Single-nucleus transcriptome unveils the role of ferroptosis in ischemic stroke.

Multiple cell death pathways are involved in neuronal death in ischemic stroke (IS). However, the role of different cell death pathways in different cell types has not been elucidated. By analyzing three single-nucleus RNA sequencing (snRNA-seq) data of IS, we first found that a variety of programmed cell death (PCD) -related genes were significantly changed in different cell types. Based on machine learning and virtual gene knockout, we found that ferroptosis related genes, ferritin heavy chain 1 (Fth1) and ferritin light chain (Ftl1), play a key role in IS. Ftl1 and Fth1 can promote microglia activation, as well as the production of inflammatory factors and chemokines. Cell communication analysis showed that activated microglia could enhance chemotactic peripheral leukocyte infiltration, such as macrophages and neutrophils, through Spp1-Cd44 and App-Cd74 signaling, thereby aggravating brain tissue damage. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) showed that P2ry12 and Mef2c were significantly decreased in oxygen-glucose deprivation (OGD) group, while Ftl1, Fth1, Apoe, Ctsb, Cd44 and Cd74 were significantly increased in OGD group. Collectively, our findings suggested targeted therapy against microglia Ftl1 and Fth1 might improve the state of microglia, reduce the infiltration of peripheral immune cells and tissue inflammation, and then improve the ischemic brain injury in mouse.

Single Nucleus Total RNA Sequencing of Formalin-Fixed Paraffin-Embedded Gliomas.

Gliomas, the predominant form of brain cancer, comprise diverse malignant subtypes with limited curative therapies available. The insufficient understanding of their molecular diversity and evolutionary processes hinders the advancement of new treatments. Technical complexities associated with formalin-fixed paraffin-embedded (FFPE) clinical samples hinder molecular-level analyses of gliomas. Current single-cell RNA sequencing (scRNA-seq) platforms are inadequate for large-scale clinical applications. In this study, automated snRandom-seq is developed, a high-throughput single-nucleus total RNA sequencing platform optimized for archival FFPE samples. This platform integrates automated single-nucleus isolation and droplet barcoding systems with the random primer-based scRNA-seq chemistry, accommodating a broad spectrum of sample types. The automated snRandom-seq is applied to analyze 116 492 single nuclei from 17 FFPE samples of various glioma subtypes, including rare clinical samples and matched primary-recurrent glioblastomas (GBMs). The study provides comprehensive insights into the molecular characteristics of gliomas at the single-cell level. Abundant non-coding RNAs (ncRNAs) with distinct expression profiles across different glioma clusters and uncovered promising recurrence-related targets and pathways in primary-recurrent GBMs are identified. These findings establish automated snRandom-seq as a robust tool for scRNA-seq of FFPE samples, enabling exploration of molecular diversities and tumor evolution. This platform holds significant implications for large-scale integrative and retrospective clinical research.

Single nucleus RNA-sequencing integrated into risk variant colocalization discovers 17 cell-type-specific abdominal obesity genes for metabolic dysfunction-associated steatotic liver disease.

BACKGROUND: Abdominal obesity increases the risk for non-alcoholic fatty liver disease (NAFLD), now known as metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: To elucidate the directional cell-type level biological mechanisms underlying the association between abdominal obesity and MASLD, we integrated adipose and liver single nucleus RNA-sequencing and bulk cis-expression quantitative trait locus (eQTL) data with the UK Biobank genome-wide association study (GWAS) data using colocalization. Then we used colocalized cis-eQTL variants as instrumental variables in Mendelian randomization (MR) analyses, followed by functional validation experiments on the target genes of the cis-eQTL variants. FINDINGS: We identified 17 colocalized abdominal obesity GWAS variants, regulating 17 adipose cell-type marker genes. Incorporating these 17 variants into MR discovers a putative tissue-of-origin, cell-type-aware causal effect of abdominal obesity on MASLD consistently with multiple MR methods without significant evidence for pleiotropy or heterogeneity. Single cell data confirm the adipocyte-enriched mean expression of the 17 genes. Our cellular experiments across human adipogenesis identify risk variant -specific epigenetic and transcriptional mechanisms. Knocking down two of the 17 genes, PPP2R5A and SH3PXD2B, shows a marked decrease in adipocyte lipidation and significantly alters adipocyte function and adipogenesis regulator genes, including DGAT2, LPL, ADIPOQ, PPARG, and SREBF1. Furthermore, the 17 genes capture a characteristic MASLD expression signature in subcutaneous adipose tissue. INTERPRETATION: Overall, we discover a significant cell-type level effect of abdominal obesity on MASLD and trace its biological effect to adipogenesis. FUNDING: NIH grants R01HG010505, R01DK132775, and R01HL170604; the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant No. 802825), Academy of Finland (Grants Nos. 333021), the Finnish Foundation for Cardiovascular Research the Sigrid Jusélius Foundation and the Jane and Aatos Erkko Foundation; American Association for the Study of Liver Diseases (AASLD) Advanced Transplant Hepatology award and NIH/NIDDK (P30DK41301) Pilot and Feasibility award; NIH/NIEHS F32 award (F32ES034668); Finnish Diabetes Research Foundation, Kuopio University Hospital Project grant (EVO/VTR grants 2005-2021), the Academy of Finland grant (Contract no. 138006); Academy of Finland (Grant Nos 335443, 314383, 272376 and 266286), Sigrid Jusélius Foundation, Finnish Medical Foundation, Finnish Diabetes Research Foundation, Novo Nordisk Foundation (#NNF20OC0060547, NNF17OC0027232, NNF10OC1013354) and Government Research Funds to Helsinki University Hospital; Orion Research Foundation, Maud Kuistila Foundation, Finish Medical Foundation, and University of Helsinki.

Dentate gyrus granule cells are a locus of pathology in Scn8a developmental encephalopathy.

Gain-of-function mutations in SCN8A cause developmental and epileptic encephalopathy (DEE), a disorder characterized by early-onset refractory seizures, deficits in motor and intellectual functions, and increased risk of sudden unexpected death in epilepsy. Altered activity of neurons in the corticohippocampal circuit has been reported in mouse models of DEE. We examined the effect of chronic seizures on gene expression in the hippocampus by single-nucleus RNA sequencing in mice expressing the patient mutation SCN8A-p.Asn1768Asp (N1768D). One hundred and eighty four differentially expressed genes were identified in dentate gyrus granule cells, many more than in other cell types. Electrophysiological recording from dentate gyrus granule cells demonstrated an elevated firing rate. Targeted reduction of Scn8a expression in the dentate gyrus by viral delivery of an shRNA resulted in doubling of median survival time from 4 months to 8 months, whereas delivery of shRNA to the CA1 and CA3 regions did not result in lengthened survival. These data indicate that granule cells of the dentate gyrus are a specific locus of pathology in SCN8A-DEE.

Xenografted human iPSC-derived neurons with the familial Alzheimer's disease APPV717I mutation reveal dysregulated transcriptome signatures linked to synaptic function and implicate LINGO2 as a disease signaling mediator.

Alzheimer's disease (AD) is the most common cause of dementia, and disease mechanisms are still not fully understood. Here, we explored pathological changes in human induced pluripotent stem cell (iPSC)-derived neurons carrying the familial AD APPV717I mutation after cell injection into the mouse forebrain. APPV717I mutant iPSCs and isogenic controls were differentiated into neurons revealing enhanced Aß42 production, elevated phospho-tau, and impaired neurite outgrowth in APPV717I neurons. Two months after transplantation, APPV717I and control neural cells showed robust engraftment but at 12 months post-injection, APPV717I grafts were smaller and demonstrated impaired neurite outgrowth compared to controls, while plaque and tangle pathology were not seen. Single-nucleus RNA-sequencing of micro-dissected grafts, performed 2 months after cell injection, identified significantly altered transcriptome signatures in APPV717I iPSC-derived neurons pointing towards dysregulated synaptic function and axon guidance. Interestingly, APPV717I neurons showed an increased expression of genes, many of which are also upregulated in postmortem neurons of AD patients including the transmembrane protein LINGO2. Downregulation of LINGO2 in cultured APPV717I neurons rescued neurite outgrowth deficits and reversed key AD-associated transcriptional changes related but not limited to synaptic function, apoptosis and cellular senescence. These results provide important insights into transcriptional dysregulation in xenografted APPV717I neurons linked to synaptic function, and they indicate that LINGO2 may represent a potential therapeutic target in AD.

Cell-type-specific effects of autism-associated chromosome 15q11.2-13.1 duplications in human brain.

Recurrent copy number variation represents one of the most well-established genetic drivers in neurodevelopmental disorders, including autism spectrum disorder (ASD). Duplication of 15q11.2-13.1 (dup15q) is a well-described neurodevelopmental syndrome that increases the risk of ASD by over 40-fold. However, the effects of this duplication on gene expression and chromatin accessibility in specific cell types in the human brain remain unknown. To identify the cell-type-specific transcriptional and epigenetic effects of dup15q in the human frontal cortex we conducted single-nucleus RNA-sequencing and multi-omic sequencing on dup15q cases (n=6) as well as non-dup15q ASD (n=7) and neurotypical controls (n=7). Cell-type-specific differential expression analysis identified significantly regulated genes, critical biological pathways, and differentially accessible genomic regions. Although there was overall increased gene expression across the duplicated genomic region, cellular identity represented an important factor mediating gene expression changes. Neuronal subtypes, showed greater upregulation of gene expression across a critical region within the duplication as compared to other cell types. Genes within the duplicated region that had high baseline expression in control individuals showed only modest changes in dup15q, regardless of cell type. Of note, dup15q and ASD had largely distinct signatures of chromatin accessibility, but shared the majority of transcriptional regulatory motifs, suggesting convergent biological pathways. However, the transcriptional binding factor motifs implicated in each condition implicated distinct biological mechanisms; neuronal JUN/FOS networks in ASD vs. an inflammatory transcriptional network in dup15q microglia. This work provides a cell-type-specific analysis of how dup15q changes gene expression and chromatin accessibility in the human brain and finds evidence of marked cell-type-specific effects of this genetic driver. These findings have implications for guiding therapeutic development in dup15q syndrome, as well as understanding the functional effects CNVs more broadly in neurodevelopmental disorders.

Cell-specific regulation of the circadian clock by BMAL1 in the paraventricular nucleus: Implications for regulation of systemic biological rhythms.

Circadian rhythms are internal biological rhythms driving temporal tissue-specific, metabolic programs. Loss of the circadian transcription factor BMAL1 in the paraventricular nucleus (PVN) of the hypothalamus reveals its importance in metabolic rhythms, but its functions in individual PVN cells are poorly understood. Here, loss of BMAL1 in the PVN results in arrhythmicity of processes controlling energy balance and alters peripheral diurnal gene expression. BMAL1 chromatin immunoprecipitation sequencing (ChIP-seq) and single-nucleus RNA sequencing (snRNA-seq) reveal its temporal regulation of target genes, including oxytocin (OXT), and restoring circulating OXT peaks in BMAL1-PVN knockout (KO) mice rescues absent activity rhythms. While glutamatergic neurons undergo day/night changes in expression of genes involved in cell morphogenesis, astrocytes and oligodendrocytes show gene expression changes in cytoskeletal organization and oxidative phosphorylation. Collectively, our findings show diurnal gene regulation in neuronal and non-neuronal PVN cells and that BMAL1 contributes to diurnal OXT secretion, which is important for systemic diurnal rhythms.

Opto-seq reveals input-specific immediate-early gene induction in ventral tegmental area cell types.

The ventral tegmental area (VTA) is a critical node in circuits governing motivated behavior and is home to diverse populations of neurons that release dopamine, gamma-aminobutyric acid (GABA), glutamate, or combinations of these neurotransmitters. The VTA receives inputs from many brain regions, but a comprehensive understanding of input-specific activation of VTA neuronal subpopulations is lacking. To address this, we combined optogenetic stimulation of select VTA inputs with single-nucleus RNA sequencing (snRNA-seq) and highly multiplexed in situ hybridization to identify distinct neuronal clusters and characterize their spatial distribution and activation patterns. Quantification of immediate-early gene (IEG) expression revealed that different inputs activated select VTA subpopulations, which demonstrated cell-type-specific transcriptional programs. Within dopaminergic subpopulations, IEG induction levels correlated with differential expression of ion channel genes. This new transcriptomics-guided circuit analysis reveals the diversity of VTA activation driven by distinct inputs and provides a resource for future analysis of VTA cell types.

Single-nucleus RNA sequencing and lipidomics reveal characteristics of transcriptional and lipid composition in porcine longissimus dorsi muscle.

BACKGROUND: Global per capita meat consumption continues to rise, especially pork. Meat quality is influenced by the content of intramuscular fat (IMF) as a key factor. The longissimus dorsi muscle of Dahe pigs (DHM, IMF: 7.98% ± 1.96%) and Dahe black pigs (DHBM, IMF: 3.30% ± 0.64%) was studied to explore cellular heterogeneity and differentially expressed genes (DEGs) associated with IMF deposition using single-nucleus RNA sequencing (snRNA-seq). The lipid composition was then analyzed using non-targeted lipidomics. RESULTS: A total of seven cell subpopulations were identified, including myocytes, fibroblast/fibro/adipogenic progenitors (FAPs), satellite cells, endothelial cells, macrophages, pericytes, and adipocytes. Among them, FAPs and adipocytes were more focused because they could be associated with lipid deposition. 1623 DEGs in the FAPs subpopulation of DHBM were up-regulated compared with DHM, while 1535 were down-regulated. These DEGs enriched in the glycolysis/gluconeogenesis pathway. 109 DEGs were up-regulated and 806 were down-regulated in the adipocyte subpopulation of DHBM compared with DHM, which were mainly enriched in the PPAR signaling pathway and fatty acid (FA) biosynthesis. The expression level of PPARG, ABP4, LEP, and ACSL1 genes in DHM was higher than that in DHBM. Lipidomics reveals porcine lipid composition characteristics of muscle tissue. A total of 41 lipid classes and 2699 lipid species were identified in DHM and DHBM groups. The top ten relative peak areas of lipid classes in DHM and DHBM were triglyceride (TG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), diglyceride (DG), cardiolipin (CL), ceramides (Cer), Simple Glc series (Hex1Cer), sphingomyelin (phSM), and phosphatidylinositol (PI). The relative peak areas of 35 lipid species in DHM were lower than DHBM, and 28 lipid species that were higher. There was a significant increase in the TG fatty acyl chains C6:0, C17:0, and C11:4, and a significant decrease in C16:0, C18:1, C18:2, and C22:4 in DHBM (p < 0.05). CONCLUSIONS: C16:0 FA may downregulate the expression level of PPARG gene, which leads to the downregulation of fat metabolism-related genes such as ACSL, PLIN2, and FABP4 in DHBM compared with DHM. This may be the reason that the lipid deposition ability of Dahe pigs is stronger than that of Dahe black pigs, which need further investigation.

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.

Single-nucleus RNA transcriptome profiling reveals murine adipose tissue endothelial cell proliferation gene networks involved in obesity development.

Endothelial cells play an important role in the metabolism of adipose tissue (AT). This study aimed to analyze the changes that adipose tissue in AT endothelial cells undergo during the development of obesity, using single-nucleus RNA sequence (snRNA-seq). Mouse paraepididymal AT cells were subjected to snRNA-seq with the 10X Genomics platform. The cell types were then clustered using t-distributed stochastic neighbor embedding and unbiased computational informatics analyses. Protein-protein interactions network was established using the STRING database and visualized using Cytoscape. The dataset was subjected to differential gene enrichment analysis. In total, 21,333 cells acquired from 24 mouse paraepididymal AT samples were analyzed using snRNA-seq. This study identified 18 distinct clusters and annotated macrophages, fibroblasts, epithelial cells, T cells, endothelial cells, stem cells, neutrophil cells, and neutrophil cell types based on representative markers. Cluster 12 was defined as endothelial cells. The proportion of endothelial cells decreased with the development of obesity. Inflammatory factors, such as Vegfa and Prdm16 were upregulated in the medium obesity group but downregulated in the obesity group. Genes, such as Prox1, Erg, Flt4, Kdr, Flt1, and Pecam1 promoted the proliferation of AT endothelial cells and maintained the internal environment of AT. This study established a reference model and general framework for studying the mechanisms, biomarkers, and therapeutic targets of endothelial cell dysfunction-related diseases at the single-cell level.

Benchmarking mapping algorithms for cell-type annotating in mouse brain by integrating single-nucleus RNA-seq and Stereo-seq data.

Limited gene capture efficiency and spot size of spatial transcriptome (ST) data pose significant challenges in cell-type characterization. The heterogeneity and complexity of cell composition in the mammalian brain make it more challenging to accurately annotate ST data from brain. Many algorithms attempt to characterize subtypes of neuron by integrating ST data with single-nucleus RNA sequencing (snRNA-seq) or single-cell RNA sequencing. However, assessing the accuracy of these algorithms on Stereo-seq ST data remains unresolved. Here, we benchmarked 9 mapping algorithms using 10 ST datasets from four mouse brain regions in two different resolutions and 24 pseudo-ST datasets from snRNA-seq. Both actual ST data and pseudo-ST data were mapped using snRNA-seq datasets from the corresponding brain regions as reference data. After comparing the performance across different areas and resolutions of the mouse brain, we have reached the conclusion that both robust cell-type decomposition and SpatialDWLS demonstrated superior robustness and accuracy in cell-type annotation. Testing with publicly available snRNA-seq data from another sequencing platform in the cortex region further validated our conclusions. Altogether, we developed a workflow for assessing suitability of mapping algorithm that fits for ST datasets, which can improve the efficiency and accuracy of spatial data annotation.

Single-Cell Data Analysis Reveals Critical Hepatic Cells Subpopulations in the Progression of Non-alcoholic Fatty Liver Disease to Non-Alcoholic Steatohepatitis.

AIMS: The aim of this study was to reveal the hepatic cell landscape and function in the progression of NAFLD to NASH. BACKGROUND: Non-alcoholic steatohepatitis (NASH) is the progressive form and turning point of nonalcoholic fatty liver disease (NAFLD), which severely causes irreversible cirrhosis as well as hepatocellular carcinoma. The mechanism underlying the progression of NAFLD to NASH has not been revealed. Unraveling the mechanism of action of NAFLD-NASH is an important goal in improving the survival of patients with liver disease. OBJECTIVE: The aim of this study is to discover heterogeneous hepatic cells during the progression of NAFLD to NASH. METHODS: Single-nucleus RNA-seq (snRNA-seq) data containing NASH in NAFLD samples were obtained from the Gene Expression Omnibus (GEO) database. Cell types in liver tissues from NASH and NAFLD were identified after dimensionality reduction analysis, cluster analysis, and cell annotation. The cell pathways in which differences existed were identified by analyzing metabolic pathways in Hepatic cells. We also identified cell subpopulations in Hepatic cells. The developmental trajectories of Hepatic cells were characterized by pseudotime trajectory analysis. Single-cell regulatory network inference and clustering analysis identified key transcription factors and gene regulatory networks in Hepatic cells. Moreover, cell communication analysis determined the potential interactions between Hepatic cells and immune cells, and heapatic stellate cells. RESULTS: Seven cell types were identified in NAFLD and NASH. The proportion of Hepatic cells was lower in NASH and showed greater energy metabolism and glucose metabolism activity. Hepatic cells exhibited heterogeneity, showing two cell subpopulations, Hepatic cells 1 and Hepatic cells 2. Dysregulation of lipid metabolism in Hepatic Cell 2 resulted in lipid accumulation in the liver, which might be involved in the progression of NAFLD. Four key transcription factors, BHLHE40, NFEL2L, RUNX1, and INF4A, were primarily found in Hepatic cells 2. The transcription factors within the hepatic cells 2 subpopulation mainly regulated genes related to lipid metabolism, energy metabolism, and inflammatory response. The cell communication analysis showed that hepatocyte interactions with immune cells were associated with inflammatory responses, while interactions with hepatic astrocytes were associated with liver injury and hepatocyte fibrosis. CONCLUSION: The hepatic cells 2 might promote the progression of NAFLD to NASH by regulating metabolic activity, which might contribute to liver injury through inflammation.

Asporin and CD109, expressed in the injured neonatal spinal cord, attenuate axonal re-growth in vitro.

Axonal regeneration is restricted in adults and causes irreversible motor dysfunction following spinal cord injury (SCI). In contrast, neonates have prominent regenerative potential and can restore their neural function. Although the distinct cellular responses in neonates have been studied, how they contribute to neural recovery remains unclear. To assess whether the secreted molecules in neonatal SCI can enhance neural regeneration, we re-analyzed the previously performed single-nucleus RNA-seq (snRNA-seq) and focused on Asporin and Cd109, the highly expressed genes in the injured neonatal spinal cord. In the present study, we showed that both these molecules were expressed in the injured spinal cords of adults and neonates. We treated the cortical neurons with recombinant Asporin or CD109 to observe their direct effects on neurons in vitro. We demonstrated that these molecules enhance neurite outgrowth in neurons. However, these molecules did not enhance re-growth of severed axons. Our results suggest that Asporin and CD109 influence neurites at the lesion site, rather than promoting axon regeneration, to restore neural function in neonates after SCI.

Empagliflozin and liraglutide ameliorate HFpEF in mice via augmenting the Erbb4 signaling pathway.

Heart failure with preserved ejection fraction (HFpEF) is closely associated with metabolic derangement. Sodium glucose cotransporter-2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1RA) exert anti-HFpEF effects, but the underlying mechanisms remain unclear. In this study, we explored the anti-HFpEF effects of empagliflozin and liraglutide and the underlying molecular mechanisms in a mouse model of HFpEF. This model was established by high-fat diet (HFD) feeding plus Nω-nitro-L-arginine methyl ester (L-NAME) treatment. The mice were treated with empagliflozin (20 mg·kg-1·d-1, i.g.) or liraglutide (0.3 mg·kg-1·d-1, i.p.) or their combination for 4 weeks. At the end of the experimental protocol, cardiac function was measured using ultrasound, then mice were euthanized and heart, liver, and kidney tissues were collected. Nuclei were isolated from frozen mouse ventricular tissue for single-nucleus RNA-sequencing (snRNA-seq). We showed that administration of empagliflozin or liraglutide alone or in combination significantly improved diastolic function, ameliorated cardiomyocyte hypertrophy and cardiac fibrosis, as well as exercise tolerance but no synergism was observed in the combination group. Furthermore, empagliflozin and/or liraglutide lowered body weight, improved glucose metabolism, lowered blood pressure, and improved liver and kidney function. After the withdrawal of empagliflozin or liraglutide for 1 week, these beneficial effects tended to diminish. The snRNA-seq analysis revealed a subcluster of myocytes, in which Erbb4 expression was down-regulated under HFpEF conditions, and restored by empagliflozin or liraglutide. Pseudo-time trajectory analysis and cell-to-cell communication studies confirmed that the Erbb4 pathway was a prominent pathway essential for both drug actions. In the HFpEF mouse model, both empagliflozin and liraglutide reversed Erbb4 down-regulation. In rat h9c2 cells, we showed that palmitic acid- or high glucose-induced changes in PKCα and/or ERK1/2 phosphorylation at least in part through Erbb4. Collectively, the single-cell atlas reveals the anti-HFpEF mechanism of empagliflozin and liraglutide, suggesting that Erbb4 pathway represents a new therapeutic target for HFpEF. Effects and mechanisms of action of empagliflozin and liraglutide in HFpEF mice. HFpEF was induced with a high-fat diet and L-NAME for 15 weeks, and treatment with empagliflozin and liraglutide improved the HFpEF phenotype. Single nucleus RNA sequencing (snRNA-seq) was used to reveal the underlying mechanism of action of empagliflozin and liraglutide.

Single-Cell Nuclear Sequencing Atlas Revealed the Induction of Parkinson's Disease by RELN+ Neuron 3 and the Gene Regulatory Network of MSRA.

AIMS: To determine the cell types that promoted the progression of Parkinson's disease (PD) using the substantia nigra in the brain tissues derived from patients with PD and normal controls. BACKGROUND: PD is an incurable neurodegenerative disease that threatens the physical activity of the aging population, and the complex molecular mechanisms remain be comprehensively elucidated. OBJECTIVE: To describe potential disease-promoting cell types in PD and to provide a theoretical basis. METHODS: Single-cell nuclear sequencing data of nine PD samples and control samples from Gene Expression Omnibus (GEO) were included, and heterogeneous cell subpopulations in the substantia nigra were identified by annotation analysis. Potential pathogenic cell subpopulations of PD were determined based on the expression data of marker genes. Cell differentiation trajectories and communication networks were generated by Pseudotime trajectory analysis and cell communication analysis. Furthermore, single-- cell regulatory network inference and clustering (SCENIC) analysis was conducted to determine the regulatory network of transcription factor-target genes in PD. RESULTS: Among the nine cell subpopulations classified, RELN+neuron 3 showed reduced abundance and dopamine secretion capacity in PD and was therefore considered as a promoter of PD pathogenesis and progression. The regulatory network of MSRA action was involved in the developmental process of cells in the central nervous system, indicating that MSRA and its targets might serve as potential therapeutic targets for PD. RELN+neuron 3 had two directions of differentiation, specifically, branch 1 exhibited a high apoptotic profile and branch 2 exhibited a high cell death profile. In addition, the intensity of EPHA and EPHB signaling was attenuated between RELN+neuron 3 and other cell subpopulations. CONCLUSION: To conclude, this study identified a subpopulation of RELN+neuron 3 cells with markedly reduced abundance in the brain substantia nigra in PD. The MSRA-involved gene regulatory networks was considered as a novel therapeutic network for PD.

ARDS and aging: TYMS emerges as a promising biomarker and therapeutic target.

Background: Acute Respiratory Distress Syndrome (ARDS) is a common condition in the intensive care unit (ICU) with a high mortality rate, yet the diagnosis rate remains low. Recent studies have increasingly highlighted the role of aging in the occurrence and progression of ARDS. This study is committed to investigating the pathogenic mechanisms of cellular and genetic changes in elderly ARDS patients, providing theoretical support for the precise treatment of ARDS. Methods: Gene expression profiles for control and ARDS samples were obtained from the Gene Expression Omnibus (GEO) database, while aging-related genes (ARGs) were sourced from the Human Aging Genomic Resources (HAGR) database. Differentially expressed genes (DEGs) were subjected to functional enrichment analysis to understand their roles in ARDS and aging. The Weighted Gene Co-expression Network Analysis (WGCNA) and machine learning pinpointed key modules and marker genes, with ROC curves illustrating their significance. The expression of four ARDS-ARDEGs was validated in lung samples from aged mice with ARDS using qRT-PCR. Gene set enrichment analysis (GSEA) investigated the signaling pathways and immune cell infiltration associated with TYMS expression. Single-nucleus RNA sequencing (snRNA-Seq) explored gene-level differences among cells to investigate intercellular communication during ARDS onset and progression. Results: ARDEGs are involved in cellular responses to DNA damage stimuli, inflammatory reactions, and cellular senescence pathways. The MEmagenta module exhibited a significant correlation with elderly ARDS patients. The LASSO, RRF, and XGBoost algorithms were employed to screen for signature genes, including CKAP2, P2RY14, RBP2, and TYMS. Further validation emphasized the potential role of TYMS in the onset and progression of ARDS. Immune cell infiltration indicated differential proportion and correlations with TYMS expression. SnRNA-Seq and cell-cell communication analysis revealed that TYMS is highly expressed in endothelial cells, and the SEMA3 signaling pathway primarily mediates cell communication between endothelial cells and other cells. Conclusion: Endothelial cell damage associated with aging could contribute to ARDS progression by triggering inflammation. TYMS emerges as a promising diagnostic biomarker and potential therapeutic target for ARDS.

Osteopontin drives neuroinflammation and cell loss in MAPT-N279K frontotemporal dementia patient neurons.

Frontotemporal dementia (FTD) is an incurable group of early-onset dementias that can be caused by the deposition of hyperphosphorylated tau in patient brains. However, the mechanisms leading to neurodegeneration remain largely unknown. Here, we combined single-cell analyses of FTD patient brains with a stem cell culture and transplantation model of FTD. We identified disease phenotypes in FTD neurons carrying the MAPT-N279K mutation, which were related to oxidative stress, oxidative phosphorylation, and neuroinflammation with an upregulation of the inflammation-associated protein osteopontin (OPN). Human FTD neurons survived less and elicited an increased microglial response after transplantation into the mouse forebrain, which we further characterized by single nucleus RNA sequencing of microdissected grafts. Notably, downregulation of OPN in engrafted FTD neurons resulted in improved engraftment and reduced microglial infiltration, indicating an immune-modulatory role of OPN in patient neurons, which may represent a potential therapeutic target in FTD.

Functional specialisation and coordination of myonuclei.

Myofibres serve as the functional unit for locomotion, with the sarcomere as fundamental subunit. Running the entire length of this structure are hundreds of myonuclei, located at the periphery of the myofibre, juxtaposed to the plasma membrane. Myonuclear specialisation and clustering at the centre and ends of the fibre are known to be essential for muscle contraction, yet the molecular basis of this regionalisation has remained unclear. While the 'myonuclear domain hypothesis' helped explain how myonuclei can independently govern large cytoplasmic territories, novel technologies have provided granularity on the diverse transcriptional programs running simultaneously within the syncytia and added a new perspective on how myonuclei communicate. Building upon this, we explore the critical cellular and molecular sources of transcriptional and functional heterogeneity within myofibres, discussing the impact of intrinsic and extrinsic factors on myonuclear programs. This knowledge provides new insights for understanding muscle development, repair, and disease, but also opens avenues for the development of novel and precise therapeutic approaches.

Perirenal adipose tissue contains a subpopulation of cold-inducible adipocytes derived from brown-to-white conversion.

Perirenal adipose tissue (PRAT) is a unique visceral depot that contains a mixture of brown and white adipocytes. The origin and plasticity of such cellular heterogeneity remains unknown. Here, we combine single-nucleus RNA sequencing with genetic lineage tracing to reveal the existence of a distinct subpopulation of Ucp1-&Cidea+ adipocytes that arises from brown-to-white conversion during postnatal life in the periureter region of mouse PRAT. Cold exposure restores Ucp1 expression and a thermogenic phenotype in this subpopulation. These cells have a transcriptome that is distinct from subcutaneous beige adipocytes and may represent a unique type of cold-recruitable adipocytes. These results pave the way for studies of PRAT physiology and mechanisms controlling the plasticity of brown/white adipocyte phenotypes.