, ascending limbs of the loop of Henle, distal convoluted tubule, and T cell) in kidneys were found to be directly interacted with HKC. The interactions regulated 8 downstream regulons. The docking receptors in T cell led to transcriptional event differences in the regulons such as Cebpb, Rel, Tbx21 and Klf2 and consequently affected the activation, differentiation, and infiltration of T cell, while the receptors Tgfbr1 and Ldlr in stromal cells of kidneys were closely associated with the downstream transcriptional events of renal injury and proteinuria in DKD. ConclusionThe current study provides novel information of the key receptors and regulons in renal cells for a better understanding of the cell type specific molecular mechanisms of A. manihot in treatment of DKD.

Cell-Type Specific Connectivity of Whisker-Related Sensory and Motor Cortical Input to Dorsal Striatum.

The anterior dorsolateral striatum (DLS) is heavily innervated by convergent excitatory projections from the primary motor (M1) and sensory cortex (S1) and considered an important site of sensorimotor integration. M1 and S1 corticostriatal synapses have functional differences in their connection strength with striatal spiny projection neurons (SPNs) and fast-spiking interneurons (FSIs) in the DLS and, as a result, exert distinct influences on sensory-guided behaviors. In the present study, we tested whether M1 and S1 inputs exhibit differences in the subcellular anatomical distribution of striatal neurons. We injected adeno-associated viral vectors encoding spaghetti monster fluorescent proteins (sm.FPs) into M1 and S1 in male and female mice and used confocal microscopy to generate 3D reconstructions of corticostriatal inputs to single identified SPNs and FSIs obtained through ex vivo patch clamp electrophysiology. We found that M1 and S1 dually innervate SPNs and FSIs; however, there is a consistent bias towards the M1 input in SPNs that is not found in FSIs. In addition, M1 and S1 inputs were distributed similarly across the proximal, medial, and distal regions of SPN and FSI dendrites. Notably, closely localized M1 and S1 clusters of inputs were more prevalent in SPNs than FSIs, suggesting that cortical inputs are integrated through cell-type specific mechanisms. Our results suggest that the stronger functional connectivity from M1 to SPNs compared to S1, as previously observed, is due to a higher quantity of synaptic inputs. Our results have implications for how sensorimotor integration is performed in the striatum through cell-specific differences in corticostriatal connections.

Single-cell transcriptomic atlas of goat ovarian aging

BackgroundThe ovaries are one of the first organs that undergo degenerative changes earlier in the aging process, and ovarian aging is shown by a decrease in the number and quality of oocytes. However, little is known about the molecular mechanisms of female age-related fertility decline in different types of ovarian cells during aging, especially in goats. Therefore, the aim of this study was to reveal the mechanisms driving ovarian aging in goats at single-cell resolution.ResultsFor the first time, we surveyed the single-cell transcriptomic landscape of over 27,000 ovarian cells from newborn, young and aging goats, and identified nine ovarian cell types with distinct gene-expression signatures. Functional enrichment analysis showed that ovarian cell types were involved in their own unique biological processes, such as Wnt beta-catenin signalling was enriched in germ cells, whereas ovarian steroidogenesis was enriched in granulosa cells (GCs). Further analysis showed that ovarian aging was linked to GCs-specific changes in the antioxidant system, oxidative phosphorylation, and apoptosis. Subsequently, we identified a series of dynamic genes, such as AMH, CRABP2, THBS1 and TIMP1, which determined the fate of GCs. Additionally, FOXO1, SOX4, and HIF1A were identified as significant regulons that instructed the differentiation of GCs in a distinct manner during ovarian aging.ConclusionsThis study revealed a comprehensive aging-associated transcriptomic atlas characterizing the cell type-specific mechanisms during ovarian aging at the single-cell level and offers new diagnostic biomarkers and potential therapeutic targets for age-related goat ovarian diseases.

Mapping individual cardiac cell types and their contribution in the development of heart failure in a multiple comorbidity porcine model

Abstract Background Heart failure with preserved ejection fraction (HFpEF) is characterized by impaired left ventricular (LV) relaxation and filling due to increased myocardial stiffness and hypertrophy. The development of HFpEF is associated with multiple, interrelated risk factors including obesity, chronic kidney disease (CKD), diabetes mellitus (DM), hypertension. Mechanistic studies have implied that comorbidity-driven chronic inflammation, oxidative stress, and endothelial dysfunction act as contributors to the onset and development of HFpEF. Multiple studies that support this HFpEF paradigm highlight the complexity of the disease with different pathways active in and across different cell types. A recently developed multiple comorbidity swine (MCS) model with DM, CKD hypercholesterolemia, demonstrate inflammation, microvascular endothelial dysfunction and LV diastolic dysfunction. This model mimics the slow progression of disease in patients and is therefore ideal to study the earliest mechanisms underlying HFpEF. Purpose Here we hypothesize that single nuclei RNA (snRNA) sequencing aid in mapping the contribution of each individual cardiac cell cluster to disease development, to improve our mechanistic understanding of HFpEF. Methods We performed snRNA sequencing to analyze the transcriptional landscape of the cardiac environment in the well validated MCS model. Frozen left ventricle myocardial samples of MCS (n=4) and healthy control (n=3) swine were used. Results The analysis included ∼100.000 individual nuclei and offers new insights in cell type specific mechanisms during HFpEF onset and development against the background of common comorbidities. We identified 34 different cell clusters, which together represent 6 major cell types. Besides cardiomyocytes (CMs), endothelial cells (ECs) and fibroblasts, a large population of immune cells and smaller populations of mural cells and neurons were present in both MCS and control swine. IPA pathway analysis predicts transcriptomic changes within the cell clusters in response to comorbidities including activation of semaphoring and estrogen signaling in the largest CM cluster (CM2) and activation of EIF2 signaling in ECs (Fig. 1). Gene set enrichment analysis confirmed the activation of HFpEF paradigm pathways in the MCS dataset. Overlap of human HFpEF heart bulk RNA sequencing data and the MCS dataset showed overlapping disease processes including interferon gamma (IFNG) signaling. Moreover, IFNG signaling and IFNG associated transcription factor STAT1 were activated in most of the CM and EC clusters. Conclusion These findings suggest an essential role of multiple pathways during the onset and development of HFpEF. Further improvement of our understanding of underlying molecular mechanisms and crosstalk between cell populations, could pinpoint drug targets and aid the development of new treatments for HFpEF.Top 5 IPA predicted canonical pathways

Integrative Transcriptome and Proteome Profiling of Insulin-Resistant Kidney Cell Models and Patient Biopsies Reveals Common and Cell Type-Specific Mechanisms Underpinning Diabetic Kidney Disease

Integrative Transcriptome and Proteome Profiling of Insulin-Resistant Kidney Cell Models and Patient Biopsies Reveals Common and Cell Type-Specific Mechanisms Underpinning Diabetic Kidney Disease

Cell type-specific molecular mechanisms and implications of necroptosis in inflammatory respiratory diseases.

Necroptosis is generally considered as an inflammatory cell death form. The core regulators of necroptotic signaling are receptor-interacting serine-threonine protein kinases 1 (RIPK1) and RIPK3, and the executioner, mixed lineage kinase domain-like pseudokinase (MLKL). Evidence demonstrates that necroptosis contributes profoundly to inflammatory respiratory diseases that are common public health problem. Necroptosis occurs in nearly all pulmonary cell types in the settings of inflammatory respiratory diseases. The influence of necroptosis on cells varies depending upon the type of cells, tissues, organs, etc., which is an important factor to consider. Thus, in this review, we briefly summarize the current state of knowledge regarding the biology of necroptosis, and focus on the key molecular mechanisms that define the necroptosis status of specific cell types in inflammatory respiratory diseases. We also discuss the clinical potential of small molecular inhibitors of necroptosis in treating inflammatory respiratory diseases, and describe the pathological processes that engage cross talk between necroptosis and other cell death pathways in the context of respiratory inflammation. The rapid advancement of single-cell technologies will help understand the key mechanisms underlying cell type-specific necroptosis that are critical to effectively treat pathogenic lung infections and inflammatory respiratory diseases.

Impaired autophagy in the lower airways and lung parenchyma in stable COPD.

There is increasing evidence of autophagy activation in COPD, but its role is complex and probably regulated through cell type-specific mechanisms. This study aims to investigate the autophagic process at multiple levels within the respiratory system, using different methods to clarify conflicting results reported so far. This cross-sectional study was performed on bronchial biopsies and peripheral lung samples obtained from COPD patients (30 and 12 per sample type, respectively) and healthy controls (25 and 22 per sample type, respectively), divided by smoking history. Subjects were matched for age and smoking history. We analysed some of the most important proteins involved in autophagosome formation, such as LC3 and p62, as well as some molecules essential for lysosome function, such as lysosome-associated membrane protein 1 (LAMP1). Immunohistochemistry was used to assess the autophagic process in both sample types. ELISA and transcriptomic analysis were performed on lung samples. We found increased autophagic stimulus in smoking subjects, regardless of respiratory function. This was revealed by immunohistochemistry through a significant increase in LC3 (p<0.01) and LAMP1 (p<0.01) in small airway bronchiolar epithelium, alveolar septa and alveolar macrophages. Similar results were obtained in bronchial biopsy epithelium by evaluating LC3B (p<0.05), also increased in homogenate lung tissue using ELISA (p<0.05). Patients with COPD, unlike the others, showed an increase in p62 by ELISA (p<0.05). No differences were found in transcriptomics analysis. Different techniques, applied at post-transcriptional level, confirm that cigarette smoke stimulates autophagy at multiple levels inside the respiratory system, and that autophagy failure may characterise COPD.

A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.

Communication and contact between neurons and astrocytes is important for proper brain physiology. How neuron/astrocyte crosstalk is affected by intraneuronal tau aggregation in neurodegenerative tauopathies is largely elusive. Human induced pluripotent stem cell (iPSC)-derived neurons provide the opportunity to model tau pathology in a translationally relevant in vitro context. However, current iPSC models inefficiently develop tau aggregates, and co-culture models of tau pathology have thus far utilized rodent astrocytes. In this article, we describe the co-culture of human iPSC-derived neurons with primary human astrocytes in a 96-well format compatible with high-content microscopy. By lentiviral overexpression of different mutated tau variants, this protocol can be flexibly adapted for the efficient induction of seeded or spontaneous tau aggregation. We used this novel co-culture model to identify cell type-specific disease mechanisms and to provide proof of concept for intervention by antisense therapy. These results show that this human co-culture model provides a highly translational tool for target discovery and drug development for human tauopathies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Human neuron/astrocyte co-culture for seeded and spontaneous intraneuronal tau aggregation Support Protocol 1: Human induced pluripotent stem cell culture Support Protocol 2: Human primary astrocyte culture.

Uncovering specific mechanisms across cell types in dynamical models.

Ordinary differential equations are frequently employed for mathematical modeling of biological systems. The identification of mechanisms that are specific to certain cell types is crucial for building useful models and to gain insights into the underlying biological processes. Regularization techniques have been proposed and applied to identify mechanisms specific to two cell types, e.g., healthy and cancer cells, including the LASSO (least absolute shrinkage and selection operator). However, when analyzing more than two cell types, these approaches are not consistent, and require the selection of a reference cell type, which can affect the results. To make the regularization approach applicable to identifying cell-type specific mechanisms in any number of cell types, we propose to incorporate the clustered LASSO into the framework of ordinary differential equation modeling by penalizing the pairwise differences of the logarithmized fold-change parameters encoding a specific mechanism in different cell types. The symmetry introduced by this approach renders the results independent of the reference cell type. We discuss the necessary adaptations of state-of-the-art numerical optimization techniques and the process of model selection for this method. We assess the performance with realistic biological models and synthetic data, and demonstrate that it outperforms existing approaches. Finally, we also exemplify its application to published biological models including experimental data, and link the results to independent biological measurements.