Transcriptional Alterations Research Articles

Single-neuron analysis of aging-associated changes in learning reveals impairments in transcriptional plasticity.

The molecular mechanisms underlying age-related declines in learning and long-term memory are still not fully understood. To address this gap, our study focused on investigating the transcriptional landscape of a singularly identified motor neuron L7 in Aplysia, which is pivotal in a specific type of nonassociative learning known as sensitization of the siphon-withdraw reflex. Employing total RNAseq analysis on a single isolated L7 motor neuron after short-term or long-term sensitization (LTS) training of Aplysia at 8, 10, and 12 months (representing mature, late mature, and senescent stages), we uncovered aberrant changes in transcriptional plasticity during the aging process. Our findings specifically highlight changes in the expression of messenger RNAs (mRNAs) that encode transcription factors, translation regulators, RNA methylation participants, and contributors to cytoskeletal rearrangements during learning and long noncoding RNAs (lncRNAs). Furthermore, our comparative gene expression analysis identified distinct transcriptional alterations in two other neurons, namely the motor neuron L11 and the giant cholinergic neuron R2, whose roles in LTS are not yet fully elucidated. Taken together, our analyses underscore cell type-specific impairments in the expression of key components related to learning and memory within the transcriptome as organisms age, shedding light on the complex molecular mechanisms driving cognitive decline during aging.

Single-cell transcriptional profile of CD34+ hematopoietic progenitor cells from del(5q) myelodysplastic syndromes and impact of lenalidomide

While myelodysplastic syndromes with del(5q) (del(5q) MDS) comprises a well-defined hematological subgroup, the molecular basis underlying its origin remains unknown. Using single cell RNA-seq (scRNA-seq) on CD34+ progenitors from del(5q) MDS patients, we have identified cells harboring the deletion, characterizing the transcriptional impact of this genetic insult on disease pathogenesis and treatment response. Interestingly, both del(5q) and non-del(5q) cells present similar transcriptional lesions, indicating that all cells, and not only those harboring the deletion, may contribute to aberrant hematopoietic differentiation. However, gene regulatory network (GRN) analyses reveal a group of regulons showing aberrant activity that could trigger altered hematopoiesis exclusively in del(5q) cells, pointing to a more prominent role of these cells in disease phenotype. In del(5q) MDS patients achieving hematological response upon lenalidomide treatment, the drug reverts several transcriptional alterations in both del(5q) and non-del(5q) cells, but other lesions remain, which may be responsible for potential future relapses. Moreover, lack of hematological response is associated with the inability of lenalidomide to reverse transcriptional alterations. Collectively, this study reveals transcriptional alterations that could contribute to the pathogenesis and treatment response of del(5q) MDS.

DIPG-86. INVESTIGATING THE TUMOR ENVIRONMENT AND SIGNALING DEPENDENCIES DRIVING DIFFUSE MIDLINE GLIOMA TUMOR INITIATION, PROGRESSION, AND RESISTANCE: A COMPREHENSIVE APPROACH LEVERAGING BULK MULTIOMICS DECONVOLUTION AND SINGLE-CELL OMICS VALIDATION

Abstract BACKGROUND Diffuse Midline Glioma (DMG) is a incurable tumor affecting children. Recent genomic investigations have identified a recurrent H3K27M mutation which induces global alterations in histone methylation patterns and DNA methylation. These epigenetic modifications hint at pivotal roles in DMG pathogenesis, yet effective therapeutic strategies remain elusive, with median survival rates stagnant at approximately one year. This shortfall stems from two main factors: 1) Inadequate multiomics studies hindering our understanding of DMG evolution and tumor progression, and 2) Limited comprehension of the tumor microenvironment in DMG. METHODS To elucidate DMG’s clonal evolution, we adopted a comprehensive multi-region sampling approach, acquiring 33 specimens from seven patients. Whole-exome and transcriptome sequencing, alongside DNA methylation profiling, were performed. Additionally, we generated one of the most extensive single-cell multiomics datasets (>300,000 cells from 21 additional samples), with a focus on pre- and post-treatment, and dissemination effects in DMG. RESULTS Our analysis delineated a tumor-promoting microenvironment characterized by hypoxia and pro-inflammatory conditions, nurturing genomic alterations and specialized biological processes such as proliferation and epithelial-mesenchymal transition. Notably, a majority of immune cells exhibited M1-like polarization, bolstering pro-inflammatory programs within the tumor milieu. Subclones within the DMG cohort demonstrated dissemination potential, often exhibiting up-regulation of NOTCH, P53, and WNT beta-catenin signaling pathways. These findings suggest that DMG clones harboring dissemination capabilities outside the pons acquire additional phenotypic features, possibly mediated by epigenetic or transcriptional alterations, contributing to enhanced migratory and aggressive behaviors. CONCLUSIONS Our study unveils the parallel evolution of DMG at genetic, epigenetic, and transcriptional levels, unveiling novel subclonal phenotypes governing tumor behavior. Crucially, we identify critical environmental shifts such as hypoxia and inflammatory changes, coupled with specialized signaling programs, driving dissemination and resistance phenotypes. These insights may pave the way for generating accurate genetically mouse models and targeted therapeutic interventions to combat this challenging malignancy.

DIPG-82. RADIOSENSITIZATION WITH RAD52 INHIBITION IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline glioma (DMG) is one of the most devastating childhood cancers that limited response to radiation therapy (RT). There is a critical need for new therapeutics that enhance the radiation effect. We, therefore, tested the hypothesis that targeting RAD52 activity sensitizes the radiation response of DMG. METHODS Genome-wide CRISPR/Cas9 screening was performed to identify the potential therapeutic target. RAD52 inhibition was used by shRNA-mediated RAD52 depletion and treatment with RAD52 inhibitor, D-I03. The protein expression, cell proliferation, DNA damage marker expression, DNA repair pathway, and transcriptional alteration were analyzed by western blotting, MTS assay, colony formation assay, immunocytochemistry, DNA repair pathway assay, and RNA sequencing. Mice with DMG patient-derived xenograft (PDX) models were treated with RAD52 inhibitor alone or combination with RT. RESULTS Genome-wide CRISPR/Cas9 screening identified RAD52 as a potential therapeutic target in DMG cells. RAD52 inhibition suppressed DMG cell proliferation. Importantly, RAD52 inhibition in combination with RT further increased the radiosensitivity of DMG cells. Immunocytochemistry of DNA double-strand breaks (DSB) marker γH2AX and repair marker 53BP1 showed that RAD52 inhibition sustained DNA damage with high levels of γH2AX at 24 hours following radiation while the level of 53BPI was decreased, thereby inhibiting DNA DSB repair. Western blotting also revealed that RAD52 inhibition causes a sustained level of phosphorylated Rad50 and γH2AX in irradiated DIPG cells over 24 hours. DNA repair assay showed that RAD52 inhibition suppressed homologous recombination DNA repair pathway. RNA sequencing showed that RAD52 inhibition downregulated genes associated with the DNA repair pathway. Finally, the combination therapy of RAD52 inhibitor and RT further suppressed tumor growth and increased survival of mice bearing DMG PDXs, outperforming either monotherapy. CONCLUSIONS these results highlight RAD52 inhibition as a potential radiosensitization and provide a rationale for developing combination therapy with radiation in the treatment of DMG.

Adrenal gland macrophages regulate glucocorticoid production through Trem2 and TGF-β.

Glucocorticoid synthesis by adrenal glands (AGs) is regulated by the hypothalamic-pituitary-adrenal axis to facilitate stress responses when the host is exposed to stimuli. Recent studies implicate macrophages as potential steroidogenic regulators, but the molecular mechanisms by which AG macrophages exert such influence remain unclear. In this study, we investigated the role of AG macrophages in response to cold challenge or atherosclerotic inflammation as physiologic models of acute or chronic stress. Using single-cell RNA sequencing, we observed dynamic AG macrophage polarization toward classical activation and lipid-associated phenotypes following acute or chronic stimulation. Among transcriptional alterations induced in macrophages, triggering receptor expressed on myeloid cells 2 (Trem2) was highlighted because of its upregulation following stress. Conditional deletion of macrophage Trem2 revealed a protective role in stress responses. Mechanistically, Trem2 deletion led to increased AG macrophage death, abolished the TGF-β-producing capacity of AG macrophages, and resulted in enhanced glucocorticoid production. In addition, enhanced glucocorticoid production was replicated by blockade of TGF-β signaling. Together, these observations suggest that AG macrophages restrict steroidogenesis through Trem2 and TGF-β, which opens potential avenues for immunotherapeutic interventions to resolve stress-related disorders.

Transcriptome profiling reveals distinct alterations in the B-cell signature and dysregulation of peripheral B-cell subsets in sickle cell anemia patients

Sickle cell anemia (SCA) is characterized by immune system activation and heightened susceptibility to infections. We hypothesized that SCA patients exhibit transcriptional alterations in B-cell-related genes, impacting their peripheral B-cell compartment and leading to dysregulated humoral immunity and increased infection susceptibility. Our objective was to conduct an in silico analysis of whole blood transcriptomes from SCA patients and healthy controls obtained from public repositories. We aimed to identify alterations in the adaptive immune system and validate these findings in our own SCA patient cohort. Bioinformatic analyses unveiled significant transcriptional alterations in B-cell signatures, developmental pathways, and signaling pathways. These results were validated in peripheral blood mononuclear cells from our SCA patient cohort and controls using real-time polymerase chain reaction and flow cytometry. Ninety genes exhibited differential expression, with 70 upregulated and 20 downregulated. Dysregulation in the B-cell compartment of SCA patients was evident, characterized by increased frequencies of immature and naive B-cells, and decreased percentages of memory B-cell subsets compared with healthy controls. Our findings highlight previously unexplored transcriptional and quantitative alterations in peripheral B-cells among SCA patients. Understanding these changes sheds light on the mechanisms contributing to the heightened infection risk in this population. Future studies should delve deeper into these molecular changes to develop targeted interventions and therapeutic strategies aimed at mitigating infection susceptibility in individuals with SCA.

Synaptic alterations in pyramidal cells following genetic manipulation of neuronal excitability in monkey prefrontal cortex.

In schizophrenia, dorsolateral prefrontal cortex (DLPFC) pyramidal neurons (PNs) have both transcriptional and structural alterations that suggest they are hypoactive. PN hypoactivity is thought to produce synaptic alterations in schizophrenia, however the effects of lower neuronal activity on synaptic function in primate DLPFC have not been examined. Here, we used, for the first time in primate neocortex, adeno-associated viral vectors (AAVs) to reduce PN excitability with Kir2.1 channel overexpression and tested if this manipulation altered the strength of synaptic inputs onto the Kir2.1-overexpressing PNs. Recordings in DLPFC slices showed that Kir2.1 overexpression depressed excitatory (but not inhibitory), synaptic currents, suggesting that, in schizophrenia, the hypoactivity of PNs might be exacerbated by reduced strength of the excitatory synapses they receive.

A Comprehensive Perspective of Huntington’s Disease

Huntington's disease (HD) is a severe genetic illness caused by a CAG expansion on chromosome 4 in the huntingtin gene. This results in an excessively long polyglutamine tract, which has negative consequences. The normal huntingtin protein serves important tasks, however the mutant version causes a variety of detrimental effects. Disruptions in cellular processes such as autophagy, decreased mitochondrial activity, lysosomal dysfunction, and others are involved in the etiology of HD. Inflammation, oxidative stress, and transcriptional alterations all contribute to neurodegeneration. Despite great progress in understanding the genetic basis of HD, there is currently no cure. The current approach to management focuses on symptomatic control, but as our understanding of genetics advances, targeted medicines might become available. Although HD is still a difficult condition to treat, there is optimism for future advancements in research. Clinical techniques mostly focus on symptom management, with genetic testing assisting in diagnosis. Promising research looks on potential disease-modifying therapies, such as ways to reduce mutant huntingtin levels and improve clearance. Ongoing clinical research provide promise for future treatments, bringing hope to HD patients and their families.

Active juvenile systemic lupus erythematosus is associated with distinct NK cell transcriptional and phenotypic alterations

While adaptive immune responses have been studied extensively in SLE (systemic lupus erythematosus), there is limited and contradictory evidence regarding the contribution of natural killer (NK) cells to disease pathogenesis. There is even less evidence about the role of NK cells in the more severe phenotype with juvenile-onset (J)SLE. In this study, analysis of the phenotype and function of NK cells in a large cohort of JSLE patients demonstrated that total NK cells, as well as perforin and granzyme A expressing NK cell populations, were significantly diminished in JSLE patients compared to age- and sex-matched healthy controls. The reduction in NK cell frequency was associated with increased disease activity, and transcriptomic analysis of NK populations from active and low disease activity JSLE patients versus healthy controls confirmed that disease activity was the main driver of differential NK cell gene expression. Pathway analysis of differentially expressed genes revealed an upregulation of interferon-α responses and a downregulation of exocytosis in active disease compared to healthy controls. Further gene set enrichment analysis also demonstrated an overrepresentation of the apoptosis pathway in active disease. This points to increased propensity for apoptosis as a potential factor contributing to NK cell deficiency in JSLE.

Regulatory roles of N6-methyladenosine (m6A) methylation in RNA processing and non-communicable diseases.

Post-transcriptional RNA modification is an emerging epigenetic control mechanism in cells that is important in many different cellular and organismal processes. N6-methyladenosine (m6A) is one of the most prevalent, prolific, and ubiquitous internal transcriptional alterations in eukaryotic mRNAs, making it an important topic in the field of Epigenetics. m6A methylation acts as a dynamical regulatory process that regulates the activity of genes and participates in multiple physiological processes, by supporting multiple aspects of essential mRNA metabolic processes, including pre-mRNA splicing, nuclear export, translation, miRNA synthesis, and stability. Extensive research has linked aberrations in m6A modification and m6A-associated proteins to a wide range of human diseases. However, the impact of m6A on mRNA metabolism and its pathological connection between m6A and other non-communicable diseases, including cardiovascular disease, neurodegenerative disorders, liver diseases, and cancer remains in fragmentation. Here, we review the existing understanding of the overall role of mechanisms by which m6A exerts its activities and address new discoveries that highlight m6A's diverse involvement in gene expression regulation. We discuss m6A deposition on mRNA and its consequences on degradation, translation, and transcription, as well as m6A methylation of non-coding chromosomal-associated RNA species. This study could give new information about the molecular process, early detection, tailored treatment, and predictive evaluation of human non-communicable diseases like cancer. We also explore more about new data that suggests targeting m6A regulators in diseases may have therapeutic advantages.

Focused cancer pathway analysis revealed unique therapeutic targets in retinoblastoma.

Retinoblastoma (RB) is a pediatric cancer of the eye that occurs in 1/15000 live births worldwide. Albeit RB is initiated by the inactivation of RB1 gene, the disease progression relies largely on transcriptional alterations. Therefore, evaluating gene expression is vital to unveil the therapeutic targets in RB management. In this study, we employed an RT2 Profiler™ PCR array for a focused analysis of 84 cancer-specific genes in RB. An interaction network was built with gene expression data to identify the dysregulated pathways in RB. The key transcript alterations identified in 13 tumors by RT2 Profiler™ PCR array was further validated in 15 tumors by independent RT-qPCR. Out of 84 cancer-specific genes, 68 were dysregulated in RB tumors. Among the 68 genes, 23 were chosen for further analysis based on statistical significance and abundance across multiple tumors. Pathway analysis of altered genes showed the frequent perturbations of cell cycle, angiogenesis and apoptotic pathways in RB. Notably, upregulation of MCM2, MKI67, PGF, WEE1, CDC20 and downregulation of COX5A were found in all the tumors. Western blot confirmed the dysregulation of identified targets at protein levels as well. These alterations were more prominent in invasive RB, correlating with the disease pathogenesis. Our molecular analysis thus identified the potential therapeutic targets for improving retinoblastoma treatment. We also suggest that PCR array can be used as a tool for rapid and cost-effective gene expression analysis.

Maternal loss-of-function of Nlrp2 results in failure of epigenetic reprogramming in mouse oocytes.

NLRP2 belongs to the subcortical maternal complex (SCMC) of mammalian oocytes and preimplantation embryos. This multiprotein complex, encoded by maternal-effect genes, plays a pivotal role in the zygote-to-embryo transition, early embryogenesis, and epigenetic (re)programming. The maternal inactivation of genes encoding SCMC proteins has been linked to infertility and subfertility in mice and humans. However, the underlying molecular mechanisms for the diverse functions of the SCMC, particularly how this cytoplasmic structure influences DNA methylation, which is a nuclear process, are not fully understood. We undertook joint transcriptome and DNA methylome profiling of pre-ovulatory germinal-vesicle oocytes from Nlrp2-null, heterozygous (Het), and wild-type (WT) female mice. We identified numerous differentially expressed genes (DEGs) in Het and Nlrp2-null when compared to WT oocytes. The genes for several crucial factors involved in oocyte transcriptome modulation and epigenetic reprogramming, such as DNMT1, UHRF1, KDM1B and ZFP57 were overexpressed in Het and Nlrp2-null oocytes. Absence or reduction of Nlrp2, did not alter the distinctive global DNA methylation landscape of oocytes, including the bimodal pattern of the oocyte methylome. Additionally, although the methylation profile of germline differentially methylated regions (gDMRs) of imprinted genes was preserved in oocytes of Het and Nlrp2-null mice, we found altered methylation in oocytes of both genotypes at a small percentage of the oocyte-characteristic hyper- and hypomethylated domains. Through a tiling approach, we identified specific DNA methylation differences between the genotypes, with approximately 1.3% of examined tiles exhibiting differential methylation in Het and Nlrp2-null compared to WT oocytes. Surprisingly, considering the well-known correlation between transcription and DNA methylation in developing oocytes, we observed no correlation between gene expression differences and gene-body DNA methylation differences in Nlrp2-null versus WT oocytes or Het versus WT oocytes. We therefore conclude that post-transcriptional changes in the stability of transcripts rather than altered transcription is primarily responsible for transcriptome differences in Nlrp2-null and Het oocytes.

Genomic signature analysis and survival outcomes in cholangiocarcinoma (CCA) using oncology research information exchange network (ORIEN) database.

4101 Background: Immunotherapy (IO) is standard of care for advanced CCA patients (pts) regardless of PD-L1 expression. Lack of biomarkers that predict prolonged benefit or resistance to therapies are unmet gaps in this field that we attempted to fill using transcriptional and survival data in the ORIEN dataset. Methods: Through an ORIEN intermember project, we accessed an avatar dataset for clinical characteristics and transcriptional data of the CCA cohort. We first identified clinical factors highly associated with transcriptional variability and conducted principal component analysis (PCA), focusing on factors linked to the first principal component (PC1). Both specimen site and type (p<0.01) were found highly associated with PC1 and were included as co-variates in subsequent models. Then, immune deconvolution using XCell was performed on the ORIEN-CCA RNA-sequencing cohort to determine the transcriptome portion attributed to effector T cell (Teff) and regulatory T cell (Treg) for each patient. Teff:Treg ratios were calculated and used to divide patients into cold (low Teff:Treg) and hot (high Teff:Treg) cohorts based on their median (mTeff:Treg = 0.0004). Differential gene expression analysis comparing hot and cold tumors yielded 72 significantly differentially expressed genes (FC <1.5, adjusted p<0.05). Gene Set Enrichment Analysis (GSEA) of transcriptomic changes revealed enrichment for immune and metabolic pathway alterations. Results: The ORIEN avatar dataset comprised of 78 pts, with 39 designated as immune hot and 39 as immune cold (demographics in Table). Median overall survival (mOS) was 3.17 years in hot and 2.65 years in cold group . These groups exhibited significantly different Teff:Treg ratio (p<0.0001). Differential gene expression identified 72 altered genes, including upregulated immune genes (e.g., IL2RA, CD80, GBP5) in the hot group and metabolic genes (e.g., AZGP1) in the cold group. GSEA of these genes revealed immune pathway enrichment in hot tumors, with increased antigen presentation, NK cell function, and B-cell activation. Surprisingly, cold tumors were enriched of pathways involved in lipogenesis, oxidation, and proliferation. Conclusions: ORIEN-CCA cohort analysis revealed immune hot and cold tumors with distinct transcriptional alterations and differences in mOS. Correlating these alterations with IO use, and further studies of enriched pathways in cold tumors are warranted. [Table: see text]

GRN knockdown regulates the expression and alternative splicing of genes associated with aphasia-related diseases in PC12 cells

BackgroundPrior research has shown that granulin precursor (GRN, also termed PGRN) is closely linked to aphasia. However, there has been little research on the mechanism of action of GRN in post-stroke aphasia (PSA). MethodsIn this study, RT-qPCR was used to identify variations in gene expression, while RNA sequencing (RNA-seq) was utilized to acquire transcriptional profiles. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were employed for bioinformatics analysis. ResultsGRN was considerably more active in PSA subjects. After silencing the GRN, 197 transcripts had differential expression, and 237 alternative splicing events (ASEs) were substantially affected. The analysis of differentially expressed genes (DEGs) using GO and KEGG approaches showed that these genes have various molecular functions and are significantly enriched in metabolic signaling pathways. Regarding Alternative Splicing (AS), the GO and KEGG analyses revealed numerous functional genes involved in transcription and metabolism. ConclusionsThe knockdown of GRN has been shown to be associated with alterations in transcription, metabolism, and ASEs, potentially impacting transcriptional and metabolic pathways through its involvement in AS. Furthermore, GRN knockdown is associated with nervous system disease-related gene transcription and AS processes, as well as its involvement in G protein-coupled receptor (GPCR) and wingless/integrated (Wnt) signaling pathways, which impact the initiation and resolution of PSA.

Toward Robust ASR System against Audio Adversarial Examples using Agitated Logit

Automatic speech recognition (ASR) systems are vulnerable to audio adversarial examples, which aim at deceiving ASR systems by adding perturbations to benign speech signals. These audio adversarial examples appear indistinguishable from benign audio waves, but the ASR system decodes them as intentional malicious commands. Previous studies have demonstrated the feasibility of such attacks in simulated environments (over-line) and have further showcased the creation of robust physical audio adversarial examples (over-air). Various defense techniques have been proposed to counter these attacks. However, most of them have either failed to handle various types of attacks effectively or have resulted in significant time overhead. In this article, we propose a novel method for detecting audio adversarial examples. Our approach involves feeding both smoothed audio and original audio inputs into the ASR system. Subsequently, we introduce noise to the logits before providing them to the decoder of the ASR. We demonstrate that carefully selected noise can considerably influence the transcription results of audio adversarial examples while having minimal impact on the transcription of benign audio waves. Leveraging this characteristic, we detect audio adversarial examples by comparing the altered transcription, resulting from logit noising, with the original transcription. The proposed method can be easily applied to ASR systems without requiring any structural modifications or additional training. Experimental results indicate that the proposed method exhibits robustness against both over-line and over-air audio adversarial examples, outperforming state-of-the-art detection methods.

GnRH-mediated suppression of S100A4 expression inhibits endometrial epithelial cell proliferation in sheep via GNAI2/MAPK signaling.

Gonadotrophin-releasing hormone (GnRH) administration significantly decreases the pregnancy rate of recipient ewes after embryo transfer, possibly because GnRH affects endometrial epithelial cell function. Therefore, this study investigated the effect of GnRH on endometrial epithelial cells. Transcriptome sequencing was used to determine the regulatory effect of GnRH on the ewe endometrium, and the S100A4 gene, which showed altered transcription, was screened as a candidate regulator of this effect. Endometrial epithelial cells were further isolated, the S100A4 protein was immunoprecipitated, and host proteins that interacted with S100A4 were identified by mass spectrometry. We further verified the effects of S100A4 and GNAI2 on the proliferation of endometrial epithelial cells via overexpression/knockdown experiments and subsequent CCK-8 and EdU assays. The effect of S100A4 deletion in endometrial cells on reproduction was verified in mice with S100A4 knockout. Our results showed that S100A4 gene transcription in endometrial cells was significantly inhibited after GnRH administration. GNAI2 was identified as a downstream interacting protein of S100A4, and S100A4 was confirmed to activate the MAPK signaling pathway to promote cell proliferation by targeting GNAI2. GnRH can suppress the expression of S100A4 in the endometrium, consequently inhibiting the proliferation of endometrial cells through the S100A4/GNAI2/MAPK signaling pathway. These findings suggest a potential explanation for the limited efficacy of GnRH in promoting embryo implantation.

Fast but not furious: neonatal cardiac fibroblasts as promoters of regeneration after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Fundação para a Ciência e Tecnologia (FCT) Introduction Mouse neonates at postnatal day 1 (P1) regenerate their hearts after myocardial infarction (MI) but this capacity is transient. Seven day-old animals (P7) develop a reparative response similar to adults, with ischemic tissue being progressively replaced by a rigid fibrotic scar mainly produced by activated cardiac fibroblasts (CF). Recent works support that CF are likely to play an important role in mediating the regeneration-to-repair transition, but the current knowledge is limited. Aims Our final goal is to unravel CF-mediated mechanisms that confer regenerative potential to the neonatal heart and later reactivate these processes in the context of adult MI. Methods Ventricles from non-manipulated mice at different developmental stages were subjected to targeted RNA-seq. CF were isolated by FACS from P1 (regenerative) and P7 (reparative) hearts and characterized at the transcriptional, phenotypic and functional level in vitro. MI was induced by permanent ligation of the left anterior descending coronary artery at P1 and P7. CF and immune cell dynamics were assessed at different timepoints after MI by flow cytometry and immunofluorescence (IF). Transcriptomics of FACS-sorted CF from sham-operated and infarcted hearts at P1 and P7 were assessed by targeted RNA-seq. Intramyocardial injections of either P1 or P7 CF in MI-operated P1 hearts were performed immediately after surgery and hearts were analysed by IF at 7 and 14 days after injury. Results Severe transcriptional alterations on extracellular matrix (ECM) genes were found between P1 (regenerative) and P7 (reparative) hearts. Concomitantly, CF were shown to progressively infiltrate the myocardium and undergo a phenotypic shift that recapitulates transcriptional alterations observed for ECM-encoding genes in the heart. In vitro, CF at P7 show increased proliferation, migration capacity and TGF-β responsiveness. After MI at P1, CF participate in the regenerative response through a fast and transient recruitment to the ischemic site, returning to basal levels at day 7, a period in which CM proliferation and neovascularization are upregulated. Contrarily, MI at P7 resulted in permanent loss of CM, impaired neovascularization and formation of aberrant fibrosis as a result of exuberant and persistent CF recruitment and activation. Whole transcriptomic analysis further showed CF from MI at P7 are highly reactive to injury and engage on the expression of ECM components. The injection of CF resulted in a reduced heart weight and accumulation of cells in the injury site at 7 and 14 days after MI, which suggests a delay in regeneration. Conclusion Physiological CF development after birth entails a shift from a pro-regenerative to a pro-fibrotic phenotype that impacts on the regenerative capacity of the heart and determines the persistent CF activation and permanent scarring after P7.

Age-Related Clusters and Favorable Immune Phenotypes in Young Breast Cancer Patients

Breast cancer (BC) patients aged <40 years at diagnosis experience aggressive disease and poorer survival compared with women diagnosed with BC at 40 to 49 years, but the age-related biology is described to little extent. Here, we explored transcriptional alterations in BC to gain better understanding of age-related tumor biology. We studied a subset of the Bergen in-house cohort (n = 127; age range, 26-49 years) and used the NanoString Breast Cancer 360 expression panel on formalin-fixed paraffin-embedded BC tissue, and publicly available global BC messenger RNA expression data (n = 204; age range, 22-49 years), to explore differentially expressed genes between the young (age <40 years) and older (age 40-49 years) patients. Unsupervised hierarchical clustering was applied to identify gene expression–based patient clusters. We applied established computational approaches to define the PAM50 subtypes, risk of recurrence scores (ROR), and risk groups and to infer the proportions of 22 immune cell types from bulk gene expression profiles of patients aged <50 years at BC diagnosis. Differentially expressed genes and gene sets were investigated using OncoEnrichR and g:Profiler to describe functional profiles and pathway enrichment. We identified 4 age-related patient clusters presenting distinct characteristics of PAM50 subtypes and ROR profiles, which demonstrated independent prognostic value when adjusted for traditional clinicopathologic variables and the known molecular subtypes. Our findings showed better survival than expected in the basal-enriched cluster 2 and in triple-negative and basal-like BC. Deconvolution analyses of immunophenotypes indicated higher levels of M0 and M1 macrophages than M2 macrophages in subsets of young BC. Our approach identifies age-based patient clusters with distinct clinicopathologic profiles, to a large extent overlapping with the PAM50 subtypes, although with independent prognostic values in multivariate survival analyses. The patient clusters provided new insight in the immune cell distribution across tumor subtypes, potentially contributing to survival differences between the clusters and the molecular subtypes and indicating age-related mechanisms improving outcome. Our study confirms the applicability of ROR as a valid prognosticator also in a young BC cohort.

Single-cell multi-cohort dissection of the schizophrenia transcriptome.

The complexity and heterogeneity of schizophrenia have hindered mechanistic elucidation and the development of more effective therapies. Here, we performed single-cell dissection of schizophrenia-associated transcriptomic changes in the human prefrontal cortex across 140 individuals in two independent cohorts. Excitatory neurons were the most affected cell group, with transcriptional changes converging on neurodevelopment and synapse-related molecular pathways. Transcriptional alterations included known genetic risk factors, suggesting convergence of rare and common genomic variants on neuronal population-specific alterations in schizophrenia. Based on the magnitude of schizophrenia-associated transcriptional change, we identified two populations of individuals with schizophrenia marked by expression of specific excitatory and inhibitory neuronal cell states. This single-cell atlas links transcriptomic changes to etiological genetic risk factors, contextualizing established knowledge within the human cortical cytoarchitecture and facilitating mechanistic understanding of schizophrenia pathophysiology and heterogeneity.

#1479 Single nucleus RNA-sequencing indicates functional heterogeneity of renal endothelial cells in chronic kidney disease

Abstract Background and Aims The burden of cardiovascular disease (CVD) in patients with chronic kidney disease (CKD) remains very high. Endothelial cell (EC) dysfunction is an important key player in the initiation and progression of both pathologies. More importantly, endothelial dysfunction occurs early in both disease courses and is potentially reversible, making it a promising target for effective preventive strategies. Recently, accumulating single-cell RNA sequencing-based evidence has highlighted EC heterogeneity in different pathophysiological contexts. However, while biological processes such as systemic inflammation, oxidative stress fibrosis are being defined as comm EC in CKD and CVD, a comprehensive understanding of at single-cell resolution is lacking. Here, we characterized renal EC subpopulations by single-nucleus RNA sequencing (snRNA-seq) to investigate alterations in CKD patients healthy controls and identify potential drivers of EC dysfunction in CKD. Method Droplet-based snRNA-seq (10X Genomics Chromium) was performed on cryopreserved kidney biopsy cores from 6 human patients with CKD (eGFR &amp;lt; 45 mL/min/1.73 m2) and 3 healthy controls. CKD patients included 2 patients with primary focal segmental glomerulosclerosis (FSGS) and 4 with secondary/maladaptive FSGS (2 patients with diabetes mellitus, 2 female patients in total); all patients had a Framingham 10-year cardiovascular risk score &amp;gt; 20, but no established CVD. Healthy control samples were pre-perfusion biopsies from patients undergoing living kidney donation (including 2 female patients). EC subclusters were annotated based on the expression of canonical EC marker genes. Differential gene expression analysis for patient samples vs. controls included all ECs and DEGs were filtered for log2FC &amp;gt; 0.25 or &amp;lt; −0.25 and adjusted P-value of &amp;lt; .05. Gene set enrichment analysis (GSEA) on the identified DEGs was performed using GO terms. Results A total of 4 081 ECs were present in the dataset, consisting of 5 EC subtypes in both the patient and control cohorts (Panel A). DEGs in CKD patients vs. controls included upregulated genes related to organogenesis and cell differentiation (MEF2C, MEIS2), vascular development (FLRT2), and angiogenesis regulators (VEGFC, PRCP, HDAC9) (Panel B). Notably, VEGFC and FLRT2, are involved in directing EC motility and vascular patterning. In accordance, GSEA showed enrichment for GO biological processes related to vascular endothelial growth factor signaling pathway, endothelial cell differentiation, endothelial development, regulation of angiogenesis, and endothelial cell migration (Panel C). This suggests a potential link between these genes and aberrant angiogenesis, analogous to previous findings in diabetic nephropathy. Some but not all of these changes were also reported for renal ECs of early diabetic nephropathy patients. Of note, the majority of patients studied in our cohort were non-diabetic, warranting further investigation into renal endothelial cell transcriptional alterations that are general to renal disease or specific to particular renal conditions such as diabetic nephropathy. Differential gene expression and GSEA analysis of the EC subclusters in CKD indicated involvement of the peritubular capillary and ascending vasa recta ECs in EC migration / angiogenesis-related responses, overlapping with the overall analysis described above. Glomerular capillary ECs showed upregulation of immune-related genes and processes, reflecting the inflammatory status of the kidney disease. Conclusion snRNA-seq analysis revealed heterogeneity and subtype-specific gene expression alterations of renal ECs from CKD patients compared to healthy controls. Differential functions include enhanced angiogenic (potentially compensatory) and immune-related responses. These findings suggest that specific biological functions may direct different roles of the renal EC subtypes in CKD and will be further validated for their cluster-specificity and for their therapeutic relevance.