Specific Transcriptomic Changes Research Articles

Perimenopausal and Menopausal Mammary Glands In A 4-Vinylcyclohexene Diepoxide Mouse Model

As both perimenopausal and menopausal periods are recognized critical windows of susceptibility for breast carcinogenesis, development of a physiologically relevant model has been warranted. The traditional ovariectomy model causes instant removal of the entire hormonal repertoire produced by the ovary, which does not accurately approximate human natural menopause with gradual transition. Here, we characterized the mammary glands of 4-vinylcyclohexene diepoxide (VCD)-treated animals at different time points, revealing that the model can provide the mammary glands with both perimenopausal and menopausal states. The perimenopausal gland showed moderate regression in ductal structure with no responsiveness to external hormones, while the menopausal gland showed severe regression with hypersensitivity to hormones. Leveraging the findings on the VCD model, effects of a major endocrine disruptor (polybrominated diphenyl ethers, PBDEs) on the mammary gland were examined during and after menopausal transition, with the two exposure modes; low-dose, chronic (environmental) and high-dose, subacute (experimental). All conditions of PBDE exposure did not augment or compromise the macroscopic ductal reorganization resulting from menopausal transition and/or hormonal treatments. Single-cell RNA sequencing revealed that the experimental PBDE exposure during the post-menopausal period caused specific transcriptomic changes in the non-epithelial compartment such as Errfi1 upregulation in fibroblasts. The environmental PBDE exposure resulted in similar transcriptomic changes to a lesser extent. In summary, the VCD mouse model provides both perimenopausal and menopausal windows of susceptibility for the breast cancer research community. PBDEs, including all tested models, may affect the post-menopausal gland including impacts on the non-epithelial compartments.

Paternal cocaine-seeking motivation defines offspring’s vulnerability to addiction by down-regulating GABAergic GABRG3 in the ventral tegmental area

Epidemiological investigations indicate that parental drug abuse experiences significantly influenced the addiction vulnerability of offspring. Studies using animal models have shown that paternal cocaine use and highly motivated drug-seeking behavior are important determinants of offspring addiction susceptibility. However, the key molecules contributing to offspring addiction susceptibility are currently unclear. The motivation for cocaine-seeking behavior in offspring of male rats was compared between those whose fathers self-administered cocaine (SA) and those who were yoked with them and received non-contingent cocaine administrations (Yoke). We found that paternal experience with cocaine-seeking behavior, but not direct cocaine exposure, could lead to increased lever-pressing behavior in male F1 offspring. This effect was observed without significant changes to the dose-response relationship. The transcriptomes of ventral tegmental area (VTA) in offspring were analyzed under both naive state and after self-administration training. Specific transcriptomic changes in response to paternal cocaine-seeking experiences were found, which mainly affected biological processes such as synaptic connections and receptor signaling pathways. Through joint analysis of these candidate genes and parental drug-seeking motivation scores, we found that gamma-aminobutyric acid receptor subunit gamma-3 (Gabrg3) was in the hub position of the drug-seeking motivation-related module network and highly correlated with parental drug-seeking motivation scores. The downregulation of Gabrg3 expression, caused by paternal motivational cocaine-seeking, mainly occurred in GABAergic neurons in the VTA. Furthermore, down-regulating GABAergic Gabrg3 in VTA resulted in an increase in cocaine-seeking behavior in the Yoke F1 group. This down-regulation also reduced transcriptome differences between the Yoke and SA groups, affecting processes related to synaptic formation and neurotransmitter transmission. Taken together, we propose that paternal cocaine-seeking behavior, rather than direct drug exposure, significantly influences offspring addiction susceptibility through the downregulation of Gabrg3 in GABAergic neurons of the VTA, highlighting the importance of understanding specific molecular pathways in the intergenerational inheritance of addiction vulnerability.

Abstract C041: Transcriptomic analysis of prostate cancer patients living with HIV

Abstract Background: Higher incidence and mortality rates of liver, lung, anal, and head and neck cancers are typically seen in patients living with human immunodeficiency virus (HIV). However, some previous studies have a decreased risk of prostate cancer in HIV+ patients, particularly in men from sexual minorities, with no clear biological or epidemiological explanation. Methods: To identify the specific transcriptomic changes and genetic pathways associated with HIV infection and prostate cancer emergence, we performed bulk RNA-Seq analysis on 18 prostate cancer samples (9 HIV+ and 9 HIV-). We first used kallisto to measure gene-level expression. We then performed principal component analysis (PCA) to determine if HIV status clearly clustered patients based on their gene expression levels. We subsequently used DESeq2 to perform differentially expressed transcriptomic analysis to identify genes that are associated with HIV and prostate cancer phenotypes. Samples were clustered based on expression levels of the top differentially expressed genes across the HIV+ and HIV- phenotypes. Gene set enrichment analysis was performed on the gene signatures from the two phenotypes. Using an existing single cell RNA-Seq prostate cancer dataset, we deconvoluted the 18 samples with CIBERSORT to approximate the cell type composition. Results: PCA highlighted no major separation between samples based on HIV status. We identified 450 significantly upregulated genes in HIV+ patients, including YAP1, MAT2A, KLF4, and ERF. We identified 409 upregulated genes in HIV- patients, including MICA, AQP2, and KRT6C. Upregulated genes found in HIV+ patients were significantly enriched in multiple immune response pathways, which is consistent with previous sequencing studies focused on HIV status. After cell type composition estimation, we found the samples to be similar in their cell type distributions - stromal and tumor cells were the largest populations, while immune cells were not well represented. Conclusions: We identified transcriptomic differences that need future studies that directly interrogate the biological pathways that affect the incidence of prostate cancer in patients living with HIV. Citation Format: Hanbing Song, Michelle Li, Jon Akutagawa, Franklin Huang. Transcriptomic analysis of prostate cancer patients living with HIV [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C041.

Cell-type brain-region specific changes in prefrontal cortex of a mouse model of alcohol dependence

The prefrontal cortex is a crucial regulator of alcohol drinking, and dependence, and other behavioral phenotypes associated with AUD. Comprehensive identification of cell-type specific transcriptomic changes in alcohol dependence will improve our understanding of mechanisms underlying the excessive alcohol use associated with alcohol dependence and will refine targets for therapeutic development. We performed single nucleus RNA sequencing (snRNA-seq) and Visium spatial gene expression profiling on the medial prefrontal cortex (mPFC) obtained from C57BL/6 J mice exposed to the two-bottle choice-chronic intermittent ethanol (CIE) vapor exposure (2BC-CIE, defined as dependent group) paradigm which models phenotypes of alcohol dependence including escalation of alcohol drinking. Gene co-expression network analysis and differential expression analysis identified highly dysregulated co-expression networks in multiple cell types. Dysregulated modules and their hub genes suggest novel understudied targets for studying molecular mechanisms contributing to the alcohol dependence state. A subtype of inhibitory neurons was the most alcohol-sensitive cell type and contained a downregulated gene co-expression module; the hub gene for this module is Cpa6, a gene previously identified by GWAS to be associated with excessive alcohol consumption. We identified an astrocytic Gpc5 module significantly upregulated in the alcohol-dependent group. To our knowledge, there are no studies linking Cpa6 and Gpc5 to the alcohol-dependent phenotype. We also identified neuroinflammation related gene expression changes in multiple cell types, specifically enriched in microglia, further implicating neuroinflammation in the escalation of alcohol drinking. Here, we present a comprehensive atlas of cell-type specific alcohol dependence mediated gene expression changes in the mPFC and identify novel cell type-specific targets implicated in alcohol dependence.

Hypomethylation-induced regulatory programs in T cells unveiled by transcriptomic analyses.

Regulatory T cells (Tregs) are essential mediators of tolerance mitigating aberrant immune responses. While naturally occurring Treg (nTreg) development and function are directed by epigenetic events, induced Treg (iTreg) identity and mechanisms of action remain elusive. Mirroring the epigenetic circuits of nTregs, we and others have used hypomethylation agents (HAs) to ex vivo convert T cells into iTregs (HA-iTregs) and further showed that the suppressive properties of the HA-iTregs are predominantly confined in an emergent population, which de novo expresses the immunomodulatory molecule HLA-G, consequently providing a surface marker for isolation of the suppressive HA-iTreg compartment (G+ cells). We isolated the HA-induced G+ cells and their G- counterparts and employed high-throughput RNA-sequencing (RNA-seq) analyses to uncover the G+-specific transcriptomic changes guiding T cells toward a regulatory trajectory upon their exposure to HA. We found a distinct transcriptional upregulation of G+ cells accompanied by enrichment of immune-response-related pathways. Although single-cell RNA-seq profiling revealed regulatory G+ cells to have molecular features akin to nTregs, when assessed in conjunction with the comparative transcriptomic analysis and profiling of secreted cytokines against the non-suppressive G- cells, FOXP3 and other T-helper signatures appear to play a minor role in their suppressive phenotype. We found an ectopic expression of IDO-1 and CCL17/22 in G+ cells, denoting that in vitro exposure of T cells to HA may well unlock myeloid suppressor genes. This report provides transcriptional data shaping the molecular identity of a highly purified and potent HA-iTreg population and hints toward ectopic myeloid-specific molecular mechanisms mediating HA-iTreg function.

Mutations in human DNA methyltransferase DNMT1 induce specific genome-wide epigenomic and transcriptomic changes in neurodevelopment.

DNA methyltransferase type 1 (DNMT1) is a major enzyme involved in maintaining the methylation pattern after DNA replication. Mutations in DNMT1 have been associated with autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN). We used fibroblasts, induced pluripotent stem cells (iPSCs) and induced neurons (iNs) generated from patients with ADCA-DN and controls, to explore the epigenomic and transcriptomic effects of mutations in DNMT1. We show cell type-specific changes in gene expression and DNA methylation patterns. DNA methylation and gene expression changes were negatively correlated in iPSCs and iNs. In addition, we identified a group of genes associated with clinical phenotypes of ADCA-DN, including PDGFB and PRDM8 for cerebellar ataxia, psychosis and dementia and NR2F1 for deafness and optic atrophy. Furthermore, ZFP57, which is required to maintain gene imprinting through DNA methylation during early development, was hypomethylated in promoters and exhibited upregulated expression in patients with ADCA-DN in both iPSC and iNs. Our results provide insight into the functions of DNMT1 and the molecular changes associated with ADCA-DN, with potential implications for genes associated with related phenotypes.

HIV-1 activates oxidative phosphorylation in infected CD4 T cells in a human tonsil explant model.

Human immunodeficiency virus type 1 (HIV-1) causes a chronic, incurable infection leading to immune activation and chronic inflammation in people with HIV-1 (PWH), even with virologic suppression on antiretroviral therapy (ART). The role of lymphoid structures as reservoirs for viral latency and immune activation has been implicated in chronic inflammation mechanisms. Still, the specific transcriptomic changes induced by HIV-1 infection in different cell types within lymphoid tissue remain unexplored. In this study, we utilized human tonsil explants from healthy human donors and infected them with HIV-1 ex vivo. We performed single-cell RNA sequencing (scRNA-seq) to analyze the cell types represented in the tissue and to investigate the impact of infection on gene expression profiles and inflammatory signaling pathways. Our analysis revealed that infected CD4+ T cells exhibited upregulation of genes associated with oxidative phosphorylation. Furthermore, macrophages exposed to the virus but uninfected showed increased expression of genes associated with the NLRP3 inflammasome pathway. These findings provide valuable insights into the specific transcriptomic changes induced by HIV-1 infection in different cell types within lymphoid tissue. The activation of oxidative phosphorylation in infected CD4+ T cells and the proinflammatory response in macrophages may contribute to the chronic inflammation observed in PWH despite ART. Understanding these mechanisms is crucial for developing targeted therapeutic strategies to eradicate HIV-1 infection in PWH.

Experimental hepatic encephalopathy causes early but sustained glial transcriptional changes

Hepatic encephalopathy (HE) is a common complication of liver cirrhosis, associated with high morbidity and mortality, for which no brain-targeted therapies exist at present. The interplay between hyperammonemia and inflammation is thought to drive HE development. As such, astrocytes, the most important ammonia-metabolizing cells in the brain, and microglia, the main immunomodulatory cells in the brain, have been heavily implicated in HE development. As insight into cellular perturbations driving brain pathology remains largely elusive, we aimed to investigate cell-type specific transcriptomic changes in the HE brain. In the recently established mouse bile duct ligation (BDL) model of HE, we performed RNA-Seq of sorted astrocytes and microglia at 14 and 28 days after induction. This revealed a marked transcriptional response in both cell types which was most pronounced in microglia. In both cell types, pathways related to inflammation and hypoxia, mechanisms commonly implicated in HE, were enriched. Additionally, astrocytes exhibited increased corticoid receptor and oxidative stress signaling, whereas microglial transcriptome changes were linked to immune cell attraction. Accordingly, both monocytes and neutrophils accumulated in the BDL mouse brain. Time-dependent changes were limited in both cell types, suggesting early establishment of a pathological phenotype. While HE is often considered a unique form of encephalopathy, astrocytic and microglial transcriptomes showed significant overlap with previously established gene expression signatures in other neuroinflammatory diseases like septic encephalopathy and stroke, suggesting common pathophysiological mechanisms. Our dataset identifies key molecular mechanisms involved in preclinical HE and provides a valuable resource for development of novel glial-directed therapeutic strategies.Graphical

Pancreatic islet cell type–specific transcriptomic changes during pregnancy and postpartum

Pancreatic β-cell mass expands during pregnancy and regresses in the postpartum period in conjunction with dynamic metabolic demands on maternal glucose homeostasis. To understand transcriptional changes driving these adaptations in β-cells and other islet cell types, we performed single-cell RNA sequencing on islets from virgin, late gestation, and early postpartum mice. We identified transcriptional signatures unique to gestation and the postpartum in β-cells, including induction of the AP-1 transcription factor subunits and other genes involved in the immediate-early response (IEGs). In addition, we found pregnancy and postpartum-induced changes differed within each endocrine cell type, and in endothelial cells and antigen-presenting cells within islets. Together, our data reveal insights into cell type-specific transcriptional changes responsible for adaptations by islet cells to pregnancy and their resolution postpartum.

The gut microbiome modulates the transformation of microglial subtypes

Clinical and animal studies have shown that gut microbiome disturbances can affect neural function and behaviors via the microbiota–gut–brain axis, and may be implicated in the pathogenesis of several brain diseases. However, exactly how the gut microbiome modulates nervous system activity remains obscure. Here, using a single-cell nucleus sequencing approach, we sought to characterize the cell type–specific transcriptomic changes in the prefrontal cortex and hippocampus derived from germ-free (GF), specific pathogen free, and colonized-GF mice. We found that the absence of gut microbiota resulted in cell-specific transcriptomic changes. Furthermore, microglia transcriptomes were preferentially influenced, which could be effectively reversed by microbial colonization. Significantly, the gut microbiome modulated the mutual transformation of microglial subpopulations in the two regions. Cross-species analysis showed that the transcriptome changes of these microglial subpopulations were mainly associated with Alzheimer’s disease (AD) and major depressive disorder (MDD), which were further supported by animal behavioral tests. Our findings demonstrate that gut microbiota mainly modulate the mutual transformation of microglial subtypes, which may lead to new insights into the pathogenesis of AD and MDD.

Single Cell Analysis of Human Thyroid Reveals the Transcriptional Signatures of Aging.

The thyroid gland plays a critical role in the maintenance of whole-body metabolism. However, aging frequently impairs homeostatic maintenance by thyroid hormones due to increased prevalence of subclinical hypothyroidism associated with mitochondrial dysfunction, inflammation, and fibrosis. To understand the specific aging-related changes of endocrine function in thyroid epithelial cells, we performed single-cell RNA sequencing (RNA-seq) of 54 726 cells derived from pathologically normal thyroid tissues from 7 patients who underwent thyroidectomy. Thyroid endocrine epithelial cells were clustered into 5 distinct subpopulations, and a subset of cells was found to be particularly vulnerable with aging, showing functional deterioration associated with the expression of metallothionein (MT) and major histocompatibility complex class II genes. We further validated that increased expression of MT family genes are highly correlated with thyroid gland aging in bulk RNAseq datasets. This study provides evidence that aging induces specific transcriptomic changes across multiple cell populations in the human thyroid gland.

ODP263 A Nonbiased Single-Cell Transcriptomic Analysis of Mouse Mammary Gland after Low Dose/Long Term vs. High Dose/Short Term Exposure to PBDEs

Abstract Introduction Polybrominated diphenyl ethers (PBDEs) are persistent environmental contaminants and associated with breast carcinogenesis. In this study, we examined the effects of PBDEs during the menopausal transition in a 4-vinylcyclohexene diepoxide (VCD)-induced mouse menopause model, which presents a follicle-depleted and ovary-intact animal that closely approximate the gradual menopause transition in humans. The aim of this study was to compare the impacts of PBDE exposures on the mammary gland at the macroscopic gland structures and the single-cell resolution under three treatment schedules. Materials and Methods We treated nine-week-old female C57BL/6 mice with VCD (160 mg/kg) for 15 days. Then, the animals had high dose PBDE exposure for 1 week during peri- (Perimenopause/High group) or post-menopausal periods (Menopause/High group). The third group was exposed to low dose PBDE (the 1/20th of the high dose) for 20 weeks during the postmenopausal period (Menopause/Low group). In each group, the mice received various combinations of 17B-estradiol and progesterone treatments simultaneously. Our experimental design allowed us to directly compare the effects of PBDE under three potential exposure conditions. Results The mammary glands significantly regressed by the VCD treatment, more evident at the postmenopausal period. All PBDE exposures did not augment or compromise the macroscopic ductal reorganization resulting from the VCD and/or hormonal treatments. Single-cell sequencing revealed that the PBDE exposure in the Menopause/High group caused specific transcriptomic changes in the non-epithelial compartment such as Errfi1 upregulation in fibroblasts and AY036118 downregulation in multiple cell types. The PBDE exposure in the Menopause/Low group resulted in similar transcriptomic changes to a lesser extent. Conclusion PBDEs may affect the postmenopausal gland through impacts on the non-epithelial compartments. Low dose PBDE exposure, which is presumably equivalent to the maximum environmental exposure in humans, can exert the influences. Presentation: No date and time listed

427 Persistence of cell type–specific transcriptomic changes in the nasal epithelium of people with cystic fibrosis receiving cystic fibrosis transmembrane conductance regulator modulators

427 Persistence of cell type–specific transcriptomic changes in the nasal epithelium of people with cystic fibrosis receiving cystic fibrosis transmembrane conductance regulator modulators

An epigenetic mechanism for over-consolidation of fear memories

Excessive fear is a hallmark of anxiety disorders, a major cause of disease burden worldwide. Substantial evidence supports a role of prefrontal cortex-amygdala circuits in the regulation of fear and anxiety, but the molecular mechanisms that regulate their activity remain poorly understood. Here, we show that downregulation of the histone methyltransferase PRDM2 in the dorsomedial prefrontal cortex enhances fear expression by modulating fear memory consolidation. We further show that Prdm2 knock-down (KD) in neurons that project from the dorsomedial prefrontal cortex to the basolateral amygdala (dmPFC-BLA) promotes increased fear expression. Prdm2 KD in the dmPFC-BLA circuit also resulted in increased expression of genes involved in synaptogenesis, suggesting that Prdm2 KD modulates consolidation of conditioned fear by modifying synaptic strength at dmPFC-BLA projection targets. Consistent with an enhanced synaptic efficacy, we found that dmPFC Prdm2 KD increased glutamatergic release probability in the BLA and increased the activity of BLA neurons in response to fear-associated cues. Together, our findings provide a new molecular mechanism for excessive fear responses, wherein PRDM2 modulates the dmPFC -BLA circuit through specific transcriptomic changes.

Mapping the cardiac vascular niche in heart failure

The cardiac vascular and perivascular niche are of major importance in homeostasis and during disease, but we lack a complete understanding of its cellular heterogeneity and alteration in response to injury as a major driver of heart failure. Using combined genetic fate tracing with confocal imaging and single-cell RNA sequencing of this niche in homeostasis and during heart failure, we unravel cell type specific transcriptomic changes in fibroblast, endothelial, pericyte and vascular smooth muscle cell subtypes. We characterize a specific fibroblast subpopulation that exists during homeostasis, acquires Thbs4 expression and expands after injury driving cardiac fibrosis, and identify the transcription factor TEAD1 as a regulator of fibroblast activation. Endothelial cells display a proliferative response after injury, which is not sustained in later remodeling, together with transcriptional changes related to hypoxia, angiogenesis, and migration. Collectively, our data provides an extensive resource of transcriptomic changes in the vascular niche in hypertrophic cardiac remodeling.

Abstract B019: Does order matter? Dissecting the single-cell differences underlying resistance to sequential drug treatment

Abstract Most cancer patients are given multiple different drugs during the course of their cancer treatment. An important but outstanding question is the role that drug order plays in the development of resistance to sequential drug treatment– that is, if cells that develop resistance to a certain first and second-line therapy are similar to cells that develop resistance to the same drugs given in the opposite order. During treatment with a first drug, both selection and transcriptional reprogramming events alter the cells within a tumor. As such, surviving cells may exhibit increased sensitivity or increased resistance to future drug therapies. Here, we zoom in to the single-cell level using cell barcoding and sequencing and explicitly test whether the order of treatment leads to different populations of cells developing resistance to a panel of different therapies. As the initial application of drug therapy changes a tumor in multiple ways, it is currently unclear which changes are most influential in the overall modification of a tumor’s sensitivity to a second-line therapy. One hypothesis is that changes in sensitivity are due to specific drug-induced modifications in the characteristics of cells that survived the first-line therapy, in which case order may matter. An alternative hypothesis is that changes in sensitivity are simply the result of a decrease in clonal diversity within the tumor, where the ability of individual subpopulations to survive the second-line therapy is unchanged by the first-line treatment, in which case order may not matter. To distinguish between these hypotheses, we have combined single-cell RNA sequencing with cellular barcoding to assess both the survival and transcriptome of cells from over 100,000 cells over the course of treatment with multiple stressors. These stressors include a combination of targeted inhibitors (Dabrafenib and Trametinib), chemotherapy (Cisplatin), and hypoxic stress (CoCl2). Through analysis of the transcriptome of cells within the same lineage after surviving each therapy in the sequential treatment scheme, we are now working to determine whether development of resistance to a first-line therapy causes individual cells within a lineage to change in ways that alter their sensitivity to subsequent therapies. Should we find that developing resistance to first-line therapy does increase resistance to specific second-line therapies, we will use our sequencing data to identify specific transcriptomic changes which lead to this induced resistance. We will then look to block this induced resistance through either small-molecule inhibitors (if available) or CRISPR-mediated knockout of genes that promote resistance to second-line therapies. Overall, this study will provide two methods for increasing the efficacy of sequential drug treatment through both the identification of ideal drug ordering and the targeting of changes that promote induced resistance, thus leading to resistance-free treatment strategies. Citation Format: Aria J. Fasse, Dylan L. Schaff, Sydney M. Shaffer. Does order matter? Dissecting the single-cell differences underlying resistance to sequential drug treatment [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B019.

Cis p‐tau underlies vascular contribution to cognitive impairment and dementia, but is effectively targeted by immunotherapy

Compelling evidence supports vascular contributions to cognitive impairment and dementia (VCID) including Alzheimer's disease (AD), but the underlying pathogenic mechanisms and treatments are not fully understood. Cis P-tau is an early driver of neurodegeneration resulting from traumatic brain injury, but its role in VCID remains unclear.We first evaluated cis P-tau induction in VCID patients with no obvious AD pathology, and in mice after bilateral carotid artery stenosis (BCAS), a surgery modeling key aspects of clinical VCID. We subsequently tested if cis P-tau elimination using cis P-tau monoclonal antibody (cis mAb) reversed the VCID relevant pathology. To interrogate the upstream mechanism, we used the transgenic mice perturbing the cis P-tau counteracting isomerase Pin1 and Pin1's inhibitory kinase DAPK1, and asked if perturbation of Pin1 and DAPK1 alters cis P-tau and VCID pathology. To investigate the mechanism underlying cis P-tau pathology, we dissected the BCAS mice cortical cell-type specific transcriptome with or without the cis mAb treatment. Finally, to test if cis P-tau is sufficient to induce the pathology, we stereotaxically injected the cis P-tau purified from the injury mice brain into injury-free mice cortex and evaluate their pathological and behavioral outcome.We surprisingly find robust and early cis P-tau despite no tau tangles in VCID patients and in mice modeling key aspects of clinical VCID, likely due to the inhibition of Pin1 by DAPK1. Elimination of cis P-tau in VCID mice using cis-targeted immunotherapy, brain-specific Pin1 overexpression or DAPK1 knockout effectively rescues VCID-like neurodegeneration and cognitive impairment in executive function. Furthermore, single-cell RNA-sequencing reveals that young VCID mice display diverse cortical cell-type specific transcriptomic changes resembling old AD patients, and the vast majority of these global changes are remarkably recovered by cis-targeted immunotherapy. Finally, purified soluble cis P-tau is sufficient to induce progressive neurodegeneration and brain dysfunction by causing axonopathy and conserved transcriptomic signature found at VCID mice and AD human patients with early pathologies.cis P-tau plays a major role in mediating VCID and its antibody may be useful for early diagnosis, prevention and treatment of cognitive impairment and dementia after neurovascular insults.

Cell type-specific gene expression changes in Alzheimer's patients with varying levels of cerebral amyloid angiopathy.

Tau neurofibrillary tangles and senile plaques comprised of insoluble amyloid beta are the major histopathological hallmarks of Alzheimer's disease (AD). More than 85% of autopsy-confirmed AD cases also exhibit some degree of cerebral amyloid angiopathy (CAA), which is characterized by amyloid beta peptide deposits predominantly in blood vessels in the meningeal and intracerebral blood vessels. Consequently, CAA predisposes individuals with AD to cerebral infarction and hemorrhages, accounting for ∼20% of cases in the elderly, which could lead to faster cognitive decline through neurovascular pathway dysfunction. We sought to identify cellular composition changes and characterize cell type-specific expression associated with CAA pathology in AD patients. Using 10x Genomics single nuclei RNA sequencing, we profiled nuclei from frozen post-mortem temporal cortex tissue of 80 individuals with neuropathologic diagnosis of AD and varying levels of CAA, ranging from zero to severe CAA pathology. Braak and Thal measures were also obtained. Downstream analyses were performed in R using Seurat v3.2.3. Quality control filtering based on percentage of genes mapped to the mitochondrial genome and number of genes and UMIs detected resulted in 135,092 nuclei for analysis. Differential gene expression analyses were performed using R package MAST. We identified 30 clusters which were annotated using established cell type marker genes. There were 3 oligodendrocyte, 14 neuronal (2 excitatory and 3 inhibitory), 5 microglial, 3 endothelial, 4 astrocyte and 1 oligodendrocyte precursor cell cluster. We observed a significant increase in microglial cell proportions with increasing CAA severity, for 3 of the microglial clusters. Conversely, the excitatory neuronal proportion were decreased in individuals with more severe CAA. Further investigation is ongoing to identify the genes underlying the dysregulation of these cell types in CAA. We observed cellular proportion changes for multiple cell types with increasing CAA severity. Additional analyses to determine the effects of CAA independent of AD severity will be performed by adjusting for Braak and Thal measures. Deeper characterization of cell type-specific expression profiles is expected to provide novel insights into cell-type specific transcriptome changes associated with vascular risk factors in AD.

Pathology-related alterations in gene expression in Alzheimer's disease: Single cell resolution.

Alzheimer's disease is pathologically defined by the presence of extracellular amyloid beta plaques and intracellular tau tangles, resulting in neurodegeneration and reactive gliosis in affected brain regions such as temporal cortex (TCX). The degree and distribution of AD pathology can be defined by Braak stage (tau) and Thal phase (amyloid). Brain tissue is comprised of multiple cell types that have different molecular profiles, the proportions of which can vary between individuals and in response to disease; creating challenges for the interpretation of bulk tissue transcriptome profiling study. We aimed to use single nucleus RNA sequencing (snRNAseq) to explore cell proportion and cell-type specific transcriptome changes associated with AD and neuropathology. We selected age and sex matched post mortem TCX from 12 AD and 12 control patients. We obtained snRNAseq profiles using 10x Genomics platform. After quality control 79,751 nuclei remained for analysis and were annotated for their type according to commonly used cellular markers. Nuclei proportions were tested for association with AD, Braak stage and Thal phase using Wilcoxon rank sum test. Differentially expressed genes (DEGs) and pathways were identified for each cell cluster using MAST and validated through in situ hybridization via RNAscope and qPCR. Annotation according to common cellular markers distinguished major and rare cell types of the brain in 35 clusters. We detected a unique excitatory neuron cluster, predominantly from control individuals that show less pathology and APOE ε4 genotype. DEG and pathway analysis revealed alterations in myelination pathways in oligodendrocytes, and synaptic signaling pathways in excitatory neurons. Analysis of the effects of sex, age, and APOE ε4 genotype on selected DEGs and pathways are conducted. In this study, we developed and optimized an snRNAseq approach for frozen human temporal cortex that captures the major brain cell types. We identified robust DEGs and pathways for these between AD vs control and demonstrated shared and unique cell-specific pathways and DEGs amongst different pathologies in AD.

Abstract 14422: Molecular Pathogenesis of Cardiac Microthrombi in Fatal Covid-19: Insights from Clinico-histopathologic and Single Nuclei RNA Sequencing Analyses

Cardiac microthrombi are postulated to underlie cardiac injury in critical COVID-19. To determine pathogenic mechanism(s) of cardiac injury in fatal COVID-19, we conducted a single-center prospective cohort study of 69 consecutive COVID-19 decedents. Microthrombi was the most commonly detected acute cardiac histopathologic feature (n=48, 70%). We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak ESR and CRP during hospitalization were independently associated with higher odds of microthrombi (ESR, P non-linearity 0.015, P association =0.008; CRP per 20mg/L increase, OR 1.17, 95%CI 1.00-1.36). Using single nuclei RNA-sequence analysis, we discovered an enrichment of prothrombotic, anti-fibrinolytic, and extracellular matrix signaling amongst cardiac fibroblasts in microthrombi-positive COVID-19 hearts, compared with microthrombi-negative COVID-19 hearts and non-COVID-19 donor hearts. Our cumulative findings identify these specific transcriptomic changes in cardiac fibroblasts as salient features of COVID-19-associated cardiac microthrombi.