Fokker-Planck diffusion maps of multiple single cell microglial transcriptomes reveals radial differentiation into substates associated with Alzheimer's pathology.

The identification of microglia subtypes is important for understanding the role of innate immunity in neurodegenerative diseases. Current methods of unsupervised cell type identification assume a small noise-to-signal ratio of transcriptome measurements that would produce well-separated cell clusters. However, identification of subtypes is obscured by gene expression noise, diminishing the distances in transcriptome space between distinct cell types and blurring boundaries. Here we use Fokker-Planck (FP) diffusion maps to model cellular differentiation as a stochastic process whereby cells settle into local minima, corresponding to cell subtypes, in a potential landscape constructed from transcriptome data using a nearest neighbor graph approach. By applying critical transition fields, we identify individual cells on the verge of transitioning between subtypes, revealing microglial cells in inactivated, homeostatic state before radially transitioning into various specialized subtypes. Specifically, we show that cells from Alzheimer's disease patients are enriched in a microglia subtype associated to antigen presentation and T-cell recruitment.

Single cell transcriptomes and multiscale networks from persons with and without Alzheimer's disease.

The emergence of single nucleus RNA sequencing (snRNA-seq) offers to revolutionize the study of Alzheimer's disease (AD). Integration with complementary multiomics data such as genetics, proteomics and clinical data provides powerful opportunities to link cell subpopulations and molecular networks with a broader disease-relevant context. We report snRNA-seq profiles from superior frontal gyrus samples from 101 well characterized subjects from the Banner Brain and Body Donation Program in combination with whole genome sequences. We report findings that link common AD risk variants with CR1 expression in oligodendrocytes as well as alterations in hematological parameters. We observed an AD-associated CD83(+) microglial subtype with unique molecular networks and which is associated with immunoglobulin IgG4 production in the transverse colon. Our major observations were replicated in two additional, independent snRNA-seq data sets. These findings illustrate the power of multi-tissue molecular profiling to contextualize snRNA-seq brain transcriptomics and reveal disease biology.

A genome scale transcriptional regulatory model of the human placenta.

Gene regulation is essential to placental function and fetal development. We built a genome-scale transcriptional regulatory network (TRN) of the human placenta using digital genomic footprinting and transcriptomic data. We integrated 475 transcriptomes and 12 DNase hypersensitivity datasets from placental samples to globally and quantitatively map transcription factor (TF)-target gene interactions. In an independent dataset, the TRN model predicted target gene expression with an out-of-sample R2 greater than 0.25 for 73% of target genes. We performed siRNA knockdowns of four TFs and achieved concordance between the predicted gene targets in our TRN and differences in expression of knockdowns with an accuracy of >0.7 for three of the four TFs. Our final model contained 113,158 interactions across 391 TFs and 7712 target genes and is publicly available. We identified 29 TFs which were significantly enriched as regulators for genes previously associated with preterm birth, and eight of these TFs were decreased in preterm placentas.

Novel TCF4:TCF12 heterodimer inhibits glioblastoma growth.

TWIST1 (TW) is a pro-oncogenic basic helix-loop-helix (bHLH) transcription factor and promotes the hallmark features of malignancy (e.g., cell invasion, cancer cell stemness, and treatment resistance), which contribute to poor prognoses of glioblastoma (GBM). We previously reported that specific TW dimerization motifs regulate unique cellular phenotypes in GBM. For example, the TW:E12 heterodimer increases periostin (POSTN) expression and promotes cell invasion. TW dimer-specific transcriptional regulation requires binding to the regulatory E-box consensus sequences, but alternative bHLH dimers that balance TW dimer activity in regulating pro-oncogenic TW target genes are unknown. We leveraged the ENCODE DNase I hypersensitivity data to identify E-box sites and tethered TW:E12 and TW:TW proteins to validate dimer binding to E-boxes in vitro. Subsequently, TW knockdown revealed a novel TCF4:TCF12 bHLH dimer occupying the same TW E-box site that, when expressed as a tethered TCF4:TCF12 dimer, markedly repressed POSTN expression and extended animal survival. These observations support TCF4:TCF12 as a novel dimer with tumor-suppressor activity in GBM that functions in part through displacement of and/or competitive inhibition of pro-oncogenic TW dimers at E-box sites.

Aß Amyloid Scaffolds the Accumulation of Matrisome and Additional Proteins in Alzheimer's Disease.

We report a highly significant correlation in brain proteome changes between Alzheimers disease (AD) and CRND8 APP695NL/F transgenic mice. However, integrating protein changes observed in the CRND8 mice with co-expression networks derived from human AD, reveals both conserved and divergent module changes. For the most highly conserved module (M42, matrisome) we find many proteins accumulate in plaques, cerebrovascular amyloid (CAA), dystrophic processes, or a combination thereof. Overexpression of two M42 proteins, midkine (Mdk) and pleiotrophin (PTN), in CRND8 mice brains leads to increased accumulation of A ß ; in plaques and in CAA; further, recombinant MDK and PTN enhance A ß ; aggregation into amyloid. Multiple M42 proteins, annotated as heparan sulfate binding proteins, bind to fibrillar A ß 42 and a non-human amyloid fibril in vitro. Supporting this binding data, MDK and PTN co-accumulate with transthyretin (TTR) amyloid in the heart and islet amyloid polypeptide (IAPP) amyloid in the pancreas. Our findings establish several critical insights. Proteomic changes in modules observed in human AD brains define an A ß ; amyloid responsome that is well conserved from mouse model to human. Further, distinct amyloid structures may serve as scaffolds, facilitating the co-accumulation of proteins with signaling functions. We hypothesize that this co-accumulation may contribute to downstream pathological sequalae. Overall, this contextualized understanding of proteomic changes and their interplay with amyloid deposition provides valuable insights into the complexity of AD pathogenesis and potential biomarkers and therapeutic targets.

A public resource of single cell transcriptomes and multiscale networks from persons with and without Alzheimer's disease.

The emergence of technologies that can support high-throughput profiling of single cell transcriptomes offers to revolutionize the study of brain tissue from persons with and without Alzheimer's disease (AD). Integration of these data with additional complementary multiomics data such as genetics, proteomics and clinical data provides powerful opportunities to link observed cell subpopulations and molecular network features within a broader disease-relevant context. We report here single nucleus RNA sequencing (snRNA-seq) profiles generated from superior frontal gyrus cortical tissue samples from 101 exceptionally well characterized, aged subjects from the Banner Brain and Body Donation Program in combination with whole genome sequences. We report findings that link common AD risk variants with CR1 expression in oligodendrocytes as well as alterations in peripheral hematological lab parameters, with these observations replicated in an independent, prospective cohort study of ageing and dementia. We also observed an AD-associated CD83(+) microglial subtype with unique molecular networks that encompass many known regulators of AD-relevant microglial biology, and which are associated with immunoglobulin IgG4 production in the transverse colon. These findings illustrate the power of multi-tissue molecular profiling to contextualize snRNA-seq brain transcriptomics and reveal novel disease biology. The transcriptomic, genetic, phenotypic, and network data resources described within this study are available for access and utilization by the scientific community.

Correction: Reproducible big data science: A case study in continuous FAIRness.

[This corrects the article DOI: 10.1371/journal.pone.0213013.].

The neuronal chromatin landscape in adult schizophrenia brains is linked to early fetal development.

Non-coding variants increase risk of neuropsychiatric disease. However, our understanding of the cell-type specific role of the non-coding genome in disease is incomplete. We performed population scale (N=1,393) chromatin accessibility profiling of neurons and non-neurons from two neocortical brain regions: the anterior cingulate cortex and dorsolateral prefrontal cortex. Across both regions, we observed notable differences in neuronal chromatin accessibility between schizophrenia cases and controls. A per-sample disease pseudotime was positively associated with genetic liability for schizophrenia. Organizing chromatin into cis- and trans-regulatory domains, identified a prominent neuronal trans-regulatory domain (TRD1) active in immature glutamatergic neurons during fetal development. Polygenic risk score analysis using genetic variants within chromatin accessibility of TRD1 successfully predicted susceptibility to schizophrenia in the Million Veteran Program cohort. Overall, we present the most extensive resource to date of chromatin accessibility in the human cortex, yielding insights into the cell-type specific etiology of schizophrenia.

The neuronal chromatin landscape in adult schizophrenia brains is linked to early fetal development.

Non-coding variants increase risk of neuropsychiatric disease. However, our understanding of the cell-type specific role of the non-coding genome in disease is incomplete. We performed population scale (N=1,393) chromatin accessibility profiling of neurons and non-neurons from two neocortical brain regions: the anterior cingulate cortex and dorsolateral prefrontal cortex. Across both regions, we observed notable differences in neuronal chromatin accessibility between schizophrenia cases and controls. A per-sample disease pseudotime was positively associated with genetic liability for schizophrenia. Organizing chromatin into cis- and trans-regulatory domains, identified a prominent neuronal trans-regulatory domain (TRD1) active in immature glutamatergic neurons during fetal development. Polygenic risk score analysis using genetic variants within chromatin accessibility of TRD1 successfully predicted susceptibility to schizophrenia in the Million Veteran Program cohort. Overall, we present the most extensive resource to date of chromatin accessibility in the human cortex, yielding insights into the cell-type specific etiology of schizophrenia.

A metabolomic signature of the APOE2 allele.

With the goal of identifying metabolites that significantly correlate with the protective e2 allele of the apolipoprotein E (APOE) gene, we established a consortium of five studies of healthy aging and extreme human longevity with 3545 participants. This consortium includes the New England Centenarian Study, the Baltimore Longitudinal Study of Aging, the Arivale study, the Longevity Genes Project/LonGenity studies, and the Long Life Family Study. We analyzed the association between APOE genotype groups E2 (e2e2 and e2e3 genotypes, N = 544), E3 (e3e3 genotypes, N = 2299), and E4 (e3e4 and e4e4 genotypes, N = 702) with metabolite profiles in the five studies and used fixed effect meta-analysis to aggregate the results. Our meta-analysis identified a signature of 19 metabolites that are significantly associated with the E2 genotype group at FDR < 10%. The group includes 10 glycerolipids and 4 glycerophospholipids that were all higher in E2 carriers compared to E3, with fold change ranging from 1.08 to 1.25. The organic acid 6-hydroxyindole sulfate, previously linked to changes in gut microbiome that were reflective of healthy aging and longevity, was also higher in E2 carriers compared to E3 carriers. Three sterol lipids and one sphingolipid species were significantly lower in carriers of the E2 genotype group. For some of these metabolites, the effect of the E2 genotype opposed the age effect. No metabolites reached a statistically significant association with the E4 group. This work confirms and expands previous results connecting the APOE gene to lipid regulation and suggests new links between the e2 allele, lipid metabolism, aging, and the gut-brain axis.

Multi-Omic analyses characterize the ceramide/sphingomyelin pathway as a therapeutic target in Alzheimer's disease.

Dysregulation of sphingomyelin and ceramide metabolism have been implicated in Alzheimer's disease. Genome-wide and transcriptome-wide association studies have identified various genes and genetic variants in lipid metabolism that are associated with Alzheimer's disease. However, the molecular mechanisms of sphingomyelin and ceramide disruption remain to be determined. We focus on the sphingolipid pathway and carry out multi-omics analyses to identify central and peripheral metabolic changes in Alzheimer's patients, correlating them to imaging features. Our multi-omics approach is based on (a) 2114 human post-mortem brain transcriptomics to identify differentially expressed genes; (b) in silico metabolic flux analysis on context-specific metabolic networks identified differential reaction fluxes; (c) multimodal neuroimaging analysis on 1576 participants to associate genetic variants in sphingomyelin pathway with Alzheimer's disease pathogenesis; (d) plasma metabolomic and lipidomic analysis to identify associations of lipid species with dysregulation in Alzheimer's; and (e) metabolite genome-wide association studies to define receptors within the pathway as a potential drug target. We validate our hypothesis in amyloidogenic APP/PS1 mice and show prolonged exposure to fingolimod alleviated synaptic plasticity and cognitive impairment in mice. Our integrative multi-omics approach identifies potential targets in the sphingomyelin pathway and suggests modulators of S1P metabolism as possible candidates for Alzheimer's disease treatment.

The Coaching for Cognition in Alzheimer's (COCOA) trial: Study design.

Comprehensive treatment of Alzheimer's disease (AD) requires not only pharmacologic treatment but also management of existing medical conditions and lifestyle modifications including diet, cognitive training, and exercise. We present the design and methodology for the Coaching for Cognition in Alzheimer's (COCOA) trial. AD and other dementias result from the interplay of multiple interacting dysfunctional biological systems. Monotherapies have had limited success. More interventional studies are needed to test the effectiveness of multimodal multi-domain therapies for dementia prevention and treatment. Multimodal therapies use multiple interventions to address multiple systemic causes and potentiators of cognitive decline and functional loss; they can be personalized, as different sets of etiologies and systems responsive to therapy may be present in different individuals. COCOA is designed to test the hypothesis that coached multimodal interventions beneficially alter the trajectory of cognitive decline for individuals on the spectrum of AD and related dementias (ADRD). COCOA is a two-arm prospective randomized controlled trial (RCT). COCOA collects psychometric, clinical, lifestyle, genomic, proteomic, metabolomic, and microbiome data at multiple timepoints across 2 years for each participant. These data enable systems biology analyses. One arm receives standard of care and generic healthy aging recommendations. The other arm receives standard of care and personalized data-driven remote coaching. The primary outcome measure is the Memory Performance Index (MPI), a measure of cognition. The MPI is a summary statistic of the MCI Screen (MCIS). Secondary outcome measures include the Functional Assessment Staging Test (FAST), a measure of function. COCOA began enrollment in January 2018. We hypothesize that multimodal interventions will ameliorate cognitive decline and that data-driven health coaching will increase compliance, assist in personalizing multimodal interventions, and improve outcomes for patients, particularly for those in the early stages of the AD spectrum. Highlights: The Coaching for Cognition in Alzheimer's (COCOA) trial tests personalized multimodal lifestyle interventions for Alzheimer's disease and related dementias.Dense longitudinal molecular data will be useful for future studies.Increased use of Hill's criteria in analyses may advance knowledge generation.Remote coaching may be an effective intervention.Because lifestyle interventions are inexpensive, they may be particularly valuable in reducing global socioeconomic disparities in dementia care.

Deletion of Abi3/Gngt2 influences age-progressive amyloid ß and tau pathologies in distinctive ways.

BACKGROUND: The S209F variant of Abelson Interactor Protein 3 (ABI3) increases risk for Alzheimer's disease (AD), but little is known about its function in relation to AD pathogenesis. METHODS: Here, we use a mouse model that is deficient in Abi3 locus to study how the loss of function of Abi3 impacts two cardinal neuropathological hallmarks of AD-amyloid ß plaques and tau pathology. Our study employs extensive neuropathological and transcriptomic characterization using transgenic mouse models and adeno-associated virus-mediated gene targeting strategies. RESULTS: Analysis of bulk RNAseq data confirmed age-progressive increase in Abi3 levels in rodent models of AD-type amyloidosis and upregulation in AD patients relative to healthy controls. Using RNAscope in situ hybridization, we localized the cellular distribution of Abi3 in mouse and human brains, finding that Abi3 is expressed in both microglial and non-microglial cells. Next, we evaluated Abi3-/- mice and document that both Abi3 and its overlapping gene, Gngt2, are disrupted in these mice. Using multiple transcriptomic datasets, we show that expression of Abi3 and Gngt2 are tightly correlated in rodent models of AD and human brains, suggesting a tight co-expression relationship. RNAseq of the Abi3-Gngt2-/- mice revealed upregulation of Trem2, Plcg2, and Tyrobp, concomitant with induction of an AD-associated neurodegenerative signature, even in the absence of AD-typical neuropathology. In APP mice, loss of Abi3-Gngt2 resulted in a gene dose- and age-dependent reduction in Aß deposition. Additionally, in Abi3-Gngt2-/- mice, expression of a pro-aggregant form of human tau exacerbated tauopathy and astrocytosis. Further, using in vitro culture assays, we show that the AD-associated S209F mutation alters the extent of ABI3 phosphorylation. CONCLUSIONS: These data provide an important experimental framework for understanding the role of Abi3-Gngt2 function and early inflammatory gliosis in AD. Our studies also demonstrate that inflammatory gliosis could have opposing effects on amyloid and tau pathology, highlighting the unpredictability of targeting immune pathways in AD.

Risk factors for severe COVID-19 differ by age for hospitalized adults.

Risk stratification for hospitalized adults with COVID-19 is essential to inform decisions about individual patients and allocation of resources. So far, risk models for severe COVID outcomes have included age but have not been optimized to best serve the needs of either older or younger adults. Models also need to be updated to reflect improvements in COVID-19 treatments. This retrospective study analyzed data from 6906 hospitalized adults with COVID-19 from a community health system across five states in the western United States. Risk models were developed to predict mechanical ventilation illness or death across one to 56 days of hospitalization, using clinical data available within the first hour after either admission with COVID-19 or a first positive SARS-CoV-2 test. For the seven-day interval, models for age ≥ 18 and < 50 years reached AUROC 0.81 (95% CI 0.71-0.91) and models for age ≥ 50 years reached AUROC 0.82 (95% CI 0.77-0.86). Models revealed differences in the statistical significance and relative predictive value of risk factors between older and younger patients including age, BMI, vital signs, and laboratory results. In addition, for hospitalized patients, sex and chronic comorbidities had lower predictive value than vital signs and laboratory results.

Manifestations of Alzheimer's disease genetic risk in the blood are evident in a multiomic analysis in healthy adults aged 18 to 90.

Genetics play an important role in late-onset Alzheimer's Disease (AD) etiology and dozens of genetic variants have been implicated in AD risk through large-scale GWAS meta-analyses. However, the precise mechanistic effects of most of these variants have yet to be determined. Deeply phenotyped cohort data can reveal physiological changes associated with genetic risk for AD across an age spectrum that may provide clues to the biology of the disease. We utilized over 2000 high-quality quantitative measurements obtained from blood of 2831 cognitively normal adult clients of a consumer-based scientific wellness company, each with CLIA-certified whole-genome sequencing data. Measurements included: clinical laboratory blood tests, targeted chip-based proteomics, and metabolomics. We performed a phenome-wide association study utilizing this diverse blood marker data and 25 known AD genetic variants and an AD-specific polygenic risk score (PGRS), adjusting for sex, age, vendor (for clinical labs), and the first four genetic principal components; sex-SNP interactions were also assessed. We observed statistically significant SNP-analyte associations for five genetic variants after correction for multiple testing (for SNPs in or near NYAP1, ABCA7, INPP5D, and APOE), with effects detectable from early adulthood. The ABCA7 SNP and the APOE2 and APOE4 encoding alleles were associated with lipid variability, as seen in previous studies; in addition, six novel proteins were associated with the e2 allele. The most statistically significant finding was between the NYAP1 variant and PILRA and PILRB protein levels, supporting previous functional genomic studies in the identification of a putative causal variant within the PILRA gene. We did not observe associations between the PGRS and any analyte. Sex modified the effects of four genetic variants, with multiple interrelated immune-modulating effects associated with the PICALM variant. In post-hoc analysis, sex-stratified GWAS results from an independent AD case-control meta-analysis supported sex-specific disease effects of the PICALM variant, highlighting the importance of sex as a biological variable. Known AD genetic variation influenced lipid metabolism and immune response systems in a population of non-AD individuals, with associations observed from early adulthood onward. Further research is needed to determine whether and how these effects are implicated in early-stage biological pathways to AD. These analyses aim to complement ongoing work on the functional interpretation of AD-associated genetic variants.

Alzheimer's disease and progressive supranuclear palsy share similar transcriptomic changes in distinct brain regions.

Vast numbers of differentially expressed genes and perturbed networks have been identified in Alzheimer's disease (AD), however, neither disease nor brain region specificity of these transcriptome alterations has been explored. Using RNA-Seq data from 231 temporal cortex and 224 cerebellum samples from patients with AD and progressive supranuclear palsy (PSP), a tauopathy, we identified a striking correlation in the directionality and magnitude of gene expression changes between these 2 neurodegenerative proteinopathies. Further, the transcriptomic changes in AD and PSP brains ware highly conserved between the temporal and cerebellar cortices, indicating that highly similar transcriptional changes occur in pathologically affected and grossly less affected, albeit functionally connected, areas of the brain. Shared up- or downregulated genes in AD and PSP are enriched in biological pathways. Many of these genes also have concordant protein changes and evidence of epigenetic control. These conserved transcriptomic alterations of 2 distinct proteinopathies in brain regions with and without significant gross neuropathology have broad implications. AD and other neurodegenerative diseases are likely characterized by common disease or compensatory pathways with widespread perturbations in the whole brain. These findings can be leveraged to develop multifaceted therapies and biomarkers that address these common, complex, and ubiquitous molecular alterations in neurodegenerative diseases.

Systems modeling of metabolic dysregulation in neurodegenerative diseases.

Neurodegenerative diseases (NDDs) encompass a wide range of conditions that arise owing to progressive degeneration and the ultimate loss of nerve cells in the brain and peripheral nervous system. NDDs such as Alzheimer's, Parkinson's, and Huntington's diseases negatively impact both length and quality of life, due to lack of effective disease-modifying treatments. Herein, we review the use of genome-scale metabolic models, network-based approaches, and integration with multiomics data to identify key biological processes that characterize NDDs. We describe powerful systems biology approaches for modeling NDD pathophysiology by leveraging in silico models that are informed by patient-derived multiomics data. These approaches can enable mechanistic insights into NDD-specific metabolic dysregulations that can be leveraged to identify potential metabolic markers of disease and predisease states.

A systems-biology clinical trial of a personalized multimodal lifestyle intervention for early Alzheimer's disease.

INTRODUCTION: There is an urgent need to develop effective interventional treatments for people with Alzheimer's disease (AD). AD results from a complex multi-decade interplay of multiple interacting dysfunctional biological systems that have not yet been fully elucidated. Epidemiological studies have linked several modifiable lifestyle factors with increased incidence for AD. Because monotherapies have failed to prevent or ameliorate AD, interventional studies should deploy multiple, targeted interventions that address the dysfunctional systems that give rise to AD. METHODS: This randomized controlled trial (RCT) will examine the efficacy of a 12-month personalized, multimodal, lifestyle intervention in 60 mild cognitive impairment (MCI) and early stage AD patients (aged 50+, amyloid positivity). Both groups receive data-driven, lifestyle recommendations designed to target multiple systemic pathways implicated in AD. One group receives these personalized recommendations without coaching. The other group receives personalized recommendations with health coaching, dietary counseling, exercise training, cognitive stimulation, and nutritional supplements. We collect clinical, proteomic, metabolomic, neuroimaging, and genetic data to fuel systems-biology analyses. We will examine effects on cognition and hippocampal volume. The overarching goal of the study is to longitudinally track biological systems implicated in AD to reveal the dynamics between these systems during the intervention to understand differences in treatment response. RESULTS: We have developed and implemented a protocol for a personalized, multimodal intervention program for early AD patients. We began enrollment in September 2019; we have enrolled a third of our target (20 of 60) with a 95% retention and 86% compliance rate. DISCUSSION: This study presents a paradigm shift in designing multimodal, lifestyle interventions to reduce cognitive decline, and how to elucidate the biological systems being targeted. Analytical efforts to explain mechanistic or causal underpinnings of individual trajectories and the interplay between multi-omic variables will inform the design of future hypotheses and development of effective precision medicine trials.