Human microglial state dynamics in Alzheimer's disease progression.

Altered microglial states affect neuroinflammation, neurodegeneration, and disease but remain poorly understood. Here, we report 194,000 single-nucleus microglial transcriptomes and epigenomes across 443 human subjects and diverse Alzheimer's disease (AD) pathological phenotypes. We annotate 12 microglial transcriptional states, including AD-dysregulated homeostatic, inflammatory, and lipid-processing states. We identify 1,542 AD-differentially-expressed genes, including both microglia-state-specific and disease-stage-specific alterations. By integrating epigenomic, transcriptomic, and motif information, we infer upstream regulators of microglial cell states, gene-regulatory networks, enhancer-gene links, and transcription-factor-driven microglial state transitions. We demonstrate that ectopic expression of our predicted homeostatic-state activators induces homeostatic features in human iPSC-derived microglia-like cells, while inhibiting activators of inflammation can block inflammatory progression. Lastly, we pinpoint the expression of AD-risk genes in microglial states and differential expression of AD-risk genes and their regulators during AD progression. Overall, we provide insights underlying microglial states, including state-specific and AD-stage-specific microglial alterations at unprecedented resolution.

Epigenomic dissection of Alzheimer's disease pinpoints causal variants and reveals epigenome erosion.

Recent work has identified dozens of non-coding loci for Alzheimer's disease (AD) risk, but their mechanisms and AD transcriptional regulatory circuitry are poorly understood. Here, we profile epigenomic and transcriptomic landscapes of 850,000 nuclei from prefrontal cortexes of 92 individuals with and without AD to build a map of the brain regulome, including epigenomic profiles, transcriptional regulators, co-accessibility modules, and peak-to-gene links in a cell-type-specific manner. We develop methods for multimodal integration and detecting regulatory modules using peak-to-gene linking. We show AD risk loci are enriched in microglial enhancers and for specific TFs including SPI1, ELF2, and RUNX1. We detect 9,628 cell-type-specific ATAC-QTL loci, which we integrate alongside peak-to-gene links to prioritize AD variant regulatory circuits. We report differential accessibility of regulatory modules in late AD in glia and in early AD in neurons. Strikingly, late-stage AD brains show global epigenome dysregulation indicative of epigenome erosion and cell identity loss.

Single-cell atlas reveals correlates of high cognitive function, dementia, and resilience to Alzheimer's disease pathology.

Alzheimer's disease (AD) is the most common cause of dementia worldwide, but the molecular and cellular mechanisms underlying cognitive impairment remain poorly understood. To address this, we generated a single-cell transcriptomic atlas of the aged human prefrontal cortex covering 2.3 million cells from postmortem human brain samples of 427 individuals with varying degrees of AD pathology and cognitive impairment. Our analyses identified AD-pathology-associated alterations shared between excitatory neuron subtypes, revealed a coordinated increase of the cohesin complex and DNA damage response factors in excitatory neurons and in oligodendrocytes, and uncovered genes and pathways associated with high cognitive function, dementia, and resilience to AD pathology. Furthermore, we identified selectively vulnerable somatostatin inhibitory neuron subtypes depleted in AD, discovered two distinct groups of inhibitory neurons that were more abundant in individuals with preserved high cognitive function late in life, and uncovered a link between inhibitory neurons and resilience to AD pathology.

Single-cell multiregion dissection of Alzheimer's disease.

Alzheimer's disease is the leading cause of dementia worldwide, but the cellular pathways that underlie its pathological progression across brain regions remain poorly understood1-3. Here we report a single-cell transcriptomic atlas of six different brain regions in the aged human brain, covering 1.3 million cells from 283 post-mortem human brain samples across 48 individuals with and without Alzheimer's disease. We identify 76 cell types, including region-specific subtypes of astrocytes and excitatory neurons and an inhibitory interneuron population unique to the thalamus and distinct from canonical inhibitory subclasses. We identify vulnerable populations of excitatory and inhibitory neurons that are depleted in specific brain regions in Alzheimer's disease, and provide evidence that the Reelin signalling pathway is involved in modulating the vulnerability of these neurons. We develop a scalable method for discovering gene modules, which we use to identify cell-type-specific and region-specific modules that are altered in Alzheimer's disease and to annotate transcriptomic differences associated with diverse pathological variables. We identify an astrocyte program that is associated with cognitive resilience to Alzheimer's disease pathology, tying choline metabolism and polyamine biosynthesis in astrocytes to preserved cognitive function late in life. Together, our study develops a regional atlas of the ageing human brain and provides insights into cellular vulnerability, response and resilience to Alzheimer's disease pathology.

Single-cell dissection of the human brain vasculature.

Despite the importance of the cerebrovasculature in maintaining normal brain physiology and in understanding neurodegeneration and drug delivery to the central nervous system1, human cerebrovascular cells remain poorly characterized owing to their sparsity and dispersion. Here we perform single-cell characterization of the human cerebrovasculature using both ex vivo fresh tissue experimental enrichment and post mortem in silico sorting of human cortical tissue samples. We capture 16,681 cerebrovascular nuclei across 11 subtypes, including endothelial cells, mural cells and three distinct subtypes of perivascular fibroblast along the vasculature. We uncover human-specific expression patterns along the arteriovenous axis and determine previously uncharacterized cell-type-specific markers. We use these human-specific signatures to study changes in 3,945 cerebrovascular cells from patients with Huntington's disease, which reveal activation of innate immune signalling in vascular and glial cell types and a concomitant reduction in the levels of proteins critical for maintenance of blood-brain barrier integrity. Finally, our study provides a comprehensive molecular atlas of the human cerebrovasculature to guide future biological and therapeutic studies.

Mechanochemical removal of ribosome biogenesis factors from nascent 60S ribosomal subunits.

The dynein-related AAA ATPase Rea1 is a preribosomal factor that triggers an unknown maturation step in 60S subunit biogenesis. Using electron microscopy, we show that Rea1's motor domain is docked to the pre-60S particle and its tail-like structure, harboring a metal ion-dependent adhesion site (MIDAS), protrudes from the preribosome. Typically, integrins utilize a MIDAS to bind extracellular ligands, an interaction that is strengthened under applied tensile force. Likewise, the Rea1 MIDAS binds the preribosomal factor Rsa4, which is located on the pre-60S subunit at a site that is contacted by the flexible Rea1 tail. The MIDAS-Rsa4 interaction is essential for ATP-dependent dissociation of a group of non-ribosomal factors from the pre-60S particle. Thus, Rea1 aligns with its interacting partners on the preribosome to effect a necessary step on the path to the export-competent 60S subunit.

Mec1 is one of multiple kinases that prime the Mcm2-7 helicase for phosphorylation by Cdc7.

Activation of the eukaryotic replicative DNA helicase, the Mcm2-7 complex, requires phosphorylation by Cdc7/Dbf4 (Dbf4-dependent kinase or DDK), which, in turn, depends on prior phosphorylation of Mcm2-7 by an unknown kinase (or kinases). We identified DDK phosphorylation sites on Mcm4 and Mcm6 and found that phosphorylation of either subunit suffices for cell proliferation. Importantly, prior phosphorylation of either S/T-P or S/T-Q motifs on these subunits is required for DDK phosphorylation of Mcm2-7 and for normal S phase passage. Phosphomimetic mutations of DDK target sites bypass both DDK function and mutation of the priming phosphorylation sites. Mrc1 facilitates Mec1 phosphorylation of the S/T-Q motifs of chromatin-bound Mcm2-7 during S phase to activate replication. Genetic interactions between priming site mutations and MRC1 or TOF1 deletion support a role for these modifications in replication fork stability. These findings identify regulatory mechanisms that modulate origin firing and replication fork assembly during cell cycle progression.

Conserved nucleosome positioning defines replication origins.

The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function.

Modular architecture of eukaryotic RNase P and RNase MRP revealed by electron microscopy.

Ribonuclease P (RNase P) and RNase MRP are closely related ribonucleoprotein enzymes, which process RNA substrates including tRNA precursors for RNase P and 5.8 S rRNA precursors, as well as some mRNAs, for RNase MRP. The structures of RNase P and RNase MRP have not yet been solved, so it is unclear how the proteins contribute to the structure of the complexes and how substrate specificity is determined. Using electron microscopy and image processing we show that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules. Such features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence. These are also the sites of greatest difference between RNase P and RNase MRP, highlighting the importance of the adaptation of this region to the different substrates.

Single-nucleus multiregion transcriptomic analysis of brain vasculature in Alzheimer's disease.

Cerebrovascular dysregulation is a hallmark of Alzheimer's disease (AD), but the changes that occur in specific cell types have not been fully characterized. Here, we profile single-nucleus transcriptomes in the human cerebrovasculature in six brain regions from 220 individuals with AD and 208 age-matched controls. We annotate 22,514 cerebrovascular cells, including 11 subtypes of endothelial, pericyte, smooth muscle, perivascular fibroblast and ependymal cells. We identify 2,676 differentially expressed genes in AD, including downregulation of PDGFRB in pericytes, and of ABCB1 and ATP10A in endothelial cells, and validate the downregulation of SLC6A1 and upregulation of APOD, INSR and COL4A1 in postmortem AD brain tissues. We detect vasculature, glial and neuronal coexpressed gene modules, suggesting coordinated neurovascular unit dysregulation in AD. Integration with AD genetics reveals 125 AD differentially expressed genes directly linked to AD-associated genetic variants. Lastly, we show that APOE4 genotype-associated differences are significantly enriched among AD-associated genes in capillary and venule endothelial cells, as well as subsets of pericytes and fibroblasts.

The tRNA aminoacylation co-factor Arc1p is excluded from the nucleus by an Xpo1p-dependent mechanism.

Arc1p, a yeast tRNA-binding protein, forms a complex with the aminoacyl-tRNA synthetases, methionyl tRNA synthetase (MetRS) and glutamyl tRNA synthetase (GluRS). Although this complex localizes normally in the cytoplasm, in the absence of Arc1p the two free synthetases are also found inside the nucleus. In this work, in order to localize free Arc1 we abolished complex assembly by deleting the appended domains from both MetRS and GluRS. Surprisingly, free Arc1p remained cytoplasmic even when fitted with a strong nuclear localization signal (NLS). However, NLS-Arc1p accumulated in the nucleus when Xpo1/Crm1, the export receptor for NES-containing cargo proteins, was mutated. Thus, the cytoplasmic location of Arc1p is maintained by Xpo1p-dependent nuclear export and Arc1p could act as an adapter in the nucleocytoplasmic trafficking of tRNA and/or the tRNA-aminoacylation machinery.

Rea1, a dynein-related nuclear AAA-ATPase, is involved in late rRNA processing and nuclear export of 60 S subunits.

Rea1, the largest predicted protein in the yeast genome, is a member of the AAA(+) family of ATPases and is associated with pre-60 S ribosomes. Here we report that Rea1 is required for maturation and nuclear export of the pre-60 S subunit. Rea1 exhibits a predominantly nucleoplasmic localization and is present in a late pre-60 S particle together with members of the Rix1 complex. To study the role of Rea1 in ribosome biogenesis, we generated a repressible GAL::REA1 strain and temperature-sensitive rea1 alleles. In vivo depletion of Rea1 results in the significant reduction of mature 60 S subunits concomitant with defects in pre-rRNA processing and late pre-60 S ribosome stability following ITS2 cleavage and prior to the generation of mature 5.8 S rRNA. Strains depleted of the components of the Rix1 complex (Rix1, Ipi1, and Ipi3) showed similar defects. Using an in vivo 60 S subunit export assay, a strong accumulation of the large subunit reporter Rpl25-GFP (green fluorescent protein) in the nucleus and at the nuclear periphery was seen in rea1 mutants at restrictive conditions.

A pre-ribosome with a tadpole-like structure functions in ATP-dependent maturation of 60S subunits.

Analyses of isolated pre-ribosomes yielded biochemical "snapshots" of the dynamic, nascent 60S and 40S subunits during their path from the nucleolus to the cytoplasm. Here, we present the structure of a pre-60S ribosomal intermediate located in the nucleoplasm. A huge dynein-related AAA-type ATPase (Rea1) and the Rix1 complex (Rix1-Ipi1-Ipi3) are components of an extended (approximately 45 nm long) pre-60S particle. Antibody crosslinking in combination with electron microscopy revealed that the Rea1 localizes to the "tail" region and ribosomal proteins to the "head" region of the elongated "tadpole-like" structure. Furthermore, in vitro treatment with ATP induces dissociation of Rea1 from the pre-60S subunits. Rea1 and the Rix1 complex could mediate ATP-dependent remodeling of 60S subunits and subsequent export from the nucleoplasm to the cytoplasm.