A cellular and spatial map of the choroid plexus across brain ventricles and ages.

The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. Here, we construct a single-cell and spatial atlas of each ChP in the developing, adult, and aged mouse brain. We delineate diverse cell types, subtypes, cell states, and expression programs in epithelial and mesenchymal cells across ages and ventricles. In the developing ChP, we predict a common progenitor pool for epithelial and neuronal cells, validated by lineage tracing. Epithelial and fibroblast cells show regionalized expression by ventricle, starting at embryonic stages and persisting with age, with a dramatic transcriptional shift with maturation, and a smaller shift in each aged cell type. With aging, epithelial cells upregulate host-defense programs, and resident macrophages upregulate interleukin-1ß (IL-1ß) signaling genes. Our atlas reveals cellular diversity, architecture and signaling across ventricles during development, maturation, and aging of the ChP-brain barrier.

Cellular communities reveal trajectories of brain ageing and Alzheimer's disease.

Alzheimer's disease (AD) has recently been associated with diverse cell states1-11, yet when and how these states affect the onset of AD remains unclear. Here we used a data-driven approach to reconstruct the dynamics of the brain's cellular environment and identified a trajectory leading to AD that is distinct from other ageing-related effects. First, we built a comprehensive cell atlas of the aged prefrontal cortex from 1.65 million single-nucleus RNA-sequencing profiles sampled from 437 older individuals, and identified specific glial and neuronal subpopulations associated with AD-related traits. Causal modelling then prioritized two distinct lipid-associated microglial subpopulations-one drives amyloid-ß proteinopathy while the other mediates the effect of amyloid-ß on tau proteinopathy-as well as an astrocyte subpopulation that mediates the effect of tau on cognitive decline. To model the dynamics of cellular environments, we devised the BEYOND methodology, which identified two distinct trajectories of brain ageing, each defined by coordinated progressive changes in certain cellular communities that lead to (1) AD dementia or (2) alternative brain ageing. Thus, we provide a cellular foundation for a new perspective on AD pathophysiology that informs personalized therapeutic development, targeting different cellular communities for individuals on the path to AD or to alternative brain ageing.

Stroke genetics informs drug discovery and risk prediction across ancestries.

Previous genome-wide association studies (GWASs) of stroke - the second leading cause of death worldwide - were conducted predominantly in populations of European ancestry1,2. Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis3, and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach4, we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry5. Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries.

High-throughput morphometric and transcriptomic profiling uncovers composition of naïve and sensory-deprived cortical cholinergic VIP/CHAT neurons.

Cortical neuronal networks control cognitive output, but their composition and modulation remain elusive. Here, we studied the morphological and transcriptional diversity of cortical cholinergic VIP/ChAT interneurons (VChIs), a sparse population with a largely unknown function. We focused on VChIs from the whole barrel cortex and developed a high-throughput automated reconstruction framework, termed PopRec, to characterize hundreds of VChIs from each mouse in an unbiased manner, while preserving 3D cortical coordinates in multiple cleared mouse brains, accumulating thousands of cells. We identified two fundamentally distinct morphological types of VChIs, bipolar and multipolar that differ in their cortical distribution and general morphological features. Following mild unilateral whisker deprivation on postnatal day seven, we found after three weeks both ipsi- and contralateral dendritic arborization differences and modified cortical depth and distribution patterns in the barrel fields alone. To seek the transcriptomic drivers, we developed NuNeX, a method for isolating nuclei from fixed tissues, to explore sorted VChIs. This highlighted differentially expressed neuronal structural transcripts, altered exitatory innervation pathways and established Elmo1 as a key regulator of morphology following deprivation.

Densely interconnected transcriptional circuits control cell states in human hematopoiesis.

Though many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly coexpressed genes, some of which are restricted to a single lineage but most of which are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed.

Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals.

Epigenetic information can be inherited through the mammalian germline and represents a plausible transgenerational carrier of environmental information. To test whether transgenerational inheritance of environmental information occurs in mammals, we carried out an expression profiling screen for genes in mice that responded to paternal diet. Offspring of males fed a low-protein diet exhibited elevated hepatic expression of many genes involved in lipid and cholesterol biosynthesis and decreased levels of cholesterol esters, relative to the offspring of males fed a control diet. Epigenomic profiling of offspring livers revealed numerous modest (∼20%) changes in cytosine methylation depending on paternal diet, including reproducible changes in methylation over a likely enhancer for the key lipid regulator Ppara. These results, in conjunction with recent human epidemiological data, indicate that parental diet can affect cholesterol and lipid metabolism in offspring and define a model system to study environmental reprogramming of the heritable epigenome.

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

Massively parallel single-nucleus RNA-seq with DroNc-seq.

Single-nucleus RNA sequencing (sNuc-seq) profiles RNA from tissues that are preserved or cannot be dissociated, but it does not provide high throughput. Here, we develop DroNc-seq: massively parallel sNuc-seq with droplet technology. We profile 39,111 nuclei from mouse and human archived brain samples to demonstrate sensitive, efficient, and unbiased classification of cell types, paving the way for systematic charting of cell atlases.

Nuclear receptors control pro-viral and antiviral metabolic responses to hepatitis C virus infection.

Viruses lack the basic machinery needed to replicate and therefore must hijack the host's metabolism to propagate. Virus-induced metabolic changes have yet to be systematically studied in the context of host transcriptional regulation, and such studies shoul offer insight into host-pathogen metabolic interplay. In this work we identified hepatitis C virus (HCV)-responsive regulators by coupling system-wide metabolic-flux analysis with targeted perturbation of nuclear receptors in primary human hepatocytes. We found HCV-induced upregulation of glycolysis, ketogenesis and drug metabolism, with glycolysis controlled by activation of HNF4α, ketogenesis by PPARα and FXR, and drug metabolism by PXR. Pharmaceutical inhibition of HNF4α reversed HCV-induced glycolysis, blocking viral replication while increasing apoptosis in infected cells showing virus-induced dependence on glycolysis. In contrast, pharmaceutical inhibition of PPARα or FXR reversed HCV-induced ketogenesis but increased viral replication, demonstrating a novel host antiviral response. Our results show that virus-induced changes to a host's metabolism can be detrimental to its life cycle, thus revealing a biologically complex relationship between virus and host.

Arboretum: reconstruction and analysis of the evolutionary history of condition-specific transcriptional modules.

Comparative functional genomics studies the evolution of biological processes by analyzing functional data, such as gene expression profiles, across species. A major challenge is to compare profiles collected in a complex phylogeny. Here, we present Arboretum, a novel scalable computational algorithm that integrates expression data from multiple species with species and gene phylogenies to infer modules of coexpressed genes in extant species and their evolutionary histories. We also develop new, generally applicable measures of conservation and divergence in gene regulatory modules to assess the impact of changes in gene content and expression on module evolution. We used Arboretum to study the evolution of the transcriptional response to heat shock in eight species of Ascomycota fungi and to reconstruct modules of the ancestral environmental stress response (ESR). We found substantial conservation in the stress response across species and in the reconstructed components of the ancestral ESR modules. The greatest divergence was in the most induced stress, primarily through module expansion. The divergence of the heat stress response exceeds that observed in the response to glucose depletion in the same species. Arboretum and its associated analyses provide a comprehensive framework to systematically study regulatory evolution of condition-specific responses.

A Single-Nucleus Transcriptome-Wide Association Study Implicates Novel Genes in Depression Pathogenesis.

BACKGROUND: Depression, a common psychiatric illness and global public health problem, remains poorly understood across different life stages, which hampers the development of novel treatments. METHODS: To identify new candidate genes for therapeutic development, we performed differential gene expression analysis of single-nucleus RNA sequencing data from the dorsolateral prefrontal cortex of older adults (n = 424) in relation to antemortem depressive symptoms. Additionally, we integrated genome-wide association study results for depression (n = 500,199) along with genetic tools for inferring the expression of 14,048 unique genes in 7 cell types and 52 cell subtypes to perform a transcriptome-wide association study of depression followed by Mendelian randomization. RESULTS: Our single-nucleus transcriptome-wide association study analysis identified 68 candidate genes for depression and showed the greatest number being in excitatory and inhibitory neurons. Of the 68 genes, 53 were novel compared to previous studies. Notably, gene expression in different neuronal subtypes had varying effects on depression risk. Traits with high genetic correlations with depression, such as neuroticism, shared more transcriptome-wide association study genes than traits that were not highly correlated with depression. Complementing these analyses, differential gene expression analysis across 52 neocortical cell subtypes showed that genes such as KCNN2, SCAI, WASF3, and SOCS6 were associated with late-life depressive symptoms in specific cell subtypes. CONCLUSIONS: These 2 sets of analyses illustrate the utility of large single-nucleus RNA sequencing data both to uncover genes whose expression is altered in specific cell subtypes in the context of depressive symptoms and to enhance the interpretation of well-powered genome-wide association studies so that we can prioritize specific susceptibility genes for further analysis and therapeutic development.

Cell subtype-specific effects of genetic variation in the Alzheimer's disease brain.

The relationship between genetic variation and gene expression in brain cell types and subtypes remains understudied. Here, we generated single-nucleus RNA sequencing data from the neocortex of 424 individuals of advanced age; we assessed the effect of genetic variants on RNA expression in cis (cis-expression quantitative trait loci) for seven cell types and 64 cell subtypes using 1.5 million transcriptomes. This effort identified 10,004 eGenes at the cell type level and 8,099 eGenes at the cell subtype level. Many eGenes are only detected within cell subtypes. A new variant influences APOE expression only in microglia and is associated with greater cerebral amyloid angiopathy but not Alzheimer's disease pathology, after adjusting for APOEε4, providing mechanistic insights into both pathologies. Furthermore, only a TMEM106B variant affects the proportion of cell subtypes. Integration of these results with genome-wide association studies highlighted the targeted cell type and probable causal gene within Alzheimer's disease, schizophrenia, educational attainment and Parkinson's disease loci.

A functional selection model explains evolutionary robustness despite plasticity in regulatory networks.

Evolutionary rewiring of regulatory networks is an important source of diversity among species. Previous evidence suggested substantial divergence of regulatory networks across species. However, systematically assessing the extent of this plasticity and its functional implications has been challenging due to limited experimental data and the noisy nature of computational predictions. Here, we introduce a novel approach to study cis-regulatory evolution, and use it to trace the regulatory history of 88 DNA motifs of transcription factors across 23 Ascomycota fungi. While motifs are conserved, we find a pervasive gain and loss in the regulation of their target genes. Despite this turnover, the biological processes associated with a motif are generally conserved. We explain these trends using a model with a strong selection to conserve the overall function of a transcription factor, and a much weaker selection over the specific genes it targets. The model also accounts for the turnover of bound targets measured experimentally across species in yeasts and mammals. Thus, selective pressures on regulatory networks mostly tolerate local rewiring, and may allow for subtle fine-tuning of gene regulation during evolution.

Non-invasive assessment of normal and impaired iron homeostasis in the brain.

Strict iron regulation is essential for normal brain function. The iron homeostasis, determined by the milieu of available iron compounds, is impaired in aging, neurodegenerative diseases and cancer. However, non-invasive assessment of different molecular iron environments implicating brain tissue's iron homeostasis remains a challenge. We present a magnetic resonance imaging (MRI) technology sensitive to the iron homeostasis of the living brain (the r1-r2* relaxivity). In vitro, our MRI approach reveals the distinct paramagnetic properties of ferritin, transferrin and ferrous iron ions. In the in vivo human brain, we validate our approach against ex vivo iron compounds quantification and gene expression. Our approach varies with the iron mobilization capacity across brain regions and in aging. It reveals brain tumors' iron homeostasis, and enhances the distinction between tumor tissue and non-pathological tissue without contrast agents. Therefore, our approach may allow for non-invasive research and diagnosis of iron homeostasis in living human brains.

Deep brain stimulation of thalamic nucleus reuniens promotes neuronal and cognitive resilience in an Alzheimer's disease mouse model.

The mechanisms that confer cognitive resilience to Alzheimer's Disease (AD) are not fully understood. Here, we describe a neural circuit mechanism underlying this resilience in a familial AD mouse model. In the prodromal disease stage, interictal epileptiform spikes (IESs) emerge during anesthesia in the CA1 and mPFC regions, leading to working memory disruptions. These IESs are driven by inputs from the thalamic nucleus reuniens (nRE). Indeed, tonic deep brain stimulation of the nRE (tDBS-nRE) effectively suppresses IESs and restores firing rate homeostasis under anesthesia, preventing further impairments in nRE-CA1 synaptic facilitation and working memory. Notably, applying tDBS-nRE during the prodromal phase in young APP/PS1 mice mitigates age-dependent memory decline. The IES rate during anesthesia in young APP/PS1 mice correlates with later working memory impairments. These findings highlight the nRE as a central hub of functional resilience and underscore the clinical promise of DBS in conferring resilience to AD pathology by restoring circuit-level homeostasis.

Claustral neurons projecting to frontal cortex restrict opioid consumption.

The synthetic opioid fentanyl is a major contributor to the current opioid addiction crisis. We report that claustral neurons projecting to the frontal cortex limit oral fentanyl self-administration in mice. We found that fentanyl transcriptionally activates frontal-projecting claustrum neurons. These neurons also exhibit a unique suppression of Ca2+ activity upon initiation of bouts of fentanyl consumption. Optogenetic stimulation of frontal-projecting claustral neurons, intervening in this suppression, decreased bouts of fentanyl consumption. In contrast, constitutive inhibition of frontal-projecting claustral neurons in the context of a novel, group-housed self-administration procedure increased fentanyl bout consumption. This same manipulation also sensitized conditioned-place preference for fentanyl and enhanced the representation of fentanyl experience in the frontal cortex. Together, our results indicate that claustrum neurons exert inhibitory control over frontal cortical neurons to restrict oral fentanyl intake. Upregulation of activity in the claustro-frontal projection may be a promising strategy for reducing human opioid addiction.

Cellular dynamics across aged human brains uncover a multicellular cascade leading to Alzheimer's disease.

Alzheimer's Disease (AD) is a progressive neurodegenerative disease seen with advancing age. Recent studies have revealed diverse AD-associated cell states, yet when and how they impact the causal chain leading to AD remains unknown. To reconstruct the dynamics of the brain's cellular environment along the disease cascade and to distinguish between AD and aging effects, we built a comprehensive cell atlas of the aged prefrontal cortex from 1.64 million single-nucleus RNA-seq profiles. We associated glial, vascular and neuronal subpopulations with AD-related traits for 424 aging individuals, and aligned them along the disease cascade using causal modeling. We identified two distinct lipid-associated microglial subpopulations, one contributed to amyloid-ß proteinopathy while the other mediated the effect of amyloid-ß in accelerating tau proteinopathy, as well as an astrocyte subpopulation that mediated the effect of tau on cognitive decline. To model the coordinated dynamics of the entire cellular environment we devised the BEYOND methodology which uncovered two distinct trajectories of brain aging that are defined by distinct sequences of changes in cellular communities. Older individuals are engaged in one of two possible trajectories, each associated with progressive changes in specific cellular communities that end with: (1) AD dementia or (2) alternative brain aging. Thus, we provide a cellular foundation for a new perspective of AD pathophysiology that could inform the development of new therapeutic interventions targeting cellular communities, while designing a different clinical management for those individuals on the path to AD or to alternative brain aging.

A single-nucleus transcriptome-wide association study implicates novel genes in depression pathogenesis.

Background: Depression is a common psychiatric illness and global public health problem. However, our limited understanding of the biological basis of depression has hindered the development of novel treatments and interventions. Methods: To identify new candidate genes for therapeutic development, we examined single-nucleus RNA sequencing (snucRNAseq) data from the dorsolateral prefrontal cortex (N=424) in relation to ante-mortem depressive symptoms. To complement these direct analyses, we also used genome-wide association study (GWAS) results for depression (N=500,199) along with genetic tools for inferring the expression of 22,159 genes in 7 cell types and 55 cell subtypes to perform transcriptome-wide association studies (TWAS) of depression followed by Mendelian randomization (MR). Results: Our single-nucleus TWAS analysis identified 71 causal genes in depression that have a role in specific neocortical cell subtypes; 59 of 71 genes were novel compared to previous studies. Depression TWAS genes showed a cell type specific pattern, with the greatest enrichment being in both excitatory and inhibitory neurons as well as astrocytes. Gene expression in different neuron subtypes have different directions of effect on depression risk. Compared to lower genetically correlated traits (e.g. body mass index) with depression, higher correlated traits (e.g., neuroticism) have more common TWAS genes with depression. In parallel, we performed differential gene expression analysis in relation to depression in 55 cortical cell subtypes, and we found that genes such as ANKRD36, MADD, TAOK3, SCAI and CHUK are associated with depression in specific cell subtypes. Conclusions: These two sets of analyses illustrate the utility of large snucRNAseq data to uncover both genes whose expression is altered in specific cell subtypes in the context of depression and to enhance the interpretation of well-powered GWAS so that we can prioritize specific susceptibility genes for further analysis and therapeutic development.