Single-nucleus analysis reveals microenvironment-specific neuron and glial cell enrichment in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a complicated neurodegenerative disease. Neuron-glial cell interactions are an important but not fully understood process in the progression of AD. We used bioinformatic methods to analyze single-nucleus RNA sequencing (snRNA-seq) data to investigate the cellular and molecular biological processes of AD. METHOD: snRNA-seq data were downloaded from Gene Expression Omnibus (GEO) datasets and reprocessed to identify 240,804 single nuclei from healthy controls and patients with AD. The cellular composition of AD was further explored using Uniform Manifold Approximation and Projection (UMAP). Enrichment analysis for the functions of the DEGs was conducted and cell development trajectory analyses were used to reveal underlying cell fate decisions. iTALK was performed to identify ligand-receptor pairs among various cell types in the pathological ecological microenvironment of AD. RESULTS: Six cell types and multiple subclusters were identified based on the snRNA-seq data. A subcluster of neuron and glial cells co-expressing lncRNA-SNHG14, myocardin-related transcription factor A (MRTFA), and MRTFB was found to be more abundant in the AD group. This subcluster was enriched in mitogen-activated protein kinase (MAPK)-, immune-, and apoptosis-related pathways. Through molecular docking, we found that lncRNA-SNHG14 may bind MRTFA and MRTFB, resulting in an interaction between neurons and glial cells. CONCLUSIONS: The findings of this study describe a regulatory relationship between lncRNA-SNHG14, MRTFA, and MRTFB in the six main cell types of AD. This relationship may contribute to microenvironment remodeling in AD and provide a theoretical basis for a more in-depth analysis of AD.

Stochastic nuclear organization and host-dependent allele contribution in Rhizophagus irregularis.

BACKGROUND: Arbuscular mycorrhizal (AM) fungi are arguably the most important symbionts of plants, offering a range of benefits to their hosts. However, the provisioning of these benefits does not appear to be uniform among AM fungal individuals, with genetic variation between fungal symbionts having a substantial impact on plant performance. Interestingly, genetic variation has also been reported within fungal individuals, which contain millions of haploid nuclei sharing a common cytoplasm. In the model AM fungus, Rhizophagus irregularis, several isolates have been reported to be dikaryotes, containing two genetically distinct types of nuclei recognized based on their mating-type (MAT) locus identity. However, their extremely coenocytic nature and lack of a known single nucleus stage has raised questions on the origin, distribution and dynamics of this genetic variation. RESULTS: Here we performed DNA and RNA sequencing at the mycelial individual, single spore and single nucleus levels to gain insight into the dynamic genetic make-up of the dikaryote-like R. irregularis C3 isolate and the effect of different host plants on its genetic variation. Our analyses reveal that parallel spore and root culture batches can have widely variable ratios of two main genotypes in C3. Additionally, numerous polymorphisms were found with frequencies that deviated significantly from the general genotype ratio, indicating a diverse population of slightly different nucleotypes. Changing host plants did not show consistent host effects on nucleotype ratio's after multiple rounds of subculturing. Instead, we found a major effect of host plant-identity on allele-specific expression in C3. CONCLUSION: Our analyses indicate a highly dynamic/variable genetic organization in different isolates of R. irregularis. Seemingly random fluctuations in nucleotype ratio's upon spore formation, recombination events, high variability of non-tandemly repeated rDNA sequences and host-dependent allele expression all add levels of variation that may contribute to the evolutionary success of these widespread symbionts.

Spatial enrichment of the type 1 interferon signature in the brain of a neuropsychiatric lupus murine model.

Among systemic lupus erythematosus (SLE) patients, neuropsychiatric symptoms are highly prevalent, being observed in up to 80% of adult and 95% of pediatric patients. Type 1 interferons, particularly interferon alpha (IFNα), have been implicated in the pathogenesis of SLE and its associated neuropsychiatric symptoms (NPSLE). However, it remains unclear how type 1 interferon signaling in the central nervous system (CNS) might result in neuropsychiatric sequelae. In this study, we validate an NPSLE mouse model and find an elevated peripheral type 1 interferon signature alongside clinically relevant NPSLE symptoms such as anxiety and fatigue. Unbiased single-nucleus sequencing of the hindbrain and hippocampus revealed that interferon-stimulated genes (ISGs) were among the most highly upregulated genes in both regions and that gene pathways involved in cellular interaction and neuronal development were generally repressed among astrocytes, oligodendrocytes, and neurons. Using image-based spatial transcriptomics, we found that the type 1 interferon signature is enriched as spatially distinct patches within the brain parenchyma of these mice. Our results suggest that type 1 interferon in the CNS may play an important mechanistic role in mediating NPSLE behavioral phenotypes by repressing general cellular communication pathways, and that type 1 interferon signaling modulators are a potential therapeutic option for NPSLE.

Inherent constraints on a polyfunctional tissue lead to a reproduction-immunity tradeoff.

BACKGROUND: Single tissues can have multiple functions, which can result in constraints, impaired function, and tradeoffs. The insect fat body performs remarkably diverse functions including metabolic control, reproductive provisioning, and systemic immune responses. How polyfunctional tissues simultaneously execute multiple distinct physiological functions is generally unknown. Immunity and reproduction are observed to trade off in many organisms but the mechanistic basis for this tradeoff is also typically not known. Here we investigate constraints and trade-offs in the polyfunctional insect fat body. RESULTS: Using single-nucleus sequencing, we determined that the Drosophila melanogaster fat body executes diverse basal functions with heterogenous cellular subpopulations. The size and identity of these subpopulations are remarkably stable between virgin and mated flies, as well as before and after infection. However, as an emergency function, the immune response engages the entire tissue and all cellular subpopulations produce induce expression of defense genes. We found that reproductively active females who were given bacterial infection exhibited signatures of ER stress and impaired capacity to synthesize new protein in response to infection, including decreased capacity to produce antimicrobial peptides. Transient provision of a reversible translation inhibitor to mated females prior to infection rescued general protein synthesis, specific production of antimicrobial peptides, and survival of infection. CONCLUSIONS: The commonly observed tradeoff between reproduction and immunity appears to be driven, in D. melanogaster, by a failure of the fat body to be able to handle simultaneous protein translation demands of reproductive provisioning and immune defense. We suggest that inherent cellular limitations in tissues that perform multiple functions may provide a general explanation for the wide prevalence of physiological and evolutionary tradeoffs.

Insights from Studies of White Adipose Tissue Using Single-Cell Approaches.

Technologies allowing studies at single-cell resolution have provided important insights into how different cell populations contribute to tissue function. Application of these methods to white adipose tissue (WAT) has revealed how various metabolic aspects of this organ, such as insulin response, inflammation and tissue expansion, are regulated by specific WAT resident cells, including different subtypes of adipocytes, adipocyte progenitors as well as immune and endothelial cells. In this chapter, we provide an overview of the different technical approaches, their strengths and weaknesses, and summarize how these studies have improved our understanding of WAT function in health and disease.

Whole genome analyses based on single, field collected spores of the arbuscular mycorrhizal fungus Funneliformis geosporum.

Arbuscular mycorrhizal (AM) fungi are ubiquitous mutualistic symbionts of most terrestrial plants and many complete their lifecycles underground. Whole genome analysis of AM fungi has long been restricted to species and strains that can be maintained under controlled conditions that facilitate collection of biological samples. There is some evidence suggesting that AM fungi can adapt to culture resulting in phenotypic and possibly also genotypic changes in the fungi. In this study, we used field isolated spores of AM fungi and identified them as Funneliformis geosporum based on morphology and phylogenetic analyses. We separately assembled the genomes of two representative spores using DNA sequences of 19 and 22 individually amplified nuclei. The genomes were compared with previously published data from other members of Glomeraceae including two strains of F. mosseae. No significant differences were observed among the species in terms of gene content, while the single nucleotide polymorphism density was higher in the strains of F. geosporum than in the strains of F. mosseae. In this study, we demonstrate that it is possible to sequence and assemble genomes from AM fungal spores sampled in the field, which opens up the possibility to include uncultured AM fungi in phylogenomic and comparative genomic analysis and to study genomic variation in natural populations of these important plant symbionts.

Sfrp2 promotes renal dysfunction of diabetic kidney disease via modulating Fzd5-induced cytosolic calcium ion concentration and CaMKII/Mek/Erk pathway in mesangial cells.

OBJECTIVE: Mesangial cells (MCs) in the kidney play central role in maintaining glomerular integrity, and their abnormal proliferation leads to major glomerular diseases including diabetic kidney disease (DKD). Although high blood glucose elicits MCs impairment, the underlying molecular mechanism is poorly understood. The present study aimed to investigate the effect of secreted frizzled-related protein 2 (Sfrp2) from single-nucleus RNA profiling on MC proliferation of DKD in vitro and in vivo and explored the specific mechanisms. RESULTS: By snRNA-seq analysis of isolated renal cells from leptin receptor-deficient db/db mice and control db/m mice, we found that Sfrp2 was increased in the MCs of DKD in comparison to other intrinsic renal cells, which was further verified in vitro and in vivo. We also found that the expression of Sfrp2 was significantly upregulated in DKD patients and correlated with renal function, demonstrating that Sfrp2 might serve as an independent biomarker for DKD patients. Functionally, we showed the loss and acquisition of Sfrp2 affected cytosolic Ca2+ concentration, cell proliferation and fibrosis of MC, albuminuria and kidney injury in vitro and in vivo. Mechanistically, we identify c-Jun as a transcription factor of Sfrp2 promoting its transcription, and the Ca2+ signaling related protein frizzled receptor 5 (Fzd5) as the binding protein of Sfrp2. And we further found Sfrp2 promoted Fzd5-induced cytosolic Ca2+ concentration and the downstream CaMKII/Mek/Erk pathway activation, leading to MC proliferation and fibrosis in DKD. CONCLUSION: Our study revealed a novel involvement for Sfrp2 in the regulation of MC function and the effect of Sfrp2 on cell proliferation and fibrosis of MC via the Fzd5/Ca2+/CaMKII/Mek/Erk pathway, implying that Sfrp2 may be a possible biomarker and therapeutic target for DKD.

Single-nucleus sequencing unveils heterogeneity in renal cell carcinomas microenvironment: Insights into pathogenic origins and treatment-responsive cellular subgroups.

BACKGROUND: Different individuals with renal cell carcinoma (RCC) exhibit substantial heterogeneity in histomorphology, genetic alterations in the proteome, immune cell infiltration patterns, and clinical behavior. OBJECTIVES: This study aims to use single-nucleus sequencing on ten samples (four normal, three clear cell renal cell carcinoma (ccRCC), and three chromophobe renal cell carcinoma (chRCC)) to uncover pathogenic origins and prognostic characteristics in patients with RCC. METHODS: By using two algorithms, inferCNV and k-means, the study explores malignant cells and compares them with the normal group to reveal their origins. Furthermore, we explore the pathogenic factors at the gene level through Summary-data-based Mendelian Randomization and co-localization methods. Based on the relevant malignant markers, a total of 212 machine-learning combinations were compared to develop a prognostic signature with high precision and stability. Finally, the study correlates with clinical data to investigate which cell subtypes may impact patients' prognosis. RESULTS & CONCLUSION: Two main origin tumor cells were identified: Proximal tubule cell B and Intercalated cell type A, which were highly differentiated in epithelial cells, and three gene loci were determined as potential pathogenic genes. The best malignant signature among the 212 prognostic models demonstrated high predictive power in ccRCC: (AUC: 0.920 (1-year), 0.920 (3-year) and 0.930 (5-year) in the training dataset; 0.756 (1-year), 0.828 (3-year), and 0.832 (5-year) in the testing dataset. In addition, we confirmed that LYVE1+ tissue-resident macrophage and TOX+ CD8 significantly impact the prognosis of ccRCC patients, while monocytes play a crucial role in the prognosis of chRCC patients.

Single-cell sequencing of the substantia nigra reveals microglial activation in a model of MPTP.

Background: N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin widely used to induce PD models, but the effect of MPTP on the cells and genes of PD has not been fully elucidated. Methods: Single-nucleus RNA sequencing was performed in the Substantia Nigra (SN) of MPTP mice. UMAP analysis was used for the dimensionality reduction visualization of the SN in the MPTP mice. Known marker genes highly expressed genes in each cluster were used to annotate most clusters. Specific Differentially Expressed Genes (DEGs) and PD risk genes analysis were used to find MPTP-associated cells. GO, KEGG, PPI network, GSEA and CellChat analysis were used to reveal cell type-specific functional alterations and disruption of cell-cell communication networks. Subset reconstruction and pseudotime analysis were used to reveal the activation status of the cells, and to find the transcription factors with trajectory characterized. Results: Initially, we observed specific DEGs and PD risk genes enrichment in microglia. Next, We obtained the functional phenotype changes in microglia and found that IGF, AGRN and PTN pathways were reduced in MPTP mice. Finally, we analyzed the activation state of microglia and revealed a pro-inflammatory trajectory characterized by transcription factors Nfe2l2 and Runx1. Conclusion: Our work revealed alterations in microglia function, signaling pathways and key genes in the SN of MPTP mice.

Combined Analysis of mRNA Expression and Open Chromatin in Microglia.

The advance of single-cell RNA-sequencing technologies in the past years has enabled unprecedented insights into the complexity and heterogeneity of microglial cell states in the homeostatic and diseased brain. This includes rather complex proteomic, metabolomic, morphological, transcriptomic, and epigenetic adaptations to external stimuli and challenges resulting in a novel concept of core microglia properties and functions. To uncover the regulatory programs facilitating the rapid transcriptomic adaptation in response to changes in the local microenvironment, the accessibility of gene bodies and gene regulatory elements can be assessed. Here, we describe the application of a previously published method for simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq) on microglia nuclei isolated from frozen mouse brain tissue.

Integrated analysis reveals the dysfunction of intercellular communication and metabolic signals in dilated cardiomyopathy.

Aims: Dilated cardiomyopathy refers to a heart muscle condition characterized by structural and functional irregularities in the myocardium that are not related to ischemia. Due to diverse etiologies such as genetic mutations, infections, and exposure to toxins, dilated cardiomyopathy can lead to substantial morbidity and mortality despite advances in the management of heart failure in dilated cardiomyopathy patients. We sought to analyze the characteristics of cell-cell communication and the metabolic signaling pathways in dilated cardiomyopathy. Methods and results: The single-nucleus sequencing data of left ventricle samples were acquired from two donor datasets and two dilated cardiomyopathy datasets. Three dilated cardiomyopathy bulk-sequencing datasets were included to determine the shared dilated cardiomyopathy-specific alterations in differentially expressed genes and signaling pathways. Using "CellChat," we analyzed intercellular communication to grasp how cell clusters interact and to map out the impaired signaling pathways in both donor and dilated cardiomyopathy conditions. Gene set enrichment analysis was applied to compare the metabolic signaling before and after dilated cardiomyopathy. We showcased how cell clusters exhibited abnormal cell-to-cell signaling transduction and how each cell type displayed dysfunctional metabolic signaling pathways through the integration of various datasets. The crucial ligand-receptor signaling contributing to outgoing or incoming signaling of dilated cardiomyopathy was identified in a cell-type dependent way, and the cell-specific metabolic alterations in glucose, lipid and amino acid were determined. The expression of gene pairs in BMP and NOTCH signal, as well as the gene expression in the arginine metabolism was validated. Conclusions: We reveal the key signals and metabolic pathways for dilated cardiomyopathy adaptation and maintenance, providing potential targets for dilated cardiomyopathy interference.

Breaking barriers: improving time and space resolution of arbuscular mycorrhizal symbiosis with single-cell sequencing approaches.

The cell and molecular bases of arbuscular mycorrhizal (AM) symbiosis, a crucial plant-fungal interaction for nutrient acquisition, have been extensively investigated by coupling traditional RNA sequencing techniques of roots sampled in bulk, with methods to capture subsets of cells such as laser microdissection. These approaches have revealed central regulators of this complex relationship, yet the requisite level of detail to effectively untangle the intricacies of temporal and spatial development remains elusive.The recent adoption of single-cell RNA sequencing (scRNA-seq) techniques in plant research is revolutionizing our ability to dissect the intricate transcriptional profiles of plant-microbe interactions, offering unparalleled insights into the diversity and dynamics of individual cells during symbiosis. The isolation of plant cells is particularly challenging due to the presence of cell walls, leading plant researchers to widely adopt nuclei isolation methods. Despite the increased resolution that single-cell analyses offer, it also comes at the cost of spatial perspective, hence, it is necessary the integration of these approaches with spatial transcriptomics to obtain a comprehensive overview.To date, few single-cell studies on plant-microbe interactions have been published, most of which provide high-resolution cell atlases that will become crucial for fully deciphering symbiotic interactions and addressing future questions. In AM symbiosis research, key processes such as the mutual recognition of partners during arbuscule development within cortical cells, or arbuscule senescence and degeneration, remain poorly understood, and these advancements are expected to shed light on these processes and contribute to a deeper understanding of this plant-fungal interaction.

Single-nucleus RNA sequencing demonstrates an autosomal dominant Alzheimer's disease profile and possible mechanisms of disease protection.

Highly penetrant autosomal dominant Alzheimer's disease (ADAD) comprises a distinct disease entity as compared to the far more prevalent form of AD in which common variants collectively contribute to risk. The downstream pathways that distinguish these AD forms in specific cell types have not been deeply explored. We compared single-nucleus transcriptomes among a set of 27 cases divided among PSEN1-E280A ADAD carriers, sporadic AD, and controls. Autophagy genes and chaperones clearly defined the PSEN1-E280A cases compared to sporadic AD. Spatial transcriptomics validated the activation of chaperone-mediated autophagy genes in PSEN1-E280A. The PSEN1-E280A case in which much of the brain was spared neurofibrillary pathology and harbored a homozygous APOE3-Christchurch variant revealed possible explanations for protection from AD pathology including overexpression of LRP1 in astrocytes, increased expression of FKBP1B, and decreased PSEN1 expression in neurons. The unique cellular responses in ADAD and sporadic AD require consideration when designing clinical trials.

Qi-Po-Sheng-Mai granule ameliorates Ach-CaCl2 -induced atrial fibrillation by regulating calcium homeostasis in cardiomyocytes.

BACKGROUND: Atrial fibrillation (AF) is one of the most common arrhythmias encountered in clinical settings. Currently, the pathophysiology of AF remains unclear, which severely limits the effectiveness and safety of medical therapies. The Chinese herbal formula Qi-Po-Sheng-Mai Granule (QPSM) has been widely used in China to treat AF. However, its pharmacological and molecular mechanisms remain unknown. PURPOSE: The purpose of this study was to investigate the molecular mechanisms and potential targets of QPSM for AF. STUDY DESIGN AND METHODS: The AF model was induced by Ach (66 µg/ml) and CaCl2 (10 mg/kg), and the dose of 0.1 ml/100 g was injected into the tail vein for 5 weeks. QPSM was administered daily at doses of 4.42 and 8.84 g/kg, and amiodarone (0.18 g/kg) was used as the positive control. The effect of QPSM on AF was assessed by electrocardiogram, echocardiography, and histopathological analysis. Then, we employed network pharmacology with single nucleus RNA sequencing (snRNA-Seq) to investigate the molecular mechanisms and potential targets of QPSM for AF. Furthermore, high performance liquid chromatography (HPLC) method was used for component analysis of QPSM, and molecular docking was used to verify the potential targets. Using the IonOptix single cell contraction and ion synchronization test equipment, single myocyte length and calcium ion variations were observed in real time. The expression levels of calcium Transporter-related proteins were detected by western blot and immunohistochemistry. RESULTS: Based on an Ach-CaCl2-induced AF model, we found that QPSM treatment significantly reduced atrial electrical remodeling-related markers, such as AF inducibility and duration, and attenuated atrial dilation and fibrosis. Network pharmacology identified 52 active ingredients and 119 potential targets for QPSM in the treatment of AF, and 45 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched, among which calcium pathway had the greatest impact. Using single nucleus sequencing (snRNA-seq), we identified cardiomyocytes as the most differentially expressed in response to drug treatment, with nine differentially expressed genes enriched in calcium signaling pathways. High performance liquid chromatography and molecular docking confirmed that the core components of QPSM strongly bind to the key factors in the calcium signaling pathway. Additional experiments have shown that QPSM increases calcium transients (CaT) and contractility in the individual cardiomyocyte. This was accomplished by increasing the expression of CACNA1C and SERCA2a and decreasing the expression of CAMK2B and NCX1. CONCLUSION: The present study has systematically elucidated the role of QPSM in maintaining calcium homeostasis in cardiomyocytes through the regulation of calcium transporters, which could lead to new drug development ideas for AF.

TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation.

The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma.

Nuclei on the Rise: When Nuclei-Based Methods Meet Next-Generation Sequencing.

In the last decade, we have witnessed an upsurge in nuclei-based studies, particularly coupled with next-generation sequencing. Such studies aim at understanding the molecular states that exist in heterogeneous cell populations by applying increasingly more affordable sequencing approaches, in addition to optimized methodologies developed to isolate and select nuclei. Although these powerful new methods promise unprecedented insights, it is important to understand and critically consider the associated challenges. Here, we provide a comprehensive overview of the rise of nuclei-based studies and elaborate on their advantages and disadvantages, with a specific focus on their utility for transcriptomic sequencing analyses. Improved designs and appropriate use of the various experimental strategies will result in acquiring biologically accurate and meaningful information.

The Single-Cell Revelation of Thermogenic Adipose Tissue.

The past two decades have witnessed an upsurge in the appreciation of adipose tissue (AT) as an immuno-metabolic hub harbouring heterogeneous cell populations that collectively fine-tune systemic metabolic homeostasis. Technological advancements, especially single-cell transcriptomics, have offered an unprecedented opportunity for dissecting the sophisticated cellular networks and compositional dynamics underpinning AT remodelling. The "re-discovery" of functional brown adipose tissue dissipating heat energy in human adults has aroused tremendous interest in exploiting the mechanisms underpinning the engagement of AT thermogenesis for combating human obesity. In this review, we aim to summarise and evaluate the use of single-cell transcriptomics that contribute to a better appreciation of the cellular plasticity and intercellular crosstalk in thermogenic AT.

A single-nucleus and spatial transcriptomic atlas of the COVID-19 liver reveals topological, functional, and regenerative organ disruption in patients.

The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

Astrocyte Heterogeneity in Multiple Sclerosis: Current Understanding and Technical Challenges.

The emergence of single cell technologies provides the opportunity to characterize complex immune/central nervous system cell assemblies in multiple sclerosis (MS) and to study their cell population structures, network activation and dynamics at unprecedented depths. In this review, we summarize the current knowledge of astrocyte subpopulations in MS tissue and discuss the challenges associated with resolving astrocyte heterogeneity with single-nucleus RNA-sequencing (snRNA-seq). We further discuss multiplexed imaging techniques as tools for defining population clusters within a spatial context. Finally, we will provide an outlook on how these technologies may aid in answering unresolved questions in MS, such as the glial phenotypes that drive MS progression and/or neuropathological differences between different clinical MS subtypes.

More Filtering on SNP Calling Does Not Remove Evidence of Inter-Nucleus Recombination in Dikaryotic Arbuscular Mycorrhizal Fungi.

Evidence for the existence of dikaryote-like strains, low nuclear sequence diversity and inter-nuclear recombination in arbuscular mycorrhizal fungi has been recently reported based on single nucleus sequencing data. Here, we aimed to support evidence of inter-nuclear recombination using an approach that filters SNP calls more conservatively, keeping only positions that are exclusively single copy and homozygous, and with at least five reads supporting a given SNP. This methodology recovers hundreds of putative inter-nucleus recombination events across publicly available sequence data from individual nuclei. Challenges related to the acquisition and analysis of sequence data from individual nuclei are highlighted and discussed, and ways to address these issues in future studies are presented.