The neurovascular unit is critical for brain health, and its dysfunction has been linked to Alzheimer's disease (AD). However, a cell-type-resolved understanding of how diverse vascular cells become dysfunctional and contribute to disease has been missing. Here, we applied Vessel Isolation and Nuclei Extraction for Sequencing (VINE-seq) to build a comprehensive transcriptomic atlas from 101 individuals along AD progression. Our analysis of over 842,646 parenchymal and vascular nuclei reveals that vascular dysfunction in AD is driven by transcriptional changes rather than shifts in cell proportions, with brain endothelial cells (BECs) and smooth muscle cells (SMCs) most affected. Strikingly, these molecular signatures emerge early at the mild cognitive impairment (MCI) stage, implicating vascular dysfunction early in AD pathogenesis. Stratifying by pathology reveals distinct vascular responses to β-amyloid and tau: β-amyloid burden primarily perturbs BECs and SMCs, while tau pathology predominantly impacts glial cells. We identify dysregulated angiopoietin signaling across multiple vascular cell types as a key axis, with antagonistic ANGPT2 in vascular cells and ANGPT1 in astrocytes becoming progressively dysregulated with AD. Together, this work provides a foundational resource that reveals early and pathology-specific pathways of vascular dysfunction in AD.

INTRODUCTION:Vervets are non-human primates that share high genetic homology with humans and develop amyloid beta (Aβ) pathology with aging. We expand current knowledge by examining Aβ pathology, aging, cognition, and biomarker proteomics. METHODS:Amyloid immunoreactivity in the frontal cortex and temporal cortex/hippocampal regions from archived vervet brain samples ranging from young adulthood to old age was quantified. We also obtained cognitive scores, plasma samples, and cerebrospinal fluid (CSF) samples in additional animals. Plasma and CSF proteins were quantified with platforms utilizing human antibodies. RESULTS:We found age-related increases in Aβ deposition in both brain regions. Bioinformatic analyses assessed associations between biomarkers and age, sex, cognition, and CSF Aβ levels, revealing changes in proteins related to immune-related inflammation, metabolism, and cellular processes. DISCUSSION:Vervets are an effective model of aging and early-stage Alzheimer's disease, and we provide translational biomarker data that both align with previous results in humans and provide a basis for future investigations. HIGHLIGHTS:We found changes in immune and metabolic plasma biomarkers associated with age and cognition. Cerebrospinal fluid (CSF) biomarkers revealed changes in cell signaling indicative of adaptative processes. TNFRSF19 (TROY) and Artemin co-localize with Alzheimer's disease pathology. Vervets are a relevant model for translational studies of early-stage Alzheimer's disease.

Although SARS-CoV-2 primarily targets the respiratory system, patients with and survivors of COVID-19 can suffer neurological symptoms1–3. However, an unbiased understanding of the cellular and molecular processes that are affected in the brains of patients with COVID-19 is missing. Here we profile 65,309 single-nucleus transcriptomes from 30 frontal cortex and choroid plexus samples across 14 control individuals (including 1 patient with terminal influenza) and 8 patients with COVID-19. Although our systematic analysis yields no molecular traces of SARS-CoV-2 in the brain, we observe broad cellular perturbations indicating that barrier cells of the choroid plexus sense and relay peripheral inflammation into the brain and show that peripheral T cells infiltrate the parenchyma. We discover microglia and astrocyte subpopulations associated with COVID-19 that share features with pathological cell states that have previously been reported in human neurodegenerative disease4–6. Synaptic signalling of upper-layer excitatory neurons—which are evolutionarily expanded in humans7 and linked to cognitive function8—is preferentially affected in COVID-19. Across cell types, perturbations associated with COVID-19 overlap with those found in chronic brain disorders and reside in genetic variants associated with cognition, schizophrenia and depression. Our findings and public dataset provide a molecular framework to understand current observations of COVID-19-related neurological disease, and any such disease that may emerge at a later date. Single-nucleus transcriptomes of frontal cortex and choroid plexus samples from patients with COVID-19 reveal pathological cell states that are similar to those associated with human neurodegenerative diseases and chronic brain disorders.

Neurofilament light chain (NfL) has emerged as a promising blood biomarker for the progression of various neurological diseases. NfL is a structural protein of nerve cells, and elevated NfL levels in blood are thought to mirror damage to the nervous system. We find that plasma NfL levels increase in humans with age (n=122; 21-107 years of age) and correlate with changes in other plasma proteins linked to neural pathways. In centenarians (n=135), plasma NfL levels are associated with mortality equally or better than previously described multi-item scales of cognitive or physical functioning, and this observation was replicated in an independent cohort of nonagenarians (n=180). Plasma NfL levels also increase in aging mice (n=114; 2-30 months of age), and dietary restriction, a paradigm that extends lifespan in mice, attenuates the age-related increase in plasma NfL levels. These observations suggest a contribution of nervous system functional deterioration to late-life mortality.

Loss of B lymphocyte regeneration in the bone marrow (BM) is an immunologic hallmark of advanced age, which impairs the replenishment of peripheral B-cell subsets and results in impaired humoral responses, thereby contributing to immune system dysfunction associated with aging. A better understanding of the mechanism behind this loss may suggest ways to restore immune competence and promote healthy aging. In this study, we uncover an immune-endocrine regulatory circuit that mediates cross-talk between peripheral B cells and progenitors in the BM, to balance B-cell lymphopoiesis in both human and mouse aging. We found that tumor necrosis factor α (TNF-α), which is increasingly produced by peripheral B cells during aging, stimulates the production of insulin-like growth factor-binding protein 1 (IGFBP-1), which binds and sequesters insulin-like growth factor 1 (IGF-1) in the circulation, thereby restraining its activity in promoting B-cell lymphopoiesis in the BM. Upon B-cell depletion in aging humans and mice, circulatory TNF-α decreases, resulting in increased IGF-1 and reactivation of B-cell lymphopoiesis. Perturbation of this circuit by administration of IGF-1 to old mice or anti-TNF-α antibodies to human patients restored B-cell lymphopoiesis in the BM. Thus, we suggest that in both human and mouse aging, peripheral B cells use the TNF-α/IGFBP-1/IGF-1 axis to repress B-cell lymphopoiesis. This trial was registered at www.clinicaltrials.govas#NCT00863187.

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 permeability 1 – 3 . Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins 4 , 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.

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.

Though SARS-CoV-2 primarily targets the respiratory system, it is increasingly appreciated that patients may suffer neurological symptoms of varied severity[1][1]–[3][2]. However, an unbiased understanding of the molecular processes across brain cell types that could contribute to these symptoms in COVID-19 patients is still missing. Here, we profile 47,678 droplet-based single-nucleus transcriptomes from the frontal cortex and choroid plexus across 10 non-viral, 4 COVID-19, and 1 influenza patient. We complement transcriptomic data with immunohistochemical staining for the presence of SARS-CoV-2. We find that all major cortex parenchymal and choroid plexus cell types are affected transcriptionally with COVID-19. This arises, in part, from SARS-CoV-2 infection of the cortical brain vasculature, meninges, and choroid plexus, stimulating increased inflammatory signaling into the brain. In parallel, peripheral immune cells infiltrate the brain, microglia activate programs mediating the phagocytosis of live neurons, and astrocytes dysregulate genes involved in neurotransmitter homeostasis. Among neurons, layer 2/3 excitatory neurons—evolutionarily expanded in humans[4][3]—show a specific downregulation of genes encoding major SNARE and synaptic vesicle components, predicting compromised synaptic transmission. These perturbations are not observed in terminal influenza. Many COVID-19 gene expression changes are shared with those in chronic brain disorders and reside in genetic variants associated with cognitive function, schizophrenia, and depression. Our findings and public dataset provide a molecular framework and new opportunities to understand COVID-19 related neurological disease.### Competing Interest StatementThe authors have declared no competing interest. [1]: #ref-1 [2]: #ref-3 [3]: #ref-4

Aging is the predominant risk factor for numerous chronic diseases that limit healthspan. Mechanisms of aging are thus increasingly recognized as therapeutic targets. Blood from young mice reverses aspects of aging and disease across multiple tissues, pointing to the intriguing possibility that age-related molecular changes in blood can provide novel insight into disease biology. We measured 2,925 plasma proteins from 4,331 young adults to nonagenarians and developed a novel bioinformatics approach which uncovered profound non-linear alterations in the human plasma proteome with age. Waves of changes in the proteome in the fourth, seventh, and eighth decades of life reflected distinct biological pathways, and revealed differential associations with the genome and proteome of age-related diseases and phenotypic traits. This new approach to the study of aging led to the identification of unexpected signatures and pathways of aging and disease and offers potential pathways for aging interventions.

Aging is the single greatest cause of disease and death worldwide, and so understanding the associated processes could vastly improve quality of life. While the field has identified major categories of aging damage such as altered intercellular communication, loss of proteostasis, and eroded mitochondrial function 1 , these deleterious processes interact with extraordinary complexity within and between organs. Yet, a comprehensive analysis of aging dynamics organism-wide is lacking. Here we performed RNA-sequencing of 17 organs and plasma proteomics at 10 ages across the mouse lifespan. We uncover previously unknown linear and non-linear expression shifts during aging, which cluster in strikingly consistent trajectory groups with coherent biological functions, including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Remarkably, these gene sets are expressed similarly across tissues, differing merely in age of onset and amplitude. Especially pronounced is widespread immune cell activation, detectable first in white adipose depots in middle age. Single-cell RNA-sequencing confirms the accumulation of adipose T and B cells, including immunoglobulin J-expressing plasma cells, which also accrue concurrently across diverse organs. Finally, we show how expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to aging of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of aging, thereby providing a foundation to track systemic sources of declining health at old age.

The plasma proteome provides a broad picture of the physiological state of a person and changes in the levels of individual plasma proteins find widespread use as indicators of disease. We and others previously showed that blood from young mice reverses aspects of aging and disease across multiple tissues, pointing to the intriguing possibility that age-related molecular changes in blood may reveal new insight into the biology of aging and disease. We measured relative levels of 1,305 proteins in 171 subjects from young adults to centenarians (21-107 years) using the SOMAscan aptamer technology. We discovered that 1/5 of the human plasma proteome changes with age, mostly in a non-linear fashion. We identified four sex-independent waves of aging in the fourth, sixth, seventh, and ninth decades, reflecting a complex shift of biological processes during aging. Plasma proteins associated with Alzheimer's Disease (AD) overlapped with the top proteins defining very old age but not with proteins in younger waves of aging. In addition, a conserved aging signature comprised of 26 proteins, or proteomic clock, accurately estimate age in healthy humans and mice. This clock, which can be modulated in mice by parabiosis, predicted that AD patients have an older proteomic age than their chronologic age. Altogether, our results support the concept of accelerated aging in AD and suggest that a deep understanding of the undulations of the aging plasma proteome and its interactions with diseases could lead to more targeted therapeutics.

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl (ScTyr(Y43G)) and a phenylalanyl (MmPhe(T413G)) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyr(Y43G) and MmPhe(T413G) label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyr(Y43G) and MmPhe(T413G) label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

The Tabula Muris ConsortiumWe have created a compendium of single cell transcriptome data from the model organism Mus musculus comprising more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, revealing gene expression in poorly characterized cell populations and allowing for direct and controlled comparison of gene expression in cell types shared between tissues, such as T-lymphocytes and endothelial cells from distinct anatomical locations. Two distinct technical approaches were used for most tissues: one approach, microfluidic droplet-based 3’-end counting, enabled the survey of thousands of cells at relatively low coverage, while the other, FACS-based full length transcript analysis, enabled characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.

Treatment with plasma of an early developmental stage, human umbilical cord, revitalizes the hippocampus and improves cognitive function in aged mice. Aging leads to changes in cognitive function that can lead to neurological disorders. Tony Wyss-Coray and colleagues show that human umbilical cord plasma is able to revitalize the hippocampus and improve cognitive function in aged mice. They find that tissue inhibitor of metalloproteinases 2 (TIMP2), a blood-borne factor, is enriched in human cord plasma, young mouse plasma and young mouse hippocampi. It enters the brain following systemic administration and is necessary for the cognitive benefits conferred by cord plasma. Systemic TIMP2 is also essential for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevents long-term potentiation, suggesting that TIMP2 has a role in normal hippocampal function. Ageing drives changes in neuronal and cognitive function, the decline of which is a major feature of many neurological disorders. The hippocampus, a brain region subserving roles of spatial and episodic memory and learning, is sensitive to the detrimental effects of ageing at morphological and molecular levels. With advancing age, synapses in various hippocampal subfields exhibit impaired long-term potentiation1, an electrophysiological correlate of learning and memory. At the molecular level, immediate early genes are among the synaptic plasticity genes that are both induced by long-term potentiation2,3,4 and downregulated in the aged brain5,6,7,8. In addition to revitalizing other aged tissues9,10,11,12,13, exposure to factors in young blood counteracts age-related changes in these central nervous system parameters14,15,16, although the identities of specific cognition-promoting factors or whether such activity exists in human plasma remains unknown17. We hypothesized that plasma of an early developmental stage, namely umbilical cord plasma, provides a reservoir of such plasticity-promoting proteins. Here we show that human cord plasma treatment revitalizes the hippocampus and improves cognitive function in aged mice. Tissue inhibitor of metalloproteinases 2 (TIMP2), a blood-borne factor enriched in human cord plasma, young mouse plasma, and young mouse hippocampi, appears in the brain after systemic administration and increases synaptic plasticity and hippocampal-dependent cognition in aged mice. Depletion experiments in aged mice revealed TIMP2 to be necessary for the cognitive benefits conferred by cord plasma. We find that systemic pools of TIMP2 are necessary for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevents long-term potentiation, arguing for previously unknown roles for TIMP2 in normal hippocampal function. Our findings reveal that human cord plasma contains plasticity-enhancing proteins of high translational value for targeting ageing- or disease-associated hippocampal dysfunction.

ABSTRACT Intact and broad immune cell effector functions and specific individual cytokines have been linked to HIV disease outcome, but their relative contribution to HIV control remains unclear. We asked whether the proteome of secreted cytokines and signaling factors in peripheral blood can be used to discover specific pathways critical for host viral control. A custom glass-based microarray, able to measure >600 plasma proteins involved in cell-to-cell communication, was used to measure plasma protein profiles in 96 HIV-infected, treatment-naive individuals with high (>50,000) or low (<10,000 HIV RNA copies/ml) viral loads. Univariate and regression model analysis demonstrate that plasma levels of soluble interleukin-27 (IL-27) are significantly elevated in individuals with high plasma viremia ( P < 0.0001) and are positively correlated with proviral HIV-DNA copy numbers in peripheral blood mononuclear cells (PBMC) (Rho = 0.4011; P = 0.0027). Moreover, soluble IL-27 plasma levels are negatively associated with the breadth and magnitude of the total virus-specific T-cell responses and directly with plasma levels of molecules involved in Wnt/β-catenin signaling. In addition to IL-27, gene expression levels of the specific IL-27 receptor ( IL27RA ) in PBMC correlated directly with both plasma viral load (Rho = 0.3531; P = 0.0218) and the proviral copy number in the peripheral blood as an indirect measure of partial viral reservoir (Rho = 0.4580; P = 0.0030). These results were validated in unrelated cohorts of early infected subjects as well as subjects before and after initiation of antiretroviral treatment, and they identify IL-27 and its specific receptor as a critical immune axis for the antiviral immune response and as robust correlates of viral load and proviral reservoir size in PBMC. IMPORTANCE The detailed knowledge of immune mechanisms that contribute to HIV control is a prerequisite for the design of effective treatment strategies to achieve HIV cure. Cells communicate with each other by secreting signaling proteins, and the blood is a key conduit for transporting such factors. Investigating the communication factors promoting effective immune responses and having potentially antiviral functions against HIV using a novel focused omics approach (“communicome”) has the potential to significantly improve our knowledge of effective host immunity and accelerate the HIV cure agenda. Including 140 subjects with variable viral loads and measuring the plasma levels of >600 soluble proteins, our data highlight the importance of Th17 cells and Wnt/β-catenin signaling in HIV control and especially identify the IL-27/IL-27 receptor subunit alpha (IL-27RA) axis as a predictor of plasma viral load and proviral copy number in the peripheral blood. These data may provide important guidance to therapeutic approaches in the HIV cure agenda.

The growing link between systemic environment and brain function opens the possibility that cellular communication and composition in blood are correlated with brain health. We tested this concept in frontotemporal dementia with novel, unbiased tools that measure hundreds of soluble signaling proteins or characterize the vast immune cell repertoire in blood. With these tools we discovered complementary abnormalities indicative of abnormal T cell populations and autoimmunity in frontotemporal dementia.

OBJECTIVE: To study the relationships between the APOE e4 allele, hippocampal subfield and stratal anatomy, and episodic memory. BACKGROUND: Apolipoprotein E4 confers an increased risk for late-onset Alzheimer’s disease (AD). Some prior studies suggest that APOE e4 carriers exhibit worse hippocampal atrophy, but the effects on hippocampal subfields and corresponding effects on memory are poorly defined. DESIGN/METHODS: We studied 11 patients with AD, 14 patients with amnestic mild cognitive impairment, and 14 age-matched healthy controls with no group differences in APOE e4 carrier status. Each subject underwent ultra-high field 7.0-Tesla MRI targeted to the hippocampus and neuropsychological assessment. RESULTS: We found a selective, dose-dependent association of APOE e4 with greater atrophy of the CA1 apical neuropil, or stratum radiatum / stratum lacunosum-moleculare (CA1-SRLM), a hippocampal subregion known to exhibit early vulnerability to neurofibrillary pathology in AD. The relationship between the e4 allele and CA1-SRLM atrophy persisted after controlling for dementia severity, and atrophy in other hippocampal subfields and the entorhinal cortex did not differ by APOE e4 carrier status. Carriers also exhibited worse episodic memory function but similar performance in other cognitive domains compared to noncarriers. In a statistical mediation analysis, we found support for the hypothesis that CA1-SRLM atrophy may link the APOE e4 allele to its phenotypic effects on memory. CONCLUSIONS: The APOE e4 allele segregated dose-dependently and selectively with worse CA1-SRLM atrophy and worse episodic memory performance in older subjects across a cognitive spectrum. These findings highlight a possible role for this gene in influencing a critical hippocampal subregion and an associated symptomatic manifestation. Study Supported by: NIH-NIA, American Federation of Aging Research, Alzheimer9s Association, and McKnight Endowment Fund for Neuroscience Disclosure: Dr. Kerchner has received personal compensation for activities with Phloronol, Inc. as an advisory board member. Dr. Berdnik has nothing to disclose. Dr. Shen has nothing to disclose. Dr. Bernstein has nothing to disclose. Dr. Fenesy has nothing to disclose. Dr. Deutsch has nothing to disclose. Dr. Wyss-Coray has nothing to disclose. Dr. Rutt has nothing to disclose.

Objectives: Using high-resolution structural MRI, we endeavored to study the relationships among APOE ε4, hippocampal subfield and stratal anatomy, and episodic memory. Methods: Using a cross-sectional design, we studied 11 patients with Alzheimer disease dementia, 14 patients with amnestic mild cognitive impairment, and 14 age-matched healthy controls with no group differences in APOE ε4 carrier status. Each subject underwent ultra-high-field 7.0-tesla MRI targeted to the hippocampus and neuropsychological assessment. Results: We found a selective, dose-dependent association of APOE ε4 with greater thinning of the CA1 apical neuropil, or stratum radiatum/stratum lacunosum-moleculare (CA1-SRLM), a hippocampal subregion known to exhibit early vulnerability to neurofibrillary pathology in Alzheimer disease. The relationship between the ε4 allele and CA1-SRLM thinning persisted after controlling for dementia severity, and the size of other hippocampal subfields and the entorhinal cortex did not differ by APOE ε4 carrier status. Carriers also exhibited worse episodic memory function but similar performance in other cognitive domains compared with noncarriers. In a statistical mediation analysis, we found support for the hypothesis that CA1-SRLM thinning may link the APOE ε4 allele to its phenotypic effects on memory. Conclusions: The APOE ε4 allele segregated dose-dependently and selectively with CA1-SRLM thinning and worse episodic memory performance in a pool of older subjects across a cognitive spectrum. These findings highlight a possible role for this gene in influencing a critical hippocampal subregion and an associated symptomatic manifestation.