ALS is imprinted in the chromatin accessibility of blood cells.

Amyotrophic Lateral Sclerosis (ALS) is a complex and incurable neurodegenerative disorder in which genetic and epigenetic factors contribute to the pathogenesis of all forms of ALS. The interplay of genetic predisposition and environmental footprints generates epigenetic signatures in the cells of affected tissues, which then alter transcriptional programs. Epigenetic modifications that arise from genetic predisposition and systemic environmental footprints should in theory be detectable not only in affected CNS tissue but also in the periphery. Here, we identify an ALS-associated epigenetic signature ('epiChromALS') by chromatin accessibility analysis of blood cells of ALS patients. In contrast to the blood transcriptome signature, epiChromALS includes also genes that are not expressed in blood cells; it is enriched in CNS neuronal pathways and it is present in the ALS motor cortex. By combining simultaneous ATAC-seq and RNA-seq with single-cell sequencing in PBMCs and motor cortex from ALS patients, we demonstrate that epigenetic changes associated with the neurodegenerative disease can be found in the periphery, thus strongly suggesting a mechanistic link between the epigenetic regulation and disease pathogenesis.

Single-Nuclei Transcriptome Profiling Reveals Intra-Tumoral Heterogeneity and Characterizes Tumor Microenvironment Architecture in a Murine Melanoma Model.

Malignant melanoma is an aggressive cancer, with a high risk of metastasis and mortality rates, characterized by cancer cell heterogeneity and complex tumor microenvironment (TME). Single cell biology is an ideal and powerful tool to address these features at a molecular level. However, this approach requires enzymatic cell dissociation that can influence cellular coverage. By contrast, single nucleus RNA sequencing (snRNA-seq) has substantial advantages including compatibility with frozen samples and the elimination of a dissociation-induced, transcriptional stress response. To better profile and understand the functional diversity of different cellular components in melanoma progression, we performed snRNA-seq of 16,839 nuclei obtained from tumor samples along the growth of murine syngeneic melanoma model carrying a BRAFV600E mutation and collected 9 days or 23 days after subcutaneous cell injection. We defined 11 different subtypes of functional cell clusters among malignant cells and 5 different subsets of myeloid cells that display distinct global transcriptional program and different enrichment in early or advanced stage of tumor growth, confirming that this approach was useful to accurately identify intratumor heterogeneity and dynamics during tumor evolution. The current study offers a deep insight into the biology of melanoma highlighting TME reprogramming through tumor initiation and progression, underlying further discovery of new TME biomarkers which may be potentially druggable.

Single-cell technologies to decipher cardiovascular diseases.

Cardiovascular disease remains the leading cause of death worldwide. A deeper understanding of the multicellular composition and molecular processes may help to identify novel therapeutic strategies. Single-cell technologies such as single-cell or single-nuclei RNA sequencing provide expression profiles of individual cells and allow for dissection of heterogeneity in tissue during health and disease. This review will summarize (i) how these novel technologies have become critical for delineating mechanistic drivers of cardiovascular disease, particularly, in humans and (ii) how they might serve as diagnostic tools for risk stratification or individualized therapy. The review will further discuss technical pitfalls and provide an overview of publicly available human and mouse data sets that can be used as a resource for research.

Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer's disease mouse model.

BACKGROUND: Deposition of amyloid beta (Aß) and hyperphosphorylated tau along with glial cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimer's disease (AD). In recent years, impairment of autophagy has been identified as another important feature contributing to AD progression. Therefore, the potential of the autophagy activator spermidine, a small body-endogenous polyamine often used as dietary supplement, was assessed on Aß pathology and glial cell-mediated neuroinflammation. RESULTS: Oral treatment of the amyloid prone AD-like APPPS1 mice with spermidine reduced neurotoxic soluble Aß and decreased AD-associated neuroinflammation. Mechanistically, single nuclei sequencing revealed AD-associated microglia to be the main target of spermidine. This microglia population was characterized by increased AXL levels and expression of genes implicated in cell migration and phagocytosis. A subsequent proteome analysis of isolated microglia confirmed the anti-inflammatory and cytoskeletal effects of spermidine in APPPS1 mice. In primary microglia and astrocytes, spermidine-induced autophagy subsequently affected TLR3- and TLR4-mediated inflammatory processes, phagocytosis of Aß and motility. Interestingly, spermidine regulated the neuroinflammatory response of microglia beyond transcriptional control by interfering with the assembly of the inflammasome. CONCLUSIONS: Our data highlight that the autophagy activator spermidine holds the potential to enhance Aß degradation and to counteract glia-mediated neuroinflammation in AD pathology.

snRNA-seq of human cutaneous neurofibromas before and after selumetinib treatment implicates role of altered Schwann cell states, inter-cellular signaling, and extracellular matrix in treatment response.

Neurofibromatosis Type 1 (NF1) is caused by loss of function variants in the NF1 gene. Most patients with NF1 develop skin lesions called cutaneous neurofibromas (cNFs). Currently the only approved therapeutic for NF1 is selumetinib, a mitogen -activated protein kinase (MEK) inhibitor. The purpose of this study was to analyze the transcriptome of cNF tumors before and on selumetinib treatment to understand both tumor composition and response. We obtained biopsy sets of tumors both pre- and on- selumetinib treatment from the same individuals and were able to collect sets from four separate individuals. We sequenced mRNA from 5844 nuclei and identified 30,442 genes in the untreated group and sequenced 5701 nuclei and identified 30,127 genes in the selumetinib treated group. We identified and quantified distinct populations of cells (Schwann cells, fibroblasts, pericytes, myeloid cells, melanocytes, keratinocytes, and two populations of endothelial cells). While we anticipated that cell proportions might change with treatment, we did not identify any one cell population that changed significantly, likely due to an inherent level of variability between tumors. We also evaluated differential gene expression based on drug treatment in each cell type. Ingenuity pathway analysis (IPA) was also used to identify pathways that differ on treatment. As anticipated, we identified a significant decrease in ERK/MAPK signaling in cells including Schwann cells but most specifically in myeloid cells. Interestingly, there is a significant decrease in opioid signaling in myeloid and endothelial cells; this downward trend is also observed in Schwann cells and fibroblasts. Cell communication was assessed by RNA velocity, Scriabin, and CellChat analyses which indicated that Schwann cells and fibroblasts have dramatically altered cell states defined by specific gene expression signatures following treatment (RNA velocity). There are dramatic changes in receptor-ligand pairs following treatment (Scriabin), and robust intercellular signaling between virtually all cell types associated with extracellular matrix (ECM) pathways (Collagen, Laminin, Fibronectin, and Nectin) is downregulated after treatment. These response specific gene signatures and interaction pathways could provide clues for understanding treatment outcomes or inform future therapies.

Molecular and spatial profiling of the paraventricular nucleus of the thalamus.

The paraventricular nucleus of the thalamus (PVT) is known to regulate various cognitive and behavioral processes. However, while functional diversity among PVT circuits has often been linked to cellular differences, the molecular identity and spatial distribution of PVT cell types remain unclear. To address this gap, here we used single nucleus RNA sequencing (snRNA-seq) and identified five molecularly distinct PVT neuronal subtypes in the mouse brain. Additionally, multiplex fluorescent in situ hybridization of top marker genes revealed that PVT subtypes are organized by a combination of previously unidentified molecular gradients. Lastly, comparing our dataset with a recently published single-cell sequencing atlas of the thalamus yielded novel insight into the PVT's connectivity with the cortex, including unexpected innervation of auditory and visual areas. This comparison also revealed that our data contains a largely non-overlapping transcriptomic map of multiple midline thalamic nuclei. Collectively, our findings uncover previously unknown features of the molecular diversity and anatomical organization of the PVT and provide a valuable resource for future investigations.

Adipose tissue at single-cell resolution.

Adipose tissue exhibits remarkable plasticity with capacity to change in size and cellular composition under physiological and pathophysiological conditions. The emergence of single-cell transcriptomics has rapidly transformed our understanding of the diverse array of cell types and cell states residing in adipose tissues and has provided insight into how transcriptional changes in individual cell types contribute to tissue plasticity. Here, we present a comprehensive overview of the cellular atlas of adipose tissues focusing on the biological insight gained from single-cell and single-nuclei transcriptomics of murine and human adipose tissues. We also offer our perspective on the exciting opportunities for mapping cellular transitions and crosstalk, which have been made possible by single-cell technologies.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration.

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

Single-cell and single-nuclei RNA sequencing as powerful tools to decipher cellular heterogeneity and dysregulation in neurodegenerative diseases.

Neurodegenerative diseases affect millions of people worldwide and there are currently no cures. Two types of common neurodegenerative diseases are Alzheimer's (AD) and Parkinson's disease (PD). Single-cell and single-nuclei RNA sequencing (scRNA-seq and snRNA-seq) have become powerful tools to elucidate the inherent complexity and dynamics of the central nervous system at cellular resolution. This technology has allowed the identification of cell types and states, providing new insights into cellular susceptibilities and molecular mechanisms underlying neurodegenerative conditions. Exciting research using high throughput scRNA-seq and snRNA-seq technologies to study AD and PD is emerging. Herein we review the recent progress in understanding these neurodegenerative diseases using these state-of-the-art technologies. We discuss the fundamental principles and implications of single-cell sequencing of the human brain. Moreover, we review some examples of the computational and analytical tools required to interpret the extensive amount of data generated from these assays. We conclude by highlighting challenges and limitations in the application of these technologies in the study of AD and PD.

Astrocytes and oligodendrocytes undergo subtype-specific transcriptional changes in Alzheimer's disease.

Resolving glial contributions to Alzheimer's disease (AD) is necessary because changes in neuronal function, such as reduced synaptic density, altered electrophysiological properties, and degeneration, are not entirely cell autonomous. To improve understanding of transcriptomic heterogeneity in glia during AD, we used single-nuclei RNA sequencing (snRNA-seq) to characterize astrocytes and oligodendrocytes from apolipoprotein (APOE) Ɛ2/3 human AD and age- and genotype-matched non-symptomatic (NS) brains. We enriched astrocytes before sequencing and characterized pathology from the same location as the sequenced material. We characterized baseline heterogeneity in both astrocytes and oligodendrocytes and identified global and subtype-specific transcriptomic changes between AD and NS astrocytes and oligodendrocytes. We also took advantage of recent human and mouse spatial transcriptomics resources to localize heterogeneous astrocyte subtypes to specific regions in the healthy and inflamed brain. Finally, we integrated our data with published AD snRNA-seq datasets, highlighting the power of combining datasets to resolve previously unidentifiable astrocyte subpopulations.

In-depth Phylogenomic Analysis of Arbuscular Mycorrhizal Fungi Based on a Comprehensive Set of de novo Genome Assemblies.

Morphological characters and nuclear ribosomal DNA (rDNA) phylogenies have so far been the basis of the current classifications of arbuscular mycorrhizal (AM) fungi. Improved understanding of the evolutionary history of AM fungi requires extensive ortholog sampling and analyses of genome and transcriptome data from a wide range of taxa. To circumvent the need for axenic culturing of AM fungi we gathered and combined genomic data from single nuclei to generate de novo genome assemblies covering seven families of AM fungi. We successfully sequenced the genomes of 15 AM fungal species for which genome data was not previously available. Comparative analysis of the previously published Rhizophagus irregularis DAOM197198 assembly confirm that our novel workflow generates genome assemblies suitable for phylogenomic analysis. Predicted genes of our assemblies, together with published protein sequences of AM fungi and their sister clades, were used for phylogenomic analyses. We evaluated the phylogenetic placement of Glomeromycota in relation to its sister phyla (Mucoromycota and Mortierellomycota), and found no support to reject a polytomy. Finally, we explored the phylogenetic relationships within Glomeromycota. Our results support family level classification from previous phylogenetic studies, and the polyphyly of the order Glomerales with Claroideoglomeraceae as the sister group to Glomeraceae and Diversisporales.