ABSTRACT
Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFCâPAG (periaqueductal gray). By comparing Ca2+ activity of the molecularly homogeneous PFCâPAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics.
Subject(s)
Cognition/physiology , Neurons/physiology , Prefrontal Cortex/physiology , Task Performance and Analysis , Animals , Calcium/metabolism , Choice Behavior , Cues , Imaging, Three-Dimensional , Integrases/metabolism , Mice, Inbred C57BL , Odorants , Optogenetics , Periaqueductal Gray/physiology , Reward , Single-Cell Analysis , Transcriptome/geneticsABSTRACT
Severe dengue (SD) is a major cause of morbidity and mortality. To define dengue virus (DENV) target cells and immunological hallmarks of SD progression in children's blood, we integrated two single-cell approaches capturing cellular and viral elements: virus-inclusive single-cell RNA sequencing (viscRNA-Seq 2) and targeted proteomics with secretome analysis and functional assays. Beyond myeloid cells, in natural infection, B cells harbor replicating DENV capable of infecting permissive cells. Alterations in cell type abundance, gene and protein expression and secretion as well as cell-cell communications point towards increased immune cell migration and inflammation in SD progressors. Concurrently, antigen-presenting cells from SD progressors demonstrate intact uptake yet impaired interferon response and antigen processing and presentation signatures, which are partly modulated by DENV. Increased activation, regulation and exhaustion of effector responses and expansion of HLA-DR-expressing adaptive-like NK cells also characterize SD progressors. These findings reveal DENV target cells in human blood and provide insight into SD pathogenesis beyond antibody-mediated enhancement.
Subject(s)
Dengue Virus , Dengue , Severe Dengue , Child , Humans , B-Lymphocytes , Killer Cells, NaturalABSTRACT
Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.
Subject(s)
Olfactory Pathways/metabolism , Olfactory Receptor Neurons/metabolism , Proteomics/methods , Animals , Axons/metabolism , Brain/metabolism , Dendrites/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental/genetics , Membrane Proteins/metabolism , Neurogenesis/physiology , Olfactory Nerve/metabolism , Olfactory Pathways/cytology , Olfactory Pathways/physiology , Receptors, Lipoprotein/metabolism , Smell/physiologyABSTRACT
The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of "orphan" T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A∗02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile indentification of tumor antigens through unbiased screening.
Subject(s)
Adenocarcinoma/immunology , Antigens, Neoplasm/immunology , Colorectal Neoplasms/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Receptors, Antigen, T-Cell/immunology , Aged , Animals , Antigens, Neoplasm/chemistry , Cell Line, Tumor , Cells, Cultured , HEK293 Cells , HLA-A Antigens/chemistry , HLA-A Antigens/immunology , Humans , Male , Middle Aged , Peptide Library , Sf9 Cells , SpodopteraABSTRACT
As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue.
Subject(s)
Aging/pathology , Cellular Senescence , Mutation , Pancreas/pathology , Single-Cell Analysis , Adult , Child , Child, Preschool , Humans , Infant , Middle Aged , Pancreas/cytology , Pancreas/physiology , Polymorphism, Single Nucleotide , Sequence Analysis, RNA , Transcription, GeneticABSTRACT
The definition of neuronal type and how it relates to the transcriptome are open questions. Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: different PN classes target dendrites to distinct olfactory glomeruli, while PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomes of most PN classes and unequivocally mapped transcriptomes to specific olfactory function for six classes. Transcriptomes of closely related PN classes exhibit the largest differences during circuit assembly but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Transcription factors and cell-surface molecules are the most differentially expressed genes between classes and are highly informative in encoding cell identity, enabling us to identify a new lineage-specific transcription factor that instructs PN dendrite targeting. These findings establish that neuronal transcriptomic identity corresponds with anatomical and physiological identity defined by connectivity and function.
Subject(s)
Drosophila melanogaster/metabolism , Neurons/metabolism , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Animals , Brain/cytology , Brain/metabolism , Cluster Analysis , Dendrites/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/growth & development , Gene Expression Profiling , Olfactory Bulb/cytology , Olfactory Bulb/metabolism , Organ Specificity , Pupa/cytology , Pupa/metabolism , Transcription Factors/metabolismABSTRACT
Social communication guides decision-making, which is essential for survival. Social transmission of food preference (STFP) is an ecologically relevant memory paradigm in which an animal learns a desirable food odour from another animal in a social context, creating a long-term memory1,2. How food-preference memory is acquired, consolidated and stored is unclear. Here we show that the posteromedial nucleus of the cortical amygdala (COApm) serves as a computational centre in long-term STFP memory consolidation by integrating social and sensory olfactory inputs. Blocking synaptic signalling by the COApm-based circuit selectively abolished STFP memory consolidation without impairing memory acquisition, storage or recall. COApm-mediated STFP memory consolidation depends on synaptic inputs from the accessory olfactory bulb and on synaptic outputs to the anterior olfactory nucleus. STFP memory consolidation requires protein synthesis, suggesting a gene-expression mechanism. Deep single-cell and spatially resolved transcriptomics revealed robust but distinct gene-expression signatures induced by STFP memory formation in the COApm that are consistent with synapse restructuring. Our data thus define a neural circuit for the consolidation of a socially communicated long-term memory, thereby mechanistically distinguishing protein-synthesis-dependent memory consolidation from memory acquisition, storage or retrieval.
Subject(s)
Amygdala , Food Preferences , Memory Consolidation , Memory, Long-Term , Social Behavior , Animals , Male , Mice , Amygdala/physiology , Amygdala/cytology , Memory Consolidation/physiology , Memory, Long-Term/physiology , Mice, Inbred C57BL , Odorants/analysis , Olfactory Bulb/physiology , Olfactory Bulb/cytology , Single-Cell Analysis , Synapses/metabolism , Transcriptome , Food Preferences/physiology , Food Preferences/psychologyABSTRACT
The assembly and specification of synapses in the brain is incompletely understood1-3. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion G-protein-coupled receptor-mediates synapse formation in the hippocampus4 but the mechanisms involved remain unclear. Here we show in mice that LPHN3 organizes synapses through a convergent dual-pathway mechanism: activation of Gαs signalling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell-type-specific alternative splicing of Lphn3 controls the LPHN3 G-protein-coupling mode, resulting in LPHN3 variants that predominantly signal through Gαs or Gα12/13. CRISPR-mediated manipulation of Lphn3 alternative splicing that shifts LPHN3 from a Gαs- to a Gα12/13-coupled mode impaired synaptic connectivity as severely as the overall deletion of Lphn3, suggesting that Gαs signalling by LPHN3 splice variants mediates synapse formation. Notably, Gαs-coupled, but not Gα12/13-coupled, splice variants of LPHN3 also recruit phase-transitioned postsynaptic protein scaffold condensates, such that these condensates are clustered by binding of presynaptic teneurin and FLRT ligands to LPHN3. Moreover, neuronal activity promotes alternative splicing of the synaptogenic Gαs-coupled variant of LPHN3. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways: Gαs signalling and clustered phase separation of postsynaptic protein scaffolds.
Subject(s)
Alternative Splicing , Receptors, G-Protein-Coupled , Receptors, Peptide , Synapses , Animals , Mice , Alternative Splicing/genetics , GTP-Binding Protein alpha Subunits, G12-G13 , GTP-Binding Protein alpha Subunits, Gs , Ligands , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Receptors, Peptide/deficiency , Receptors, Peptide/genetics , Receptors, Peptide/metabolism , Synapses/metabolism , Signal TransductionABSTRACT
Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory.
Subject(s)
Astrocytes , Cell Communication , Gene Expression Profiling , Memory, Long-Term , Neurons , Astrocytes/cytology , Astrocytes/metabolism , Astrocytes/physiology , Basolateral Nuclear Complex/cytology , Basolateral Nuclear Complex/metabolism , Basolateral Nuclear Complex/physiology , Brain-Derived Neurotrophic Factor/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Memory, Long-Term/physiology , Mitogen-Activated Protein Kinases/metabolism , Neurons/cytology , Neurons/metabolism , Neurons/physiology , Sequence Analysis, RNA , Single Molecule Imaging , Single-Cell Gene Expression Analysis , UbiquitinationABSTRACT
There are few substantive methods to measure the health of the immune system, and the connection between immune strength and the viral component of the microbiome is poorly understood. Organ transplant recipients are treated with posttransplant therapies that combine immunosuppressive and antiviral drugs, offering a window into the effects of immune modulation on the virome. We used sequencing of cell-free DNA in plasma to investigate drug-virome interactions in a cohort of organ transplant recipients (656 samples, 96 patients) and find that antivirals and immunosuppressants strongly affect the structure of the virome in plasma. We observe marked virome compositional dynamics at the onset of the therapy and find that the total viral load increases with immunosuppression, whereas the bacterial component of the microbiome remains largely unaffected. The data provide insight into the relationship between the human virome, the state of the immune system, and the effects of pharmacological treatment and offer a potential application of the virome state to predict immunocompetence.
Subject(s)
Antiviral Agents/therapeutic use , Blood/virology , Heart Transplantation , Immunosuppressive Agents/therapeutic use , Lung Transplantation , Viruses/isolation & purification , Adult , Antibiotic Prophylaxis , Blood/microbiology , Child , DNA/blood , DNA/genetics , Humans , Viruses/classificationABSTRACT
The ability to slow or reverse biological ageing would have major implications for mitigating disease risk and maintaining vitality1. Although an increasing number of interventions show promise for rejuvenation2, their effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. Here we performed single-cell RNA sequencing on 20 organs to reveal cell-type-specific responses to young and aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, haematopoietic stem cells and hepatocytes are among those cell types that are especially responsive. On the pathway level, young blood invokes new gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. We observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it in select cell types. Together, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.
Subject(s)
Parabiosis , Single-Cell Analysis , Adipocytes , Aging/genetics , Electron Transport/genetics , Hematopoietic Stem Cells , Hepatocytes , Mesenchymal Stem Cells , Mitochondria , Organ Specificity/genetics , RNA-Seq , RejuvenationABSTRACT
Liquid biopsies that measure circulating cell-free RNA (cfRNA) offer an opportunity to study the development of pregnancy-related complications in a non-invasive manner and to bridge gaps in clinical care1-4. Here we used 404 blood samples from 199 pregnant mothers to identify and validate cfRNA transcriptomic changes that are associated with preeclampsia, a multi-organ syndrome that is the second largest cause of maternal death globally5. We find that changes in cfRNA gene expression between normotensive and preeclamptic mothers are marked and stable early in gestation, well before the onset of symptoms. These changes are enriched for genes specific to neuromuscular, endothelial and immune cell types and tissues that reflect key aspects of preeclampsia physiology6-9, suggest new hypotheses for disease progression and correlate with maternal organ health. This enabled the identification and independent validation of a panel of 18 genes that when measured between 5 and 16 weeks of gestation can form the basis of a liquid biopsy test that would identify mothers at risk of preeclampsia long before clinical symptoms manifest themselves. Tests based on these observations could help predict and manage who is at risk for preeclampsia-an important objective for obstetric care10,11.
Subject(s)
Cell-Free Nucleic Acids , Early Diagnosis , Pre-Eclampsia , RNA , Blood Pressure , Cell-Free Nucleic Acids/blood , Cell-Free Nucleic Acids/genetics , Female , Humans , Mothers , Pre-Eclampsia/diagnosis , Pre-Eclampsia/genetics , Pregnancy , RNA/blood , RNA/genetics , TranscriptomeABSTRACT
Maternal morbidity and mortality continue to rise, and pre-eclampsia is a major driver of this burden1. Yet the ability to assess underlying pathophysiology before clinical presentation to enable identification of pregnancies at risk remains elusive. Here we demonstrate the ability of plasma cell-free RNA (cfRNA) to reveal patterns of normal pregnancy progression and determine the risk of developing pre-eclampsia months before clinical presentation. Our results centre on comprehensive transcriptome data from eight independent prospectively collected cohorts comprising 1,840 racially diverse pregnancies and retrospective analysis of 2,539 banked plasma samples. The pre-eclampsia data include 524 samples (72 cases and 452 non-cases) from two diverse independent cohorts collected 14.5 weeks (s.d., 4.5 weeks) before delivery. We show that cfRNA signatures from a single blood draw can track pregnancy progression at the placental, maternal and fetal levels and can robustly predict pre-eclampsia, with a sensitivity of 75% and a positive predictive value of 32.3% (s.d., 3%), which is superior to the state-of-the-art method2. cfRNA signatures of normal pregnancy progression and pre-eclampsia are independent of clinical factors, such as maternal age, body mass index and race, which cumulatively account for less than 1% of model variance. Further, the cfRNA signature for pre-eclampsia contains gene features linked to biological processes implicated in the underlying pathophysiology of pre-eclampsia.
Subject(s)
Cell-Free Nucleic Acids , Pre-Eclampsia , RNA , Cell-Free Nucleic Acids/blood , Female , Humans , Pre-Eclampsia/diagnosis , Pre-Eclampsia/genetics , Predictive Value of Tests , Pregnancy , RNA/blood , Retrospective Studies , Sensitivity and SpecificityABSTRACT
Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual human's genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individual's gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.
Subject(s)
Mutation Rate , Single-Cell Analysis , Spermatozoa/metabolism , Adult , Gene Conversion , Genome-Wide Association Study , Genomic Instability , Humans , Male , Meiosis , Microfluidic Analytical Techniques , Recombination, Genetic , Spermatozoa/cytologyABSTRACT
Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival1,2. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms3-5. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. Here we show that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. Our findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies.
Subject(s)
Aging/metabolism , Cytokines/metabolism , Drosophila melanogaster/metabolism , Intestines , Neuroglia/metabolism , Smell/physiology , Animals , Avoidance Learning , Drosophila Proteins/metabolism , Drosophila melanogaster/microbiology , Female , Inflammation/metabolism , Inflammation/microbiology , Intestines/microbiology , Janus Kinases/metabolism , Lactic Acid/metabolism , Lipid Metabolism , Neurons/metabolism , Pectobacterium carotovorum , STAT Transcription Factors/metabolism , Signal Transduction , Survival Rate , Transcription Factors/metabolismABSTRACT
The role of gene expression during learning and in short-term memories has been studied extensively1-3, but less is known about remote memories, which can persist for a lifetime4. Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex. We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning. Out of a vast plasticity-coding space, we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory. Unexpectedly, astrocytes and microglia also acquired persistent gene expression signatures that were associated with remote memory, suggesting that they actively contribute to memory circuits. The discovery of gene expression programmes associated with remote memory engrams adds an important dimension of activity-dependent cellular states to existing brain taxonomy atlases and sheds light on the elusive mechanisms of remote memory storage.
Subject(s)
Gene Expression Regulation , Memory Consolidation , Memory, Long-Term/physiology , Transcription, Genetic , Animals , Astrocytes/metabolism , Biomarkers/analysis , Exocytosis , Fear/physiology , Male , Mice , Microglia/metabolism , Neurons/classification , Neurons/cytology , Neurons/metabolism , Prefrontal Cortex/cytology , Prefrontal Cortex/physiology , Single-Cell Analysis , Time FactorsABSTRACT
Although single-cell RNA sequencing studies have begun to provide compendia of cell expression profiles1-9, it has been difficult to systematically identify and localize all molecular cell types in individual organs to create a full molecular cell atlas. Here, using droplet- and plate-based single-cell RNA sequencing of approximately 75,000 human cells across all lung tissue compartments and circulating blood, combined with a multi-pronged cell annotation approach, we create an extensive cell atlas of the human lung. We define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 out of 45 previously known cell types and 14 previously unknown ones. This comprehensive molecular atlas identifies the biochemical functions of lung cells and the transcription factors and markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signalling interactions and immune cell homing; and identifies cell types that are directly affected by lung disease genes and respiratory viruses. By comparing human and mouse data, we identified 17 molecular cell types that have been gained or lost during lung evolution and others with substantially altered expression profiles, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions and interactions are achieved in development and tissue engineering and altered in disease and evolution.
Subject(s)
Cells/classification , Cells/metabolism , Immunity , Lung/cytology , Sequence Analysis, RNA , Single-Cell Analysis , Transcriptome/genetics , Aged , Animals , Atlases as Topic , Biomarkers , Cell Communication , Cells/immunology , Chemokines/metabolism , Endothelial Cells/metabolism , Epithelial Cells/metabolism , Female , Humans , Lung/immunology , Male , Mice , Middle Aged , Receptors, Lymphocyte Homing/metabolism , Signal Transduction , Stromal Cells/metabolismABSTRACT
The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1-3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.
Subject(s)
Aging/metabolism , Aging/pathology , Blood-Brain Barrier/metabolism , Transcytosis , Alkaline Phosphatase/metabolism , Animals , Antibodies/metabolism , Biological Transport , Blood Proteins/administration & dosage , Blood Proteins/metabolism , Blood Proteins/pharmacokinetics , Brain/blood supply , Brain/metabolism , Drug Delivery Systems , Health , Humans , Male , Mice , Mice, Inbred C57BL , Plasma/metabolism , Proteome/administration & dosage , Proteome/metabolism , Proteome/pharmacokinetics , Receptors, Transferrin/immunology , Transcription, Genetic , Transferrin/metabolismABSTRACT
Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function1-these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis2-or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris3. We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.
Subject(s)
Aging/genetics , Aging/physiology , Gene Expression Regulation , Organ Specificity/genetics , Animals , Blood Proteins/analysis , Blood Proteins/genetics , Female , Immunoglobulin J-Chains/genetics , Immunoglobulin J-Chains/metabolism , Male , Mice , Plasma Cells/cytology , Plasma Cells/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA-Seq , Single-Cell Analysis , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Time Factors , TranscriptomeABSTRACT
In recent years there has been tremendous progress towards deep molecular characterization of cell types using single-cell transcriptome sequencing, creating so-called 'cell atlases'. These atlases provide a basic understanding of how different cell types of the same organism - which all share the genome - make distinct use of subsets of genes from the genome to create a variety of distinct cell types across tissues with specialized functions. In this opinion article I discuss some of the history and technological innovations that led to the development of whole-organism atlases.