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Grant, R. A.

Publications and source records attributed to Grant, R. A..

17 recordsLinked to original sources

ExPert infers causal perturbations from cell states across biological and clinical contexts

Pooled single-cell CRISPR screens have driven substantial effort toward models predicting the response to a perturbation. We posed the inverse question: which perturbation produced an observed cell state? Here we developed ExPert (Extracting Perturbations), which learns one prototype per perturbation in a latent space shared by 12 cellular contexts, trained on 8,964 perturbations and 19.5 million cells, and ranks 19,895 candidate genes per query, over half absent from training. ExPert more than doubled held-out-context recall over the best existing method with 1,000-fold less compute, and transferred without retraining to drugs, protein degradation and combinations. In alcohol-associated liver disease it implicated mRNA export, unchanged in expression, and we experimentally confirmed nuclear poly(A) retention and isoform switching. In primary mitochondrial disease it recovered the affected complex from blood, localized the signal to monocytes, and flagged a misdiagnosed control. Perturbation atlases can thus be queried in reverse, from state to likely driver.

systems biology↗

Apigenin Improves Healthspan and Protects Against Alzheimer's Disease-Associated Proteotoxicity in C. elegans

Apigenin is an abundant, bioactive phytochemical with reported therapeutic potential and neuroprotective effects in a variety of age-related disease models. However, the extent to which apigenin may promote healthspan and protect against neurodegeneration-associated proteotoxicity has not been thoroughly investigated, and the biological processes/mechanisms that underlie its protective effects remain unclear. To determine if apigenin increases healthspan, we performed lifespan assays in conjunction with measures of aging-relevant physiological function in wild-type C. elegans. We found that chronic exposure to apigenin extended median lifespan and improved multiple readouts of healthspan, including pharyngeal pumping, locomotor activity, and age-related macromolecular damage accumulation. Additional analyses of transgenic reporter strains and transcriptomic profiling (RNA-seq) indicated that apigenin primarily modulated redox-sensitive stress signaling and pathways associated with cellular homeostasis in aging animals. Finally, in transgenic C. elegans strains expressing the pathological Alzheimer's disease-associated proteins amyloid-beta or tau, we found that apigenin had similar beneficial effects on lifespan, healthspan, and proteotoxicity-associated functional impairments. Together, our results support apigenin as a potential healthspan-promoting intervention that acts in part through reductions in oxidative stress-associated signaling, with beneficial effects that extend to models of Alzheimer's disease-associated proteotoxicity.

neuroscience↗

Identity, ontogeny, and age-related changes in splenic white pulp macrophages in mouse and human spleen

The spleen contains diverse macrophage subsets that remove aged erythrocytes, prevent the dissemination of circulating pathogens, and shape the adaptive immune response1-3. The mouse spleen hosts red pulp macrophages (RPM), marginal zone macrophages (MZM), marginal zone metallophilic macrophages (MMM), and tingible body macrophages (TBM). However, their transcriptomic identity, ontogeny, and dynamics during aging are unknown. Furthermore, it is not known whether homologous populations of macrophages exist in the human spleen. We find that in mice, MZM and MMM are tissue-resident macrophages that maintain their population via local proliferation, while TBM are slowly replaced by circulating monocytes. Lineage tracing shows that MMM maintain the MZM pool, and that after MMM depletion, circulating monocytes restore MMM. We show that a decrease in MMM abundance in aging precedes changes in other cellular populations and splenic niches. In human spleen, we identify TBM and perifollicular zone macrophages (PFZM) as a single macrophage population homologous to MMM and MZM in mice. We show that in both mouse and human TBM become more abundant during aging. Our results suggest age-related changes in the splenic microenvironment drive changes in tissue-resident splenic macrophage populations with potential importance for the loss of immunologic function in older individuals.

immunology↗

Influenza A infection accelerates disease-associated microglia formation during physiological aging

Severe pneumonia is associated with an increased risk of cognitive decline and dementia, particularly in the elderly. Changes in microglia, the most abundant immune cell population in the brain, are also associated with cognitive decline and dementia, including the emergence of a transcriptional cell state referred to as disease-associated microglia (DAM). We sought to test the hypothesis that non-neuroinvasive influenza A virus (IAV) pneumonia results in transcriptional responses in brain microglia that drive premature expansion of DAM. Using bulk and single-cell RNA-sequencing, metabolomics, and spatial transcriptomics, we profiled neuroimmune populations in young, middle-aged, and old male mice during IAV infection and recovery. We observed an increased abundance of DAM, interferon-responsive microglia (IRM), CD4+ T cells, and CD8+ T cells in white matter regions beginning in middle age and persisting in old animals, irrespective of IAV infection. DAM exhibited a metabolic shift toward aerobic glycolysis with disrupted TCA cycling, citrulline depletion, and an elevated itaconate/-ketoglutarate ratio. Spatial transcriptomic profiling of the human middle frontal gyrus (MFG) in normal agers, SuperAgers, and patients with dementia revealed an analogous accumulation of DAM and CD8+ T cells in white matter. IAV pneumonia induced a transient immunosenescent-like response in microglia, marked by glucocorticoid-responsive gene expression and Ccnd3 upregulation. In response to IAV pneumonia, DAM expanded in middle-aged mice, whereas old mice were elevated at baseline and were largely unaffected by IAV infection. The age-related expansion of DAM was unaffected by pharmacological depletion and repopulation of microglia with a CSF1R antagonist or genetic gain or loss of function of the phagocytic receptor MERTK, suggesting the DAM phenotype is driven by the CNS microenvironment, rather than cell-intrinsic mechanisms. Our findings suggest that IAV pneumonia induces an acute immunosenescence response in microglia and accelerates the age-dependent expansion of DAM in white matter.

neuroscience↗

Tissue-resident skeletal muscle macrophages promote recovery from viral pneumonia-induced sarcopenia in normal aging.

Sarcopenia, which diminishes lifespan and healthspan in the elderly, is commonly exacerbated by viral pneumonia, including influenza and COVID-19. In a study of influenza A pneumonia in mice, young mice fully recovered from sarcopenia, while older mice did not. We identified a population of tissue-resident skeletal muscle macrophages that form a spatial niche with satellite cells and myofibers in young mice but are lost with age. Mice with a gain-of-function mutation in the Mertk receptor maintained this macrophage-myofiber interaction during aging and fully recovered from influenza-induced sarcopenia. In contrast, deletion of Mertk in macrophages or loss of Cx3cr1 disrupted this niche, preventing muscle regeneration. Heterochronic parabiosis did not restore the niche in old mice. These findings suggest that age-related loss of Mertk in muscle tissue-resident macrophages disrupts the cellular signaling necessary for muscle regeneration after viral pneumonia, offering a potential target to mitigate sarcopenia in aging.

immunology↗

A distinctive evolution of alveolar T cell responses is associated with clinical outcomes in unvaccinated patients with SARS-CoV-2 pneumonia

Pathogen clearance and resolution of inflammation in patients with pneumonia require an effective local T cell response. Nevertheless, local T cell activation may drive lung injury, particularly during prolonged episodes of respiratory failure characteristic of severe SARS-CoV-2 pneumonia. While T cell responses in the peripheral blood are well described, the evolution of T cell phenotypes and molecular signatures in the distal lung of patients with severe pneumonia caused by SARS-CoV-2 or other pathogens is understudied. Accordingly, we serially obtained 432 bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia and respiratory failure, including 74 unvaccinated patients with COVID-19, and performed flow cytometry, transcriptional, and T cell receptor profiling on sorted CD8+ and CD4+ T cell subsets. In patients with COVID-19 but not pneumonia secondary to other pathogens, we found that early and persistent enrichment in CD8+ and CD4+ T cell subsets correlated with survival to hospital discharge. Activation of interferon signaling pathways early after intubation for COVID-19 was associated with favorable outcomes, while activation of NF-{kappa}B-driven programs late in disease was associated with poor outcomes. Patients with SARS-CoV-2 pneumonia whose alveolar T cells preferentially targeted the Spike and Nucleocapsid proteins tended to experience more favorable outcomes than patients whose T cells predominantly targeted the ORF1ab polyprotein complex. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize patients who recover, yet these responses progress to NF-{kappa}B activation against non-structural proteins in patients who go on to experience poor clinical outcomes.

systems biology↗

The Immune-Evasive Proline 283 Substitution in Influenza Nucleoprotein Increases Aggregation Propensity Without Altering the Native Structure

Nucleoprotein (NP) is a key structural protein of influenza ribonucleoprotein complexes and is central to viral RNA packing and trafficking. In human cells, the interferon induced Myxovirus resistance protein 1 (MxA) binds to NP and restricts influenza replication. This selection pressure has caused NP to evolve a few critical MxA-resistant mutations, particularly the highly conserved Pro283 substitution. Previous work showed that this essential Pro283 substitution impairs influenza growth, and the fitness defect becomes particularly prominent at febrile temperature (39 {degrees}C) when host chaperones are depleted. Here, we biophysically characterize Pro283 NP and Ser283 NP to test if the fitness defect is owing to Pro283 substitution introducing folding defects. We show that the Pro283 substitution changes the folding pathway of NP without altering the native structure, making NP more aggregation prone during folding. These findings suggest that influenza has evolved to hijack host chaperones to promote the folding of otherwise biophysically incompetent viral proteins that enable innate immune system escape. TeaserPro283 substitution in flu nucleoprotein introduces folding defects, and makes influenza uniquely dependent on host chaperones.

biophysics↗

Expansion of profibrotic monocyte-derived alveolar macrophages in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19

As many as 10-30% of the over 760 million survivors of COVID-19 develop persistent symptoms, of which respiratory symptoms are among the most common. To understand the cellular and molecular basis for respiratory PASC, we combined a machine learning based analysis of lung computed tomography (CT) with flow cytometry, single-cell RNA-sequencing analysis of bronchoalveolar lavage fluid and nasal curettage samples, and alveolar cytokine profiling in a cohort of thirty-five patients with respiratory symptoms and radiographic abnormalities more than 90 days after infection with COVID-19. CT images from patients with PASC revealed abnormalities involving 73% of the lung, which improved on subsequent imaging. Interstitial abnormalities suggestive of fibrosis on CT were associated with the increased numbers of neutrophils and presence of profibrotic monocyte-derived alveolar macrophages in BAL fluid, reflecting unresolved epithelial injury. Persistent infection with SARS-CoV-2 was identified in six patients and secondary bacterial or viral infections in two others. These findings suggest that despite its heterogenous clinical presentations, respiratory PASC with radiographic abnormalities results from a common pathobiology characterized by the ongoing recruitment of neutrophils and profibrotic monocyte-derived alveolar macrophages driving lung fibrosis with implications for diagnosis and therapy.

immunology↗

Prolonged exposure to lung-derived cytokines is associated with inflammatory activation of microglia in patients with COVID-19

Neurological impairment is the most common finding in patients with post-acute sequelae of COVID-19. Furthermore, survivors of pneumonia from any cause have an elevated risk of dementia1-4. Dysfunction in microglia, the primary immune cell in the brain, has been linked to cognitive impairment in murine models of dementia and in humans5. Here, we report a transcriptional response in human microglia collected from patients who died following COVID-19 suggestive of their activation by TNF-[a] and other circulating pro-inflammatory cytokines. Consistent with these findings, the levels of 55 alveolar and plasma cytokines were elevated in a cohort of 341 patients with respiratory failure, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. While peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, cumulative cytokine exposure was higher in patients with COVID-19. Corticosteroid treatment, which has been shown to be beneficial in patients with COVID-196, was associated with lower levels of CXCL10, CCL8, and CCL2--molecules that sustain inflammatory circuits between alveolar macrophages harboring SARS-CoV-2 and activated T cells7. These findings suggest that corticosteroids may break this cycle and decrease systemic exposure to lung-derived cytokines and inflammatory activation of microglia in patients with COVID-19.

immunology↗

Nuclear receptor signaling via NHR-49/MDT-15 regulates stress resilience and proteostasis in response to reproductive and metabolic cues

The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience in C. elegans. We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, a functional homolog of mammalian peroxisome proliferator-activated receptor alpha (PPAR), regulates stress resilience and proteostasis downstream of embryo integrity and other pathways that influence lipid homeostasis, and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing inter-tissue pathway in somatic tissues, also triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49 together with its co-activator MDT-15 contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer as well as by other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting. Further, we show that increased NHR-49 activity is sufficient to suppress polyglutamine aggregation and improve stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.

genetics↗

Discovery of diverse human BH3-only and non-native peptide binders of pro-apoptotic BAK indicate that activators and inhibitors use asimilar binding mode and are not distinguished by binding affinity or kinetics

Apoptosis is a programmed form of cell death important for the development and maintenance of tissue homeostasis. The BCL-2 protein family controls key steps in apoptosis, dysregulation of which can lead to a wide range of human diseases. BCL-2 proteins comprise three groups: anti-apoptotic proteins, pro-apoptotic proteins, and BH3-only proteins. BAK is one of two pro-apoptotic proteins, and previous work has shown that binding of certain BH3-only proteins such as truncated BID (tBID), BIM, or PUMA to BAK leads to mitochondrial outer membrane permeabilization, the release of cytochrome c, and ultimately cell death. This process, referred to as activation, involves the BH3-stimulated conversion of BAK from monomer to dimer and then to oligomers that promote membrane disruption. Crystal structures of putative intermediates in this pathway, crosslinking data, and in vitro functional tests have provided insights into the activation event, yet the sequence-function relationships that make some but not all BH3-only proteins function as activators remain largely unexamined. In this work, we used computational protein design, yeast surface-display screening of candidate BH3-like peptides, and structure-based energy scoring to identify ten new binders of BAK that span a large sequence space. Among the new binders are two peptides from human proteins BNIP5 and PXT1 that promote BAK activation in liposome assays and induce cytochrome-c release from mitochondria, expanding current views of how BAK-mediated cell death may be triggered in cells. High-resolution crystal structures and binding experiments revealed a high degree of similarity in binding geometry, affinity, and association kinetics between peptide activators and inhibitors, including peptides described previously and those identified in this work. We propose a model for BAK activation that is based on differential engagement of BAK monomers vs. the BAK activation transition state that integrates our observations with previous reports of BAK binders, activators, and inhibitors.

biochemistry↗

A distributed residue network permits conformational binding specificity in a conserved family of actin remodelers

Metazoan proteomes contain many paralogous proteins that have evolved distinct functions. The Ena/VASP family of actin regulators consists of three members that share an EVH1 interaction domain with a 100% conserved binding site. A proteome-wide screen revealed ciliary protein PCARE as a high-affinity ligand for ENAH EVH1. Here we report the surprising observation that PCARE is ~100-fold specific for ENAH over paralogs VASP and EVL and can selectively bind and inhibit ENAH-dependent adhesion in cells. Specificity arises from a mechanism whereby PCARE stabilizes a conformation of the ENAH EVH1 domain that is inaccessible to family members VASP and EVL. Structure-based modeling rapidly identified seven residues distributed throughout EVL that are sufficient to differentiate binding by ENAH vs. EVL. By exploiting the ENAH-specific conformation, we rationally designed the tightest and most selective ENAH binder to date. Our work uncovers a conformational mechanism of interaction specificity that distinguishes highly similar paralogs and establishes tools for dissecting specific Ena/VASP functions in processes including cancer cell invasion.

biochemistry↗

A proteome-wide screen uncovers diverse roles for sequence context surrounding proline-rich motifs in Ena/VASP molecular recognition

The human proteome is replete with short linear motifs (SLiMs) of 4-6 residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to systematically examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of ENAH, an actin regulator that is upregulated in invasive cancers, we screened 36-residue proteome-derived peptides for binding. We discovered a pocket on the ENAH EVH1 domain that diverged from its orthologs to recognize extended SLiMs, and we found that proteins with two EVH1-binding SLiMs can wrap around a single domain. We also found that the ciliary protein PCARE uses an extended 23-residue region to obtain higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of broader proteomic context on motif-mediated interactions, revealing diverse mechanisms of contextual control over EVH1 interactions and establishing that SLiMs cant be fully understood outside of their native context.

biochemistry↗

Alveolitis in severe SARS-CoV-2 pneumonia is driven by self-sustaining circuits between infected alveolar macrophages and T cells

Some patients infected with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) develop severe pneumonia and the acute respiratory distress syndrome (ARDS) [1]. Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from other types of pneumonia [2]. We collected bronchoalveolar lavage fluid samples from 86 patients with SARS-CoV-2-induced respiratory failure and 252 patients with known or suspected pneumonia from other pathogens and subjected them to flow cytometry and bulk transcriptomic profiling. We performed single cell RNA-Seq in 5 bronchoalveolar lavage fluid samples collected from patients with severe COVID-19 within 48 hours of intubation. In the majority of patients with SARS-CoV-2 infection at the onset of mechanical ventilation, the alveolar space is persistently enriched in alveolar macrophages and T cells without neutrophilia. Bulk and single cell transcriptomic profiling suggest SARS-CoV-2 infects alveolar macrophages that respond by recruiting T cells. These T cells release interferon-gamma to induce inflammatory cytokine release from alveolar macrophages and further promote T cell recruitment. Our results suggest SARS-CoV-2 causes a slowly unfolding, spatially-limited alveolitis in which alveolar macrophages harboring SARS-CoV-2 transcripts and T cells form a positive feedback loop that drives progressive alveolar inflammation. This manuscript is accompanied by an online resource: https://www.nupulmonary.org/covid-19/ One sentence summarySARS-CoV-2-infected alveolar macrophages form positive feedback loops with T cells in patients with severe COVID-19.

immunology↗

Resetting proteostasis with ISRIB prevents pulmonary fibrosis

Aging is among the most important risk factors for the development of pulmonary fibrosis. We found that a small molecule that specifically inhibits translational inhibition induced by activation of the integrated stress response (ISRIB) attenuated the severity of pulmonary fibrosis in young and old mice. The more severe fibrosis in old compared to young mice was associated with increased recruitment of pathogenic monocyte-derived alveolar macrophages. Using genetic lineage tracing and transcriptomic profiling we found that ISRIB modulates stress response signaling in alveolar epithelial cells resulting in decreased apoptosis and decreased recruitment of pathogenic monocyte-derived alveolar macrophages. These data support multicellular model of fibrosis involving epithelial cells, pathogenic monocyte-derived alveolar macrophages and fibroblasts. Inhibition of the integrated stress response in the aging lung epithelium ameliorates pulmonary fibrosis by preventing the prolonged recruitment of monocyte-derived alveolar macrophages.

immunology↗

Proteostasis and Energetics as Proteome Hallmarks of Aging and Influenza Challenge in Pulmonary Disease

Aging is associated with an increased risk for the development of many diseases. This is exemplified by the increased incidence of lung injury, muscle dysfunction and cognitive impairment in the elderly following influenza infection. Because the infectious cycle of flu is dependent upon the properties of the host, we examined the proteome of alveolar macrophages (AM) and type 2 cells (AT2) obtained from young (3 months) and old (18 months) naive mice and mice exposed to influenza A. Our proteomics data show that there is a maladaptive collapse of the proteostasis network (PN) and changes in mitochondrial pathways in the aged naive AM and AT2 proteomes. The mitochondrial imbalance and proteostatic collapse seen in aged cells places an excessive folding burden on these cells, which is further exacerbated following exposure to influenza A. Specifically, we see an imbalance in Hsp70 co-chaperones involved in protein folding and Hsp90 co-chaperones important for stress signaling pathways that are essential for cellular protection during aging. The acute challenge of influenza A infection of young and aged AM and AT2 cells reveals that age-associated changes in the chaperome affect the ability of these cells to properly manage the infection and post-infection biology, contributing to cytotoxicity. We posit that proteomic profiling of individual cell type specific responses provides a high impact approach to pinpoint fundamental molecular relationships that may contribute to the susceptibility to aging and environmental stress, providing a platform to identify new targets for therapeutic intervention to improve resiliency in the elderly.

cell biology↗

Aging is associated with a systemic length-driven transcriptome imbalance

Aging manifests itself through a decline in organismal homeostasis and a multitude of cellular and physiological functions1. Efforts to identify a common basis for vertebrate aging face many challenges; for example, while there have been documented changes in the expression of many hundreds of mRNAs, the results across tissues and species have been inconsistent2. We therefore analyzed age-resolved transcriptomic data from 17 mouse organs and 51 human organs using unsupervised machine learning3-5 to identify the architectural and regulatory characteristics most informative on the differential expression of genes with age. We report a hitherto unknown phenomenon, a systemic age-dependent length-driven transcriptome imbalance that for older organisms disrupts the homeostatic balance between short and long transcript molecules for mice, rats, killifishes, and humans. We also demonstrate that in a mouse model of healthy aging, length-driven transcriptome imbalance correlates with changes in expression of splicing factor proline and glutamine rich (Sfpq), which regulates transcriptional elongation according to gene length6. Furthermore, we demonstrate that length-driven transcriptome imbalance can be triggered by environmental hazards and pathogens. Our findings reinforce the picture of aging as a systemic homeostasis breakdown and suggest a promising explanation for why diverse insults affect multiple age-dependent phenotypes in a similar manner.

systems biology↗