Single-cell genomics and regulatory networks for 388 human brains.

Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet, little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multi-omics datasets into a resource comprising >2.8M nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550K cell-type-specific regulatory elements and >1.4M single-cell expression-quantitative-trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized ~250 disease-risk genes and drug targets with associated cell types.

Structurally divergent and recurrently mutated regions of primate genomes.

We sequenced and assembled using multiple long-read sequencing technologies the genomes of chimpanzee, bonobo, gorilla, orangutan, gibbon, macaque, owl monkey, and marmoset. We identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. We estimate that 819.47 Mbp or ∼27% of the genome has been affected by SVs across primate evolution. We identify 1,607 structurally divergent regions wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (e.g., CARD, C4, and OLAH gene families) and additional lineage-specific genes are generated (e.g., CKAP2, VPS36, ACBD7, and NEK5 paralogs), becoming targets of rapid chromosomal diversification and positive selection (e.g., RGPD gene family). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species.

Enhancer-AAVs allow genetic access to oligodendrocytes and diverse populations of astrocytes across species.

Proper brain function requires the assembly and function of diverse populations of neurons and glia. Single cell gene expression studies have mostly focused on characterization of neuronal cell diversity; however, recent studies have revealed substantial diversity of glial cells, particularly astrocytes. To better understand glial cell types and their roles in neurobiology, we built a new suite of adeno-associated viral (AAV)-based genetic tools to enable genetic access to astrocytes and oligodendrocytes. These oligodendrocyte and astrocyte enhancer-AAVs are highly specific (usually > 95% cell type specificity) with variable expression levels, and our astrocyte enhancer-AAVs show multiple distinct expression patterns reflecting the spatial distribution of astrocyte cell types. To provide the best glial-specific functional tools, several enhancer-AAVs were: optimized for higher expression levels, shown to be functional and specific in rat and macaque, shown to maintain specific activity in epilepsy where traditional promoters changed activity, and used to drive functional transgenes in astrocytes including Cre recombinase and acetylcholine-responsive sensor iAChSnFR. The astrocyte-specific iAChSnFR revealed a clear reward-dependent acetylcholine response in astrocytes of the nucleus accumbens during reinforcement learning. Together, this collection of glial enhancer-AAVs will enable characterization of astrocyte and oligodendrocyte populations and their roles across species, disease states, and behavioral epochs.

Comparative transcriptomics reveals human-specific cortical features.

The cognitive abilities of humans are distinctive among primates, but their molecular and cellular substrates are poorly understood. We used comparative single-nucleus transcriptomics to analyze samples of the middle temporal gyrus (MTG) from adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets to understand human-specific features of the neocortex. Human, chimpanzee, and gorilla MTG showed highly similar cell-type composition and laminar organization as well as a large shift in proportions of deep-layer intratelencephalic-projecting neurons compared with macaque and marmoset MTG. Microglia, astrocytes, and oligodendrocytes had more-divergent expression across species compared with neurons or oligodendrocyte precursor cells, and neuronal expression diverged more rapidly on the human lineage. Only a few hundred genes showed human-specific patterning, suggesting that relatively few cellular and molecular changes distinctively define adult human cortical structure.

Interindividual variation in human cortical cell type abundance and expression.

Single-cell transcriptomic studies have identified a conserved set of neocortical cell types from small postmortem cohorts. We extended these efforts by assessing cell type variation across 75 adult individuals undergoing epilepsy and tumor surgeries. Nearly all nuclei map to one of 125 robust cell types identified in the middle temporal gyrus. However, we found interindividual variance in abundances and gene expression signatures, particularly in deep-layer glutamatergic neurons and microglia. A minority of donor variance is explainable by age, sex, ancestry, disease state, and cell state. Genomic variation was associated with expression of 150 to 250 genes for most cell types. This characterization of cellular variation provides a baseline for cell typing in health and disease.

Transcriptomic cytoarchitecture reveals principles of human neocortex organization.

Variation in cytoarchitecture is the basis for the histological definition of cortical areas. We used single cell transcriptomics and performed cellular characterization of the human cortex to better understand cortical areal specialization. Single-nucleus RNA-sequencing of 8 areas spanning cortical structural variation showed a highly consistent cellular makeup for 24 cell subclasses. However, proportions of excitatory neuron subclasses varied substantially, likely reflecting differences in connectivity across primary sensorimotor and association cortices. Laminar organization of astrocytes and oligodendrocytes also differed across areas. Primary visual cortex showed characteristic organization with major changes in the excitatory to inhibitory neuron ratio, expansion of layer 4 excitatory neurons, and specialized inhibitory neurons. These results lay the groundwork for a refined cellular and molecular characterization of human cortical cytoarchitecture and areal specialization.

Morphoelectric and transcriptomic divergence of the layer 1 interneuron repertoire in human versus mouse neocortex.

Neocortical layer 1 (L1) is a site of convergence between pyramidal-neuron dendrites and feedback axons where local inhibitory signaling can profoundly shape cortical processing. Evolutionary expansion of human neocortex is marked by distinctive pyramidal neurons with extensive L1 branching, but whether L1 interneurons are similarly diverse is underexplored. Using Patch-seq recordings from human neurosurgical tissue, we identified four transcriptomic subclasses with mouse L1 homologs, along with distinct subtypes and types unmatched in mouse L1. Subclass and subtype comparisons showed stronger transcriptomic differences in human L1 and were correlated with strong morphoelectric variability along dimensions distinct from mouse L1 variability. Accompanied by greater layer thickness and other cytoarchitecture changes, these findings suggest that L1 has diverged in evolution, reflecting the demands of regulating the expanded human neocortical circuit.

Target cell-specific synaptic dynamics of excitatory to inhibitory neuron connections in supragranular layers of human neocortex.

Rodent studies have demonstrated that synaptic dynamics from excitatory to inhibitory neuron types are often dependent on the target cell type. However, these target cell-specific properties have not been well investigated in human cortex, where there are major technical challenges in reliably obtaining healthy tissue, conducting multiple patch-clamp recordings on inhibitory cell types, and identifying those cell types. Here, we take advantage of newly developed methods for human neurosurgical tissue analysis with multiple patch-clamp recordings, post-hoc fluorescent in situ hybridization (FISH), machine learning-based cell type classification and prospective GABAergic AAV-based labeling to investigate synaptic properties between pyramidal neurons and PVALB- vs. SST-positive interneurons. We find that there are robust molecular differences in synapse-associated genes between these neuron types, and that individual presynaptic pyramidal neurons evoke postsynaptic responses with heterogeneous synaptic dynamics in different postsynaptic cell types. Using molecular identification with FISH and classifiers based on transcriptomically identified PVALB neurons analyzed by Patch-seq, we find that PVALB neurons typically show depressing synaptic characteristics, whereas other interneuron types including SST-positive neurons show facilitating characteristics. Together, these data support the existence of target cell-specific synaptic properties in human cortex that are similar to rodent, thereby indicating evolutionary conservation of local circuit connectivity motifs from excitatory to inhibitory neurons and their synaptic dynamics.

A comparison of anatomic and cellular transcriptome structures across 40 human brain diseases.

Genes associated with risk for brain disease exhibit characteristic expression patterns that reflect both anatomical and cell type relationships. Brain-wide transcriptomic patterns of disease risk genes provide a molecular-based signature, based on differential co-expression, that is often unique to that disease. Brain diseases can be compared and aggregated based on the similarity of their signatures which often associates diseases from diverse phenotypic classes. Analysis of 40 common human brain diseases identifies 5 major transcriptional patterns, representing tumor-related, neurodegenerative, psychiatric and substance abuse, and 2 mixed groups of diseases affecting basal ganglia and hypothalamus. Further, for diseases with enriched expression in cortex, single-nucleus data in the middle temporal gyrus (MTG) exhibits a cell type expression gradient separating neurodegenerative, psychiatric, and substance abuse diseases, with unique excitatory cell type expression differentiating psychiatric diseases. Through mapping of homologous cell types between mouse and human, most disease risk genes are found to act in common cell types, while having species-specific expression in those types and preserving similar phenotypic classification within species. These results describe structural and cellular transcriptomic relationships of disease risk genes in the adult brain and provide a molecular-based strategy for classifying and comparing diseases, potentially identifying novel disease relationships.

Integrated gene analyses of de novo variants from 46,612 trios with autism and developmental disorders.

Most genetic studies consider autism spectrum disorder (ASD) and developmental disorder (DD) separately despite overwhelming comorbidity and shared genetic etiology. Here, we analyzed de novo variants (DNVs) from 15,560 ASD (6,557 from SPARK) and 31,052 DD trios independently and also combined as broader neurodevelopmental disorders (NDDs) using three models. We identify 615 NDD candidate genes (false discovery rate [FDR] < 0.05) supported by ≥1 models, including 138 reaching Bonferroni exome-wide significance (P < 3.64e-7) in all models. The genes group into five functional networks associating with different brain developmental lineages based on single-cell nuclei transcriptomic data. We find no evidence for ASD-specific genes in contrast to 18 genes significantly enriched for DD. There are 53 genes that show mutational bias, including enrichments for missense (n = 41) or truncating (n = 12) DNVs. We also find 10 genes with evidence of male- or female-bias enrichment, including 4 X chromosome genes with significant female burden (DDX3X, MECP2, WDR45, and HDAC8). This large-scale integrative analysis identifies candidates and functional subsets of NDD genes.

Oligodendrocyte precursor cells ingest axons in the mouse neocortex.

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.

Single nucleus multi-omics identifies human cortical cell regulatory genome diversity.

Single-cell technologies measure unique cellular signatures but are typically limited to a single modality. Computational approaches allow the fusion of diverse single-cell data types, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells, we devised single-nucleus methylcytosine, chromatin accessibility, and transcriptome sequencing (snmCAT-seq) and applied it to postmortem human frontal cortex tissue. We developed a cross-validation approach using multi-modal information to validate fine-grained cell types and assessed the effectiveness of computational data fusion methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility, and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling the prediction of cell types that are associated with diseases.

Comparative cellular analysis of motor cortex in human, marmoset and mouse.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Human neocortical expansion involves glutamatergic neuron diversification.

The neocortex is disproportionately expanded in human compared with mouse1,2, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth3. Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer's disease4,5. Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease.

Single-cell and single-nucleus RNA-seq uncovers shared and distinct axes of variation in dorsal LGN neurons in mice, non-human primates, and humans.

Abundant evidence supports the presence of at least three distinct types of thalamocortical (TC) neurons in the primate dorsal lateral geniculate nucleus (dLGN) of the thalamus, the brain region that conveys visual information from the retina to the primary visual cortex (V1). Different types of TC neurons in mice, humans, and macaques have distinct morphologies, distinct connectivity patterns, and convey different aspects of visual information to the cortex. To investigate the molecular underpinnings of these cell types, and how these relate to differences in dLGN between human, macaque, and mice, we profiled gene expression in single nuclei and cells using RNA-sequencing. These efforts identified four distinct types of TC neurons in the primate dLGN: magnocellular (M) neurons, parvocellular (P) neurons, and two types of koniocellular (K) neurons. Despite extensively documented morphological and physiological differences between M and P neurons, we identified few genes with significant differential expression between transcriptomic cell types corresponding to these two neuronal populations. Likewise, the dominant feature of TC neurons of the adult mouse dLGN is high transcriptomic similarity, with an axis of heterogeneity that aligns with core vs. shell portions of mouse dLGN. Together, these data show that transcriptomic differences between principal cell types in the mature mammalian dLGN are subtle relative to the observed differences in morphology and cortical projection targets. Finally, alignment of transcriptome profiles across species highlights expanded diversity of GABAergic neurons in primate versus mouse dLGN and homologous types of TC neurons in primates that are distinct from TC neurons in mouse.

Signature morpho-electric, transcriptomic, and dendritic properties of human layer 5 neocortical pyramidal neurons.

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The unique morpho-electric properties of these neurons have been mainly described in rodents, where retrograde tracers or transgenic lines can label them. Similar labeling strategies are infeasible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology, and morphology were assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were often subtler than differences between L5 cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy but also highlight phenotypic divergence possibly related to functional specialization of human neocortex.

Recent ultra-rare inherited variants implicate new autism candidate risk genes.

Autism is a highly heritable complex disorder in which de novo mutation (DNM) variation contributes significantly to risk. Using whole-genome sequencing data from 3,474 families, we investigate another source of large-effect risk variation, ultra-rare variants. We report and replicate a transmission disequilibrium of private, likely gene-disruptive (LGD) variants in probands but find that 95% of this burden resides outside of known DNM-enriched genes. This variant class more strongly affects multiplex family probands and supports a multi-hit model for autism. Candidate genes with private LGD variants preferentially transmitted to probands converge on the E3 ubiquitin-protein ligase complex, intracellular transport and Erb signaling protein networks. We estimate that these variants are approximately 2.5 generations old and significantly younger than other variants of similar type and frequency in siblings. Overall, private LGD variants are under strong purifying selection and appear to act on a distinct set of genes not yet associated with autism.

Multiple Alu Exonization in 3'UTR of a Primate-Specific Isoform of CYP20A1 Creates a Potential miRNA Sponge.

Alu repeats contribute to phylogenetic novelties in conserved regulatory networks in primates. Our study highlights how exonized Alus could nucleate large-scale mRNA-miRNA interactions. Using a functional genomics approach, we characterize a transcript isoform of an orphan gene, CYP20A1 (CYP20A1_Alu-LT) that has exonization of 23 Alus in its 3'UTR. CYP20A1_Alu-LT, confirmed by 3'RACE, is an outlier in length (9 kb 3'UTR) and widely expressed. Using publically available data sets, we demonstrate its expression in higher primates and presence in single nucleus RNA-seq of 15,928 human cortical neurons. miRanda predicts ∼4,700 miRNA recognition elements (MREs) for ∼1,000 miRNAs, primarily originated within these 3'UTR-Alus. CYP20A1_Alu-LT could be a potential multi-miRNA sponge as it harbors ≥10 MREs for 140 miRNAs and has cytosolic localization. We further tested whether expression of CYP20A1_Alu-LT correlates with mRNAs harboring similar MRE targets. RNA-seq with conjoint miRNA-seq analysis was done in primary human neurons where we observed CYP20A1_Alu-LT to be downregulated during heat shock response and upregulated in HIV1-Tat treatment. In total, 380 genes were positively correlated with its expression (significantly downregulated in heat shock and upregulated in Tat) and they harbored MREs for nine expressed miRNAs which were also enriched in CYP20A1_Alu-LT. MREs were significantly enriched in these 380 genes compared with random sets of differentially expressed genes (P = 8.134e-12). Gene ontology suggested involvement of these genes in neuronal development and hemostasis pathways thus proposing a novel component of Alu-miRNA-mediated transcriptional modulation that could govern specific physiological outcomes in higher primates.