Identify Gene Expression Changes Research Articles

5-Azacytidine treatment inhibits the development of lung cancer models via epigenetic reprogramming and activation of cellular pathways with anti-tumor activity

Background and aims. Non-small cell lung cancer (NSCLC) treatment is challenged by late detection and limited therapeutic options. Aberrant DNA methylation, a common epigenetic alteration in NSCLC, offers new therapeutic avenues. This study aims to evaluate the combined effects of 5-Azacytidine (5- Aza), an epigenetic modifier, and ionizing radiation (IR) on NSCLC, exploring the underlying molecular mechanisms and therapeutic potential. Methods. In this study, we examined the effects of 5-Aza combined with IR in both in vitro and in vivo models of NSCLC. Five human NSCLC cell lines were treated with 5-Aza and IR. Cell viability, colony formation, wound healing, and transwell migration assays were performed to assess treatment effects. Microarray and qPCR analyses were conducted to identify gene expression changes. Additionally, subcutaneous and orthotopic xenograft models were used to evaluate the treatment’s efficacy in vivo. Results. Treatment with 5-Aza and IR resulted in significant reductions in cell viability, colony formation, and migration in NSCLC cell lines. Microarray analysis revealed significant changes in gene expression, including the upregulation of apoptosis-related genes and the downregulation of cell proliferation-related genes. In vivo studies demonstrated a notable reduction in tumor growth and metastasis in both subcutaneous and orthotopic NSCLC models following 5-Aza and IR treatment. Histological and bioluminescent imaging confirmed the therapeutic effects of the combined treatment. Conclusions. The combination of 5-Aza and IR shows promise as an effective treatment for NSCLC, enhancing apoptosis and reducing tumor growth through epigenetic modulation.

Polyamine Pathway Inhibitor DENSPM Suppresses Lipid Metabolism in Pheochromocytoma Cell Line.

Pheochromocytomas (PCCs) are tumors arising from chromaffin cells in the adrenal medulla, and paragangliomas (PGLs) are tumors derived from extra-adrenal sympathetic or parasympathetic paraganglia; these tumors are collectively referred to as PPGL cancer. Treatment for PPGL primarily involves surgical removal of the tumor, and only limited options are available for treatment of the disease once it becomes metastatic. Human carriers of the heterozygous mutations in the succinate dehydrogenase subunit B (SDHB) gene are susceptible to the development of PPGL. A physiologically relevant PCC patient-derived cell line hPheo1 was developed, and SDHB_KD cells carrying a stable short hairpin knockdown of SDHB were derived from it. An untargeted metabolomic approach uncovered an overactive polyamine pathway in the SDHB_KD cells that was subsequently fully validated in a large set of human SDHB-mutant PPGL tumor samples. We previously reported that treatment with the polyamine metabolism inhibitor N1,N11-diethylnorspermine (DENSPM) drastically inhibited growth of these PCC-derived cells in culture as well as in xenograft mouse models. Here we explored the mechanisms underlying DENSPM action in hPheo1 and SDHB_KD cells. Specifically, by performing an RNAseq analysis, we have identified gene expression changes associated with DENSPM treatment that broadly interfere with all aspects of lipid metabolism, including fatty acid (FA) synthesis, desaturation, and import/uptake. Furthermore, by performing an untargeted lipidomic liquid chromatography-mass spectrometry (LC/MS)-based analysis we uncovered specific groups of lipids that are dramatically reduced as a result of DENSPM treatment. Specifically, the bulk of plasmanyl ether lipid species that have been recently reported as the major determinants of cancer cell fate are notably decreased. In summary, this work suggests an intersection between active polyamine and lipid pathways in PCC cells.

Antibody Mediated Rejection and T-cell Mediated Rejection Molecular Signatures Using Next-Generation Sequencing in Kidney Transplant Biopsies.

Recently, interest in transcriptomic assessment of kidney biopsies has been growing. This study investigates the use of NGS to identify gene expression changes and analyse the pathways involved in rejection. An Illumina bulk RNA sequencing on the polyadenylated RNA of 770 kidney biopsies was conducted. Differentially-expressed genes (DEGs) were determined for AMR and TCMR using DESeq2. Genes were segregated according to their previous descriptions in known panels (microarray or the Banff Human Organ Transplant (B-HOT) panel) to obtain NGS-specific genes. Pathway enrichment analysis was performed using the Reactome and Kyoto Encyclopaedia of Genes and Genomes (KEGG) public repositories. The differential gene expression using NGS analysis identified 6,141 and 8,478 transcripts associated with AMR and TCMR. While most of the genes identified were included in the microarray and the B-HOT panels, NGS analysis identified 603 (9.8%) and 1,186 (14%) new specific genes. Pathways analysis showed that the B-HOT panel was associated with the main immunological processes involved during AMR and TCMR. The microarrays specifically integrated metabolic functions and cell cycle progression processes. Novel NGS-specific based transcripts associated with AMR and TCMR were discovered, which might represent a novel source of targets for drug designing and repurposing.

Disruption of NADPH homeostasis by total flavonoids from Adinandra nitida Merr. ex Li leaves triggers ROS-dependent p53 activation leading to apoptosis in non-small cell lung cancer cells

Ethnopharmacological relevanceAdinandra nitida Merr. ex Li leaves serve as a herbal tea and hold a significant role in traditional Chinese medicine, being applied to assist in tumor treatment. Flavonoids present the primary bioactive constituents in Adinandra nitida Merr. ex Li leaves. Aim of the studyTo explore the potential of total flavonoids from Adinandra nitida Merr. ex Li Leaves (TFAN) in inhibiting non-small cell lung cancer (NSCLC) and further elucidate the underlying mechanisms. Materials and methodsHuman NSCLC cell lines and normal lung cell line were employed to assess the impact of TFAN (0–160 μg/mL for 24, 28 and 72 h) on cell proliferation in vitro. Immunofluorescence (IF) staining gauged p53 expression changes in NSCLC cells under TFAN present condition (150 μg/mL for 24 h). In vivo study utilized NSCLC cell derived xenograft tumors in nude mice, administering TFAN orally (200 and 400 mg/kg) for 14 days. Immunohistochemistry assessed Cleaved Caspase 3 expression change in A549 xenograft tumors treated with TFAN (400 mg/kg for 14 days). RNA-seq and KEGG analysis identified gene expression changes and enriched processes in A549 xenograft tumors treated with TFAN. CM-H2DCFDA and metabolomics assessed ROS level and GSH/GSSG pool changes in A549 cells under TFAN present condition. Cell viability assay and IF staining assessed A549 cell proliferation and p53 expression changes under H2O2-induced oxidative stress (0–40 μM for 24 h) and TFAN present conditions. GSEA and N-Acetyl-L-cysteine (NAC) rescue (0–1 μM for 24 h) analyzed the impact of TFAN on GSH de novo synthesis. NADPH/NADP+ pool measurement and NADPH rescue (0–10 μM for 24 h) analyzed the impact of TFAN on GSH salvage synthesis. GC-FID and HPLC-MS were utilized to detect ethanol and ethyl acetate residues, and to characterize the chemical constituents in TFAN, respectively. The total flavonoid content of TFAN was determined using a 330 nm wavelength. ResultsTFAN significantly inhibited A549 cells (wild-type p53) but not NCI–H1299 cells (p53-deficient), NCI–H596 cells (p53-mutant) or BEAS-2B in vitro. IF staining validated p53 genotype for the cell lines and revealed an increase in p53 expression in A549 cells after TFAN treatment. In vivo, TFAN selectively inhibited A549 xenograft tumor growth without discernible toxicity, inducing apoptosis evidenced by Cleaved Caspase 3 upregulation. RNA-seq and KEGG analysis suggested ROS biosynthesis was involved in TFAN-induced p53 activation in A549 cells. Elevated ROS level in TFAN-treated A549 cells were observed. Moreover, TFAN sensitized A549 cells to H2O2-induced oxidative stress, with higher p53 expression. Additionally, A549 cells compensated with GSH de novo synthesis under TFAN present condition, confirmed by GSEA and NAC rescue experiment. TFAN disrupted NADPH homeostasis to impair GSH salvage biosynthesis, supported by NADPH/NADP+ change and NADPH rescue experiment. The chemical constituents of TFAN, with acceptable limits for ethanol and ethyl acetate residues and a total flavonoid content of 68.87%, included Catechin, Epicatechin, Quercitroside, Camellianin A, and Apigenin. ConclusionThe disruption of NADPH homeostasis by TFAN triggers ROS-dependent p53 activation that leads to apoptotic cell death, ultimately suppressing NSCLC growth. These findings offer potential therapeutic implications of Adinandra nitida Merr. ex Li leaves in combating NSCLC.

Tissue-, Region-, and Gene-Specific Induction of Microsomal Epoxide Hydrolase Expression and Activity in the Mouse Intestine by Arsenic in Drinking Water.

This study aimed to characterize the effects of arsenic exposure on the expression of microsomal epoxide hydrolase (mEH or EPHX1) and soluble epoxide hydrolase (sEH or EPHX2) in the liver and small intestine. C57BL/6 mice were exposed to sodium arsenite in drinking water at various doses for up to 28 days. Intestinal, but not hepatic, mEH mRNA and protein expression was induced by arsenic at 25 ppm, in both males and females, whereas hepatic mEH expression was induced by arsenic at 50 or 100 ppm. The induction of mEH was gene specific, as the arsenic exposure did not induce sEH expression in either tissue. Within the small intestine, mEH expression was induced only in the proximal, but not the distal segments. The induction of intestinal mEH was accompanied by increases in microsomal enzymatic activities toward a model mEH substrate, cis-stilbene oxide, and an epoxide-containing drug, oprozomib, in vitro, and by increases in the levels of PR-176, the main hydrolysis metabolite of oprozomib, in the proximal small intestine of oprozomib-treated mice. These findings suggest that intestinal mEH, playing a major role in converting xenobiotic epoxides to less reactive diols, but not sEH, preferring endogenous epoxides as substrates, is relevant to the adverse effects of arsenic exposure, and that further studies of the interactions between drinking water arsenic exposure and the disposition or possible adverse effects of epoxide-containing drugs and other xenobiotic compounds in the intestine are warranted. SIGNIFICANCE STATEMENT: Consumption of arsenic-contaminated water has been associated with increased risks of various adverse health effects, such as diabetes, in humans. The small intestinal epithelial cells are the main site of absorption of ingested arsenic, but they are not well characterized for arsenic exposure-related changes. This study identified gene expression changes in the small intestine that may be mechanistically linked to the adverse effects of arsenic exposure and possible interactions between arsenic ingestion and the pharmacokinetics of epoxide-containing drugs in vivo.

The use of a complex tetra-culture alveolar model to study the biological effects induced by gold nanoparticles with different physicochemical properties

A substantial increase in engineered nanoparticles in consumer products has been observed, heightening human and environmental exposure. Inhalation represents the primary route of human exposure, necessitating a focus on lung toxicity studies. However, to avoid ethical concerns the use of in vitro models is an efficient alternative to in vivo models. This study utilized an in vitro human alveolar barrier model at air-liquid-interface with four cell lines, for evaluating the biological effects of different gold nanoparticles. Exposure to PEGylated gold nanospheres, nanorods, and nanostars did not significantly impact viability after 24 h, yet all AuNPs induced cytotoxicity in the form of membrane integrity impairment. Gold quantification revealed cellular uptake and transport. Transcriptomic analysis identified gene expression changes, particularly related to the enhancement of immune cells. Despite limited impact, distinct effects were observed, emphasizing the influence of nanoparticles physicochemical parameters while demonstrating the model’s efficacy in investigating particle biological effects.

Gene Expression Alterations in Peripheral Blood Following Sport-Related Concussion in a Prospective Cohort of Collegiate Athletes: A Concussion Assessment, Research and Education (CARE) Consortium Study

BackgroundMolecular-based approaches to understanding concussion pathophysiology provide complex biological information that can advance concussion research and identify potential diagnostic and/or prognostic biomarkers of injury.ObjectiveThe aim of this study was to identify gene expression changes in peripheral blood that are initiated following concussion and are relevant to concussion response and recovery.MethodsWe analyzed whole blood transcriptomes in a large cohort of concussed and control collegiate athletes who were participating in the multicenter prospective cohort Concussion Assessment, Research, and Education (CARE) Consortium study. Blood samples were collected from collegiate athletes at preseason (baseline), within 6 h of concussion injury, and at four additional prescribed time points spanning 24 h to 6 months post-injury. RNA sequencing was performed on samples from 230 concussed, 130 contact control, and 102 non-contact control athletes. Differential gene expression and deconvolution analysis were performed at each time point relative to baseline.ResultsCytokine and immune response signaling pathways were activated immediately after concussion, but at later time points these pathways appeared to be suppressed relative to the contact control group. We also found that the proportion of neutrophils increased and natural killer cells decreased in the blood following concussion.ConclusionsTranscriptome signatures in the blood reflect the known pathophysiology of concussion and may be useful for defining the immediate biological response and the time course for recovery. In addition, the identified immune response pathways and changes in immune cell type proportions following a concussion may inform future treatment strategies.

Cucurbitacin I Reverses Tumor-Associated Macrophage Polarization to Affect Cancer Cell Metastasis.

The tumor microenvironment plays a critical role in tumor progression and immune regulation. As one of the most important components of the tumor microenvironment, macrophages have become a new therapeutic target for inhibiting tumor progression. Despite the well-documented anticancer activity of cucurbitacin I, its effect on macrophages remains unclear. In this study, we established a coculture system of macrophages and cancer cells under hypoxic conditions to simulate the tumor-promoting environment mediated by M2-like macrophages. We determined whether cucurbitacin I modulates M2-like polarization in macrophages in vitro and conducted RNA sequencing to identify gene expression changes induced by cucurbitacin I in macrophages. The results indicated a remarkable inhibition of the M2-like polarization phenotype in macrophages following treatment with cucurbitacin I, which was accompanied by the significant downregulation of heme oxygenase-1. Moreover, we found that cucurbitacin I-treated macrophages reduced the migration of cancer cells by inhibiting the M2 polarization in vitro. These findings highlight the potential of cucurbitacin I as a therapeutic agent that targets M2-like macrophages to inhibit cancer cell metastasis. Our study provides novel insights into the intricate interplay among macrophage polarization, cucurbitacin I, and heme oxygenase-1, thereby opening new avenues for cancer treatment.

Alteration in the expression of the chemotherapy resistance‑related genes in response to chronic and acute hypoxia in pancreatic cancer.

Pancreatic cancer is currently one of the least curable types of human cancer and remains a key health problem. One of the most important characteristics of pancreatic cancer is its ability to grow under hypoxic conditions. Hypoxia is associated with resistance of cancer cells to radiotherapy and chemotherapy. It is a major contributor to pancreatic cancer genetic instability, which local and systemic resistance that may result in poor clinical outcome. Accordingly, identifying gene expression changes in cancer resistance genes that occur under hypoxic conditions may identify a new therapeutic target. The aim of the present study was to explore the association between hypoxia and resistance to chemotherapy and determine the alteration in the expression of cancer resistance-related genes in the presence of hypoxia. Pancreatic cancer cells (PANC-1) were exposed to 8 h hypoxic episodes (<1% oxygen) three times/week for a total of 20 episodes (chronic hypoxia) or 72 h hypoxic episodes twice/week for a total of 10 episodes (acute hypoxia). The alterations in gene expression were examined using reverse transcription-quantitative PCR array compared with normoxic cells. Chemoresistance of hypoxic cells toward doxorubicin was assessed using MTT cell proliferation assay. Both chronic and acute hypoxia induced chemoresistance toward doxorubicin in PANC-1 pancreatic cancer cell line. The greatest changes occurred in estrogen Receptor Alpha Gene (ESR1) and ETS Like-1 protein (ELK1) pathways, in nucleic transcription factor Peroxisome proliferator-activated receptors (PPARs) and in a cell cycle inhibitor cyclin dependent kinase inhibitor 1A (CDKN1A). The present study demonstrated that exposing cells to prolonged hypoxia results in different gene expression changes involving pleotropic pathways that serve a role in inducing resistance in pancreatic cancer.

Disruption of cortical cell type composition and function underlies diabetes-associated cognitive decline

Aims/hypothesisType 2 diabetes is associated with increased risk of cognitive decline although the pathogenic basis for this remains obscure. Deciphering diabetes-linked molecular mechanisms in cells of the cerebral cortex could uncover novel therapeutic targets.MethodsSingle-cell transcriptomic sequencing (scRNA-seq) was conducted on the cerebral cortex in a mouse model of type 2 diabetes (db/db mice) and in non-diabetic control mice in order to identify gene expression changes in distinct cell subpopulations and alterations in cell type composition. Immunohistochemistry and metabolic assessment were used to validate the findings from scRNA-seq and to investigate whether these cell-specific dysfunctions impact the neurovascular unit (NVU). Furthermore, the behavioural and cognitive alterations related to these dysfunctions in db/db mice were assessed via Morris water maze and novel object discrimination tests. Finally, results were validated in post-mortem sections and protein isolates from individuals with type 2 diabetes.ResultsCompared with non-diabetic control mice, the db/db mice demonstrated disrupted brain function as revealed by losses in episodic and spatial memory and this occurred concomitantly with dysfunctional NVU, neuronal circuitry and cerebral atrophy. scRNA-seq of db/db mouse cerebral cortex revealed cell population changes in neurons, glia and microglia linked to functional regulatory disruption including neuronal maturation and altered metabolism. These changes were validated through immunohistochemistry and protein expression analysis not just in the db/db mouse cerebral cortex but also in post-mortem sections and protein isolates from individuals with type 2 diabetes (74.3 ± 5.5 years) compared with non-diabetic control individuals (87.0 ± 8.5 years). Furthermore, metabolic and synaptic gene disruptions were evident in cortical NVU cell populations and associated with a decrease in vascular density.Conclusions/interpretationTaken together, our data reveal disruption in the cellular and molecular architecture of the cerebral cortex induced by diabetes, which can explain, at least in part, the basis for progressive cognitive decline in individuals with type 2 diabetes.Data availabilityThe single-cell sequencing data that supports this study are available at GEO accession GSE217665 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE217665).Graphical

418 A CTS Team Approach to Fetal Hyperinsulinemia in Diabetic Pregnancy and its Effects on Vasculature and Early Life Metabolism

OBJECTIVES/GOALS: Fetal glucose dynamics mediate many of the adverse outcomes seen in infants of diabetic mothers (IDM). The goals of this study are to identify: (1) rates of blood glucose change in normoglycemic and hypoglycemic IDM; (2) their relation to in-utero insulin exposure; and (3) their transcriptional impacts on placental and umbilical vasculature. METHODS/STUDY POPULATION: Using a longitudinal prospective study design, placental/umbilical cord tissue and maternal hemoglobin A1c (HbA1c) are being collected from mothers diagnosed with Type 1, Type 2, or gestational diabetes mellitus. Blood glucose levels are also collected from their infants at birth, and every 3-4 hours for up to 9 hours to determine the rate of change. Linear regression modeling will be used to determine associations between placental and umbilical endothelial RNA expression, umbilical cord insulin levels, and maternal HbA1c within each diabetic sub-type. Gene expression from endothelial specimens will be compared between diabetic sub-types and between normoglycemic and hypoglycemic infants via paired t-tests using Benjamini-Hochberg procedure for false discovery rate correction. RESULTS/ANTICIPATED RESULTS: We hypothesize the following; (1) glucose levels will have a steeper rate of change in hypoglycemic infants; (2) maternal HbA1c and in-utero insulin levels will correlate with the level of transcriptional change identified in placental and umbilical endothelial samples; (3) a negative association will exist between cord insulin levels and the rate of change in infant glucose levels; and (4) a positive association will exist between cord insulin level and transcriptional change on the placental and umbilical endothelium. DISCUSSION/SIGNIFICANCE: Identifying gene expression changes in diabetic placental/umbilical endothelium, and the role of insulin/glucose in these changes, is key to managing diabetic vasculopathy and its adverse outcomes. Understanding infant insulin response may also guide management of hypoglycemia and decrease the risk for neonatal intensive care unit admission.

Spatiotemporal transcriptomic characterization of an amyloid mouse model of Alzheimer’s disease

AbstractBackgroundTranscriptomic studies of Alzheimer's disease (AD) have identified both tissue‐level and cell‐type specific gene expression changes. However, with both "bulk"‐tissue and single‐cell approaches, we lose pertinent spatial information, such as cell‐to‐cell proximity or proximity to pathological features. Recently several techniques have emerged, aiming to profile gene expression while preserving the spatial architecture, and we have leveraged one of these techniques—spatial transcriptomics—to interrogate AD gene expression changes in the 5XFAD, an amyloid mouse model, in a spatial and temporal manner.MethodsWe generated spatial transcriptomic (10x Genomics Visium) data from 5XFAD and wildtype mice at the ages of 4, 6, 8, and 12 months (n = 80 total samples, sex‐balanced). Prior to generating libraries, we stained our tissue sections with Amylo‐glo and the conformation‐specific antibody OC to analyze gene expression changes in spatial relativity to amyloid pathology.ResultsWe profiled 18,000‐20,000 total genes per sample with 1,800‐2,700 genes per spatial spot and identified brain region‐specific transcriptionally distinct clusters. We also examined the spatial distribution of AD risk genes, identified by GWAS, throughout the disease progression. Further, we identified gene expression changes spatially related to amyloid pathology localization.ConclusionsWe have characterized the 5XFAD transcriptome, identifying spatiotemporal AD gene expression changes and providing further insight into the role of amyloid pathology in the modulation of gene expression in AD.

Glycemic control releases regenerative potential of pancreatic beta cells blocked by severe hyperglycemia.

Diabetogenic ablation of beta cells in mice triggers a regenerative response whereby surviving beta cells proliferate and euglycemia is regained. Here, we identify and characterize heterogeneity in response to beta cell ablation. Efficient beta cell elimination leading to severe hyperglycemia (>28mmol/L), causes permanent diabetes with failed regeneration despite cell cycle engagement of surviving beta cells. Strikingly,correction of glycemia via insulin, SGLT2 inhibition, or a ketogenic diet for about 3weeks allows partial regeneration of beta cell mass and recovery from diabetes, demonstrating regenerative potential masked by extreme glucotoxicity. We identify gene expression changes in beta cells exposed to extremely high glucose levels, pointing to metabolic stress and downregulation of key cell cycle genes, suggesting failure of cell cycle completion. These findings reconcile conflicting data on the impact of glucose on beta cell regeneration and identify a glucose threshold converting glycemic load from pro-regenerative to anti-regenerative.

Investigation of Transcriptome Patterns in Endometrial Cancers from Obese and Lean Women.

Endometrial cancer is the most common gynaecological malignancy in developed countries. One of the largest risk factors for endometrial cancer is obesity. The aim of this study was to determine whether there are differences in the transcriptome of endometrial cancers from obese vs. lean women. Here we investigate the transcriptome of endometrial cancer between obese and lean postmenopausal women using rRNA-depleted RNA-Seq data from endometrial cancer tissues and matched adjacent non-cancerous endometrial tissues. Differential expression analysis identified 12,484 genes (6370 up-regulated and 6114 down-regulated) in endometrial cancer tissues from obese women, and 6219 genes (3196 up-regulated and 3023 down-regulated) in endometrial cancer tissues from lean women (adjusted p-value < 0.1). A gene ontology enrichment analysis revealed that the top 1000 up-regulated genes (by adjusted p-value) were enriched for growth and proliferation pathways while the top 1000 down-regulated genes were enriched for cytoskeleton restructure networks in both obese and lean endometrial cancer tissues. In this study, we also show perturbations in the expression of protein coding genes (HIST1H2BL, HIST1H3F, HIST1H2BH, HIST1H1B, TTK, PTCHD1, ASPN, PRELP, and CDH13) and the lncRNA MBNL1-AS1 in endometrial cancer tissues. Overall, this study has identified gene expression changes that are similar and also unique to endometrial cancers from obese vs. lean women. Furthermore, some of these genes may serve as prognostic biomarkers or, possibly, therapeutic targets for endometrial cancer.

TWEAK and TNFα, Both TNF Ligand Family Members and Multiple Sclerosis-Related Cytokines, Induce Distinct Gene Response in Human Brain Microvascular Endothelial Cells.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the TNF ligand family involved in various diseases including brain inflammatory pathologies such as multiple sclerosis. It has been demonstrated that TWEAK can induce cerebrovascular permeability in an in vitro model of the blood–brain barrier. The molecular mechanisms playing a role in TWEAK versus TNFα signaling on cerebral microvascular endothelial cells are not well defined. Therefore, we aimed to identify gene expression changes in cultures of human brain microvascular endothelial cells (hCMEC/D3) to address changes initiated by TWEAK exposure. Taken together, our studies highlighted that gene involved in leukocyte extravasation, notably claudin-5, were differentially modulated by TWEAK and TNFα. We identified differential gene expression of hCMEC/D3 cells at three timepoints following TWEAK versus TNFα stimulation and also found distinct modulations of several canonical pathways including the actin cytoskeleton, vascular endothelial growth factor (VEGF), Rho family GTPases, and phosphatase and tensin homolog (PTEN) pathways. To our knowledge, this is the first study to interrogate and compare the effects of TWEAK versus TNFα on gene expression in brain microvascular endothelial cells.

Low Dopamine D2 Receptor Expression Drives Gene Networks Related to GABA, cAMP, Growth and Neuroinflammation in Striatal Indirect Pathway Neurons

BackgroundA salient effect of addictive drugs is to hijack the dopamine reward system, an evolutionarily conserved driver of goal-directed behavior and learning. Reduced dopamine type 2 receptor availability in the striatum is an important pathophysiological mechanism for addiction that is both consequential and causal for other molecular, cellular, and neuronal network differences etiologic for this disorder. Here, we sought to identify gene expression changes attributable to innate low expression of the Drd2 gene in the striatum and specific to striatal indirect medium spiny neurons (iMSNs). MethodsCre-conditional, translating ribosome affinity purification (TRAP) was used to purify and analyze the translatome (ribosome-bound messenger RNA) of iMSNs from mice with low/heterozygous or wild-type Drd2 expression in iMSNs. Complementary electrophysiological recordings and gene expression analysis of postmortem brain tissue from human cocaine users were performed. ResultsInnate low expression of Drd2 in iMSNs led to differential expression of genes involved in GABA (gamma-aminobutyric acid) and cAMP (cyclic adenosine monophosphate) signaling, neural growth, lipid metabolism, neural excitability, and inflammation. Creb1 was identified as a likely upstream regulator, among others. In human brain, expression of FXYD2, a modulatory subunit of the Na/K pump, was negatively correlated with DRD2 messenger RNA expression. In iMSN-TRAP-Drd2HET mice, increased Cartpt and reduced S100a10 (p11) expression recapitulated previous observations in cocaine paradigms. Electrophysiology experiments supported a higher GABA tone in iMSN-Drd2HET mice. ConclusionsThis study provides strong molecular evidence that, in addiction, inhibition by the indirect pathway is constitutively enhanced through neural growth and increased GABA signaling.

Abstract P2115: Differential Cardiac Remodeling Profile Of Immunosuppression Drugs

Introduction: Heart transplantation provides lifesaving therapy for patients with end-stage heart failure. The longevity of the therapy is limited by Cardiac Graft Dysfunction (CGD), which is an acquired cardiomyopathy affecting transplanted hearts associated with diastolic and/or systolic dysfunction. Some clinical risk factors for CGD have been identified, but none of them are easily modifiable. An unexplored potential contributor to CGD is the choice of immunosuppression agent used despite multiple clinical reports suggesting reduced adverse cardiac remodeling with mammalian target of rapamycin (mTOR) inhibitors compared to calcineurin inhibitors (CNI). This study examines mechanisms of differential cardiac remodeling effects of CNI versus mTOR inhibitors in a human cellular cardiac model. Methods/Results: We utilized 3D cardiac spheres composed of induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells (cardiac organoids). Cardiac organoids were treated with 5 days of vehicle, tacrolimus (CNI), or sirolimus (mTOR inhibitor). We did not observe a significant difference in surrogates of systolic or diastolic function in treated cardiac organoids. We pursued single cell-RNA sequencing of drug-treated cardiac organoids and identified gene expression changes consistent with increased extracellular matrix deposition and fibroblast activity in response to CNI treatment. In addition, CNI-treated cardiac organoids cellular composition was notable for increased proportion of fibroblasts and less cardiomyocytes compared to mTOR inhibitor-treated cardiac organoids. To validate gene expression changes observed, we treated cardiac fibroblasts with drugs and observed an increase in collagen production in response to CNI treatment and a reduction in fibroblast number and collagen production in response to mTOR inhibitor treatment. Furthermore, we observed increased ATP production in CNI-treated cardiac fibroblasts, but a reduction in mTOR-treated counterparts. Conclusion: We identify reduced extracellular matrix deposition and cardiac fibroblast proliferation in response to mTOR inhibitor as a potential mechanism for the more favorable remodeling profile observed clinically.

Cell type-specific differential expression in Alzheimer’s disease and cancer using spatial transcriptomics

Abstract Spatial transcriptomics enables spatially resolved gene expression measurements at near single-cell resolution. The detection of genes that are differentially expressed across tissue context for cell types of interest is an essential challenge for dissecting pathological mechanisms. However, changes in cell type composition across space and measurement contamination from other cell types introduce complex statistical challenges. Here, we introduce a statistical method, Generalized Linear Admixture Models for Differential Expression (GLAMDE), that estimates cell type-specific patterns of differential gene expression while accounting for localization of other cell types. By modeling spatial transcriptomics gene expression as an additive mixture, across cell types, of generalized log-linear functions, we provide a unified framework for identifying gene expression changes for a wide-range of relevant contexts: changes due to pathology, anatomical regions, physical proximity to specific cell types, and cellular microenvironment. Our approach enables statistical inference across multiple samples and replicates. We demonstrate, through simulations and validation experiments on Slide-seq and MERFISH data, that our approach accurately identifies cell type-specific differential gene expression with valid uncertainty quantification. Lastly, we apply GLAMDE to characterize spatially-localized tissue changes in the context of disease. In an Alzheimer’s mouse model Slide-seq dataset, we identify plaque-dependent patterns of cellular immune activity. We also find a putative interaction between tumor cells and myeloid immune cells in a Slide-seq tumor dataset. GLAMDE is available within the spacexr R package. D.C. was supported by a Fannie and John Hertz Foundation Fellowship and an NSF Graduate Research Fellowship. This work was supported by an NIH Early Independence Award (DP5, 1DP5OD024583 to F.C.), the NHGRI (R01, R01HG010647 to F.C.), as well as the Burroughs Wellcome Fund, the Searle Scholars Award, and the Merkin Institute to F.C.. R.A.I. was supported by NIH grants R35GM131802 and R01HG005220.

Role of Neuromedin S‐expressing Ventral Tegmental Area Neurons in Morphine Behavior

ObjectiveOpioid dependence and addiction constitute a major health and economic burden, but our limited understanding of the underlying neurobiology limits better interventions. Alteration in the activity and output of dopamine (DA) neurons in the ventral tegmental area (VTA) is known to contribute to drug effects, but the mechanisms underlying these changes in VTA DA function remain relatively unexplored. We used TRAP to identify gene expression changes in VTA DA neurons following chronic morphine and found that Neuromedin S (NMS) is enriched in VTA DA neurons, and its expression is robustly increased by morphine. Neuromedin U receptor 2 (NMUR2), the receptor with which NMS interacts, is expressed within target regions of VTA DA neurons, like the nucleus accumbens (NAc). However, whether all VTA DA neurons express NMS, and their potential functional impact has yet to be determined. We hypothesize that NMS neurons represent a novel subset of VTA neurons that contribute to morphine‐elicited behavior. Specifically in these studies, we hypothesize that activating and inhibiting VTA‐NMS neurons will promote and inhibit morphine behaviors, respectively.MethodsAdult male and female NMS‐Cre mice and wild‐type littermates were used. Cre‐dependent viral vectors (AAV‐DIO‐mCherry, AAV‐DIO‐hM3Dq‐mCherry, AAV‐DIO‐hM4Di‐mCherry) were stereotaxically injected into the VTA to allow for DREADD‐mediated activation (Dq) or inhibition (Di) of VTA‐NMS neurons. Behavioral analyses were completed two weeks after surgery. Locomotor activity was assessed following saline (d1), saline + Clozapine‐N‐oxide (CNO, 0.3mg/kg, ip, d2‐d3), and morphine (15mg/kg, ip) + CNO (d4‐d8). A morphine + CNO challenge was done 1 week following d8. Immunohistochemistry was completed to validate viral expression and specific targeting of the VTA. Repeated‐measures two‐way ANOVA was used to determine significant differences (p&lt;0.05) in locomotor behavior.ResultsWe found that a subset of VTA DA neurons express NMS (&lt;5%) and that they exhibit diverse projection targets, including the NAc. Using DREADDs, we found that activation (Dq, n=15‐17/group) or inhibition (Di, n=15‐16/group) of VTA‐NMS neurons did not affect general locomotor activity or elicit CNO‐conditioned place preference or aversion. We find that both male (n=19,20) and female Dq mice (n=17,16) exhibit increased morphine‐induced locomotor activity. Additionally, locomotor response to a challenge morphine + CNO injection was significantly increased in Dq mice compared to controls (n=20,16). In contrast, male Di mice show a trend for the opposite effect, with decreased morphine‐elicited locomotor activity (n=20,20) and decreased response to challenge (n=20,20).ConclusionsVTA‐NMS neuron activation promotes morphine‐elicited behaviors including locomotion and sensitization. Future studies will determine if NMS‐VTA neuron inhibition decreases morphine behavior and whether VTA‐NMS neuronal activity modulates morphine conditioned place preference. Our current data suggest that VTA‐NMS neurons represent a subset of VTA neurons that may be functionally relevant for morphine responses.

Quantifying p53 Regulated Genes in a Zebrafish Melanoma Model

Cancer is a prevalent disease and is one of the leading causes of death worldwide driven by mutations at the genetic level. An ideal model organism used for cancer research, specifically melanoma, is zebrafish as adult zebrafish display disappearance of their horizontal stripes and emergence of black masses. Zebrafish have highly conserved genes, comparable signaling pathways, and similar tumor development. This research project is designed to identify gene expression changes that are critical for the progression of melanoma in this model system. Wild‐type and heterozygous p53 deficient (M214K) will be exposed to ultraviolet (UV) radiation to promote tumorigenesis. The expression of multiple genes in the p53 pathway will be monitored via qPCR during progression of the melanoma. Genes that are identified as up‐regulated or down‐regulated will be targeted using CRISPR‐Cas9 to determine their role in the progression of the disease.