Early Alzheimer's disease pathology in human cortex involves transient cell states.

Cellular perturbations underlying Alzheimer's disease (AD) are primarily studied in human postmortem samples and model organisms. Here, we generated a single-nucleus atlas from a rare cohort of cortical biopsies from living individuals with varying degrees of AD pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes-which we refer to as the early cortical amyloid response-were prominent in neurons, wherein we identified a transitional hyperactive state preceding the loss of excitatory neurons, which we confirmed by acute slice physiology on independent biopsy specimens. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathology increased. Finally, both oligodendrocytes and pyramidal neurons upregulated genes associated with ß-amyloid production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.

Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro.

Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer's disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Single-Cell Multi-omic Integration Compares and Contrasts Features of Brain Cell Identity.

Defining cell types requires integrating diverse single-cell measurements from multiple experiments and biological contexts. To flexibly model single-cell datasets, we developed LIGER, an algorithm that delineates shared and dataset-specific features of cell identity. We applied it to four diverse and challenging analyses of human and mouse brain cells. First, we defined region-specific and sexually dimorphic gene expression in the mouse bed nucleus of the stria terminalis. Second, we analyzed expression in the human substantia nigra, comparing cell states in specific donors and relating cell types to those in the mouse. Third, we integrated in situ and single-cell expression data to spatially locate fine subtypes of cells present in the mouse frontal cortex. Finally, we jointly defined mouse cortical cell types using single-cell RNA-seq and DNA methylation profiles, revealing putative mechanisms of cell-type-specific epigenomic regulation. Integrative analyses using LIGER promise to accelerate investigations of cell-type definition, gene regulation, and disease states.

Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain.

The mammalian brain is composed of diverse, specialized cell populations. To systematically ascertain and learn from these cellular specializations, we used Drop-seq to profile RNA expression in 690,000 individual cells sampled from 9 regions of the adult mouse brain. We identified 565 transcriptionally distinct groups of cells using computational approaches developed to distinguish biological from technical signals. Cross-region analysis of these 565 cell populations revealed features of brain organization, including a gene-expression module for synthesizing axonal and presynaptic components, patterns in the co-deployment of voltage-gated ion channels, functional distinctions among the cells of the vasculature and specialization of glutamatergic neurons across cortical regions. Systematic neuronal classifications for two complex basal ganglia nuclei and the striatum revealed a rare population of spiny projection neurons. This adult mouse brain cell atlas, accessible through interactive online software (DropViz), serves as a reference for development, disease, and evolution.

Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics.

Patterns of gene expression can be used to characterize and classify neuronal types. It is challenging, however, to generate taxonomies that fulfill the essential criteria of being comprehensive, harmonizing with conventional classification schemes, and lacking superfluous subdivisions of genuine types. To address these challenges, we used massively parallel single-cell RNA profiling and optimized computational methods on a heterogeneous class of neurons, mouse retinal bipolar cells (BCs). From a population of ∼25,000 BCs, we derived a molecular classification that identified 15 types, including all types observed previously and two novel types, one of which has a non-canonical morphology and position. We validated the classification scheme and identified dozens of novel markers using methods that match molecular expression to cell morphology. This work provides a systematic methodology for achieving comprehensive molecular classification of neurons, identifies novel neuronal types, and uncovers transcriptional differences that distinguish types within a class.

Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets.

Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT.

Slide-tags enables single-nucleus barcoding for multimodal spatial genomics.

Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed1-6. However, missing from these measurements is the ability to routinely and easily spatially localize these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are tagged with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as an input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 µm spatial resolution and delivered whole-transcriptome data that are indistinguishable in quality from ordinary single-nucleus RNA-sequencing data. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualized receptor-ligand interactions driving B cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to almost any single-cell measurement technology. As a proof of principle, we performed multiomic measurements of open chromatin, RNA and T cell receptor (TCR) sequences in the same cells from metastatic melanoma, identifying transcription factor motifs driving cancer cell state transitions in spatially distinct microenvironments. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire.

The molecular cytoarchitecture of the adult mouse brain.

The function of the mammalian brain relies upon the specification and spatial positioning of diversely specialized cell types. Yet, the molecular identities of the cell types and their positions within individual anatomical structures remain incompletely known. To construct a comprehensive atlas of cell types in each brain structure, we paired high-throughput single-nucleus RNA sequencing with Slide-seq1,2-a recently developed spatial transcriptomics method with near-cellular resolution-across the entire mouse brain. Integration of these datasets revealed the cell type composition of each neuroanatomical structure. Cell type diversity was found to be remarkably high in the midbrain, hindbrain and hypothalamus, with most clusters requiring a combination of at least three discrete gene expression markers to uniquely define them. Using these data, we developed a framework for genetically accessing each cell type, comprehensively characterized neuropeptide and neurotransmitter signalling, elucidated region-specific specializations in activity-regulated gene expression and ascertained the heritability enrichment of neurological and psychiatric phenotypes. These data, available as an online resource ( www.BrainCellData.org ), should find diverse applications across neuroscience, including the construction of new genetic tools and the prioritization of specific cell types and circuits in the study of brain diseases.

An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes.

Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. Here, we analyzed the RNA expression patterns of more than 76,000 individual microglia in mice during development, in old age, and after brain injury. Our analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several states-including chemokine-enriched inflammatory microglia-persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human multiple sclerosis lesions. These distinct microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease.

Serotonin and the neuropeptide PDF initiate and extend opposing behavioral states in C. elegans.

Foraging animals have distinct exploration and exploitation behaviors that are organized into discrete behavioral states. Here, we characterize a neuromodulatory circuit that generates long-lasting roaming and dwelling states in Caenorhabditis elegans. We find that two opposing neuromodulators, serotonin and the neuropeptide pigment dispersing factor (PDF), each initiate and extend one behavioral state. Serotonin promotes dwelling states through the MOD-1 serotonin-gated chloride channel. The spontaneous activity of serotonergic neurons correlates with dwelling behavior, and optogenetic modulation of the critical MOD-1-expressing targets induces prolonged dwelling states. PDF promotes roaming states through a Gαs-coupled PDF receptor; optogenetic activation of cAMP production in PDF receptor-expressing cells induces prolonged roaming states. The neurons that produce and respond to each neuromodulator form a distributed circuit orthogonal to the classical wiring diagram, with several essential neurons that express each molecule. The slow temporal dynamics of this neuromodulatory circuit supplement fast motor circuits to organize long-lasting behavioral states.

Spatial genomics enables multi-modal study of clonal heterogeneity in tissues.

The state and behaviour of a cell can be influenced by both genetic and environmental factors. In particular, tumour progression is determined by underlying genetic aberrations1-4 as well as the makeup of the tumour microenvironment5,6. Quantifying the contributions of these factors requires new technologies that can accurately measure the spatial location of genomic sequence together with phenotypic readouts. Here we developed slide-DNA-seq, a method for capturing spatially resolved DNA sequences from intact tissue sections. We demonstrate that this method accurately preserves local tumour architecture and enables the de novo discovery of distinct tumour clones and their copy number alterations. We then apply slide-DNA-seq to a mouse model of metastasis and a primary human cancer, revealing that clonal populations are confined to distinct spatial regions. Moreover, through integration with spatial transcriptomics, we uncover distinct sets of genes that are associated with clone-specific genetic aberrations, the local tumour microenvironment, or both. Together, this multi-modal spatial genomics approach provides a versatile platform for quantifying how cell-intrinsic and cell-extrinsic factors contribute to gene expression, protein abundance and other cellular phenotypes.

A transcriptomic atlas of mouse cerebellar cortex comprehensively defines cell types.

The cerebellar cortex is a well-studied brain structure with diverse roles in motor learning, coordination, cognition and autonomic regulation. However,  a complete inventory of cerebellar cell types is currently lacking. Here, using recent advances in high-throughput transcriptional profiling1-3, we molecularly define cell types across individual lobules of the adult mouse cerebellum. Purkinje neurons showed considerable regional specialization, with the greatest diversity occurring in the posterior lobules. For several types of cerebellar interneuron, the molecular variation within each type was more continuous, rather than discrete. In particular, for the unipolar brush cells-an interneuron population previously subdivided into discrete populations-the continuous variation in gene expression was associated with a graded continuum of electrophysiological properties. Notably, we found that molecular layer interneurons were composed of two molecularly and functionally distinct types. Both types show a continuum of morphological variation through the thickness of the molecular layer, but electrophysiological recordings revealed marked differences between the two types in spontaneous firing, excitability and electrical coupling. Together, these findings provide a comprehensive cellular atlas of the cerebellar cortex, and outline a methodological and conceptual framework for the integration of molecular, morphological and physiological ontologies for defining brain cell types.

Molecular logic of cellular diversification in the mouse cerebral cortex.

The mammalian cerebral cortex has an unparalleled diversity of cell types, which are generated during development through a series of temporally orchestrated events that are under tight evolutionary constraint and are critical for proper cortical assembly and function1,2. However, the molecular logic that governs the establishment and organization of cortical cell types remains unknown, largely due to the large number of cell classes that undergo dynamic cell-state transitions over extended developmental timelines. Here we generate a comprehensive atlas of the developing mouse neocortex, using single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing. We sampled the neocortex every day throughout embryonic corticogenesis and at early postnatal ages, and complemented the sequencing data with a spatial transcriptomics time course. We computationally reconstruct developmental trajectories across the diversity of cortical cell classes, and infer their spatial organization and the gene regulatory programs that accompany their lineage bifurcation decisions and differentiation trajectories. Finally, we demonstrate how this developmental map pinpoints the origin of lineage-specific developmental abnormalities that are linked to aberrant corticogenesis in mutant mice. The data provide a global picture of the regulatory mechanisms that govern cellular diversification in the neocortex.

Cell type-specific inference of differential expression in spatial transcriptomics.

A central problem in spatial transcriptomics is detecting differentially expressed (DE) genes within cell types across tissue context. Challenges to learning DE include changing cell type composition across space and measurement pixels detecting transcripts from multiple cell types. Here, we introduce a statistical method, cell type-specific inference of differential expression (C-SIDE), that identifies cell type-specific DE in spatial transcriptomics, accounting for localization of other cell types. We model gene expression as an additive mixture across cell types of log-linear cell type-specific expression functions. C-SIDE's framework applies to many contexts: DE due to pathology, anatomical regions, cell-to-cell interactions and cellular microenvironment. Furthermore, C-SIDE enables statistical inference across multiple/replicates. Simulations and validation experiments on Slide-seq, MERFISH and Visium datasets demonstrate that C-SIDE accurately identifies DE with valid uncertainty quantification. Last, we apply C-SIDE to identify plaque-dependent immune activity in Alzheimer's disease and cellular interactions between tumor and immune cells. We distribute C-SIDE within the R package https://github.com/dmcable/spacexr .

Deep learning and alignment of spatially resolved single-cell transcriptomes with Tangram.

Charting an organs' biological atlas requires us to spatially resolve the entire single-cell transcriptome, and to relate such cellular features to the anatomical scale. Single-cell and single-nucleus RNA-seq (sc/snRNA-seq) can profile cells comprehensively, but lose spatial information. Spatial transcriptomics allows for spatial measurements, but at lower resolution and with limited sensitivity. Targeted in situ technologies solve both issues, but are limited in gene throughput. To overcome these limitations we present Tangram, a method that aligns sc/snRNA-seq data to various forms of spatial data collected from the same region, including MERFISH, STARmap, smFISH, Spatial Transcriptomics (Visium) and histological images. Tangram can map any type of sc/snRNA-seq data, including multimodal data such as those from SHARE-seq, which we used to reveal spatial patterns of chromatin accessibility. We demonstrate Tangram on healthy mouse brain tissue, by reconstructing a genome-wide anatomically integrated spatial map at single-cell resolution of the visual and somatomotor areas.

Insights into Neurodegeneration in Parkinson's Disease from Single-Cell and Spatial Genomics.

Parkinson's disease (PD) is pathologically defined by the death of dopaminergic (DA) neurons within the pars compacta of the substantia nigra. To date, the cause of this multifaceted disease remains largely unclear, which may contribute in part to a current lack of disease-modifying therapies. Recent advances in single-cell and spatial genomic profiling tools have provided powerful new ways to measure cellular state changes in brain diseases. Here, we describe how these tools have offered insight into these complex disorders and highlight a recently performed comprehensive study of DA neuron susceptibility in PD. The data generated by this recent work provide evidence for the role of specific pathways and common genetic variants resulting in the loss of a critical DA subtype in PD. We conclude by outlining a set of basic and translational opportunities that arise from those data and insights gathered from this work. © 2023 International Parkinson and Movement Disorder Society.

Cell diversity and network dynamics in photosensitive human brain organoids.

In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However, the cells generated within organoids and the extent to which they recapitulate the regional complexity, cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells, which are related to endogenous classes, including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months), allowing for the establishment of relatively mature features, including the formation of dendritic spines and spontaneously active neuronal networks. Finally, neuronal activity within organoids could be controlled using light stimulation of photosensitive cells, which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli.