CRISPR Base Editors enable precise single-nucleotide modifications, offering advantages over CRISPR-Cas9 knock-out in programming the desired genetic effect. However, in pooled screens targeting essential genes, discrepancies between expected genetic and phenotypic outcomes are frequent: single guide RNAs (sgRNAs), expected to be disruptive, often appear “phenotypically silent” likely due to inefficient editing rather than absence of functional impact. Here, we investigate if Cas9-based gene-level sgRNA depletion data can help to predict the probability that an sgRNA used in base editing will yield the expected fitness effect in pooled proliferation screening. We analysed proliferative effects (z-scores) from high-throughput CRISPR screens using cytosine Base Editors (BEs) and trained machine learning models to predict fitness effects. Our models integrate sequence features, edited strand, mutation type, predicted editing efficiencies and Cas9 gene essentiality scores. Our models discriminate BE sgRNAs that generate a strong phenotypic effect (depletion) in pooled screening, with AUC-ROC greater than 93% in different cell lines. We provide exhaustive analysis of feature importance highlighting the significant impact of sequence features for predicting BE-associated fitness effects. We found that editor-associated fitness predictions are primarily driven by sgRNA sequence features rather than predicted editing efficiency. Moreover, Cas9-derived gene essentiality partially contributes to predictions.

The landscape of cellular abnormalities occurring during metabolic dysfunction-associated steatotic liver disease (MASLD) is not completely clarified. We investigated cell heterogeneity involved in progressive MASLD at single cell resolution in mice fed AMLN. Single cell RNA sequencing (Sc-RNAseq) and spatial proteomics were applied to decipher cell populations and genes/pathways guiding MASLD progression. We identified 32 clusters, including hepatocytes (HEPs), hepatic stellate cells (HSCs), endothelial cells (ENDOs), Kupffer cells (KCs), and immune cells. HEPs clusters changed across disease severity, acquiring a periportal localization in MASH-fibrosis. The latter conditions were featured by ENDO clusters with higher expression of extracellular matrix (ECM) molecules, resident and recruited KCs/immune clusters with M1 polarization and HSCs with a myofibroblast phenotype. Finally, we highlight 5 hybrid populations named HSCs/ENDOs, HEPs/ENDOs, KCs/ENDOs, and HEPs/KCs which were confirmed by spatial proteomics across disease stages in mice and MASLD patients. In sum, we observed an evolution of cellular heterogeneity during MASLD and identified novel intermediate populations that feature advanced disease.

Paired mesoderm homeobox protein 2B (PHOX2B) is a transcription factor essential for autonomic nervous system development. Heterozygous mutations in the PHOX2B gene are associated with neurodevelopmental disorders, including congenital central hypoventilation syndrome and Hirschsprung's disease. Additionally, PHOX2B plays a role in the genetic landscape of neuroblastoma, with mutations detected in both familial and sporadic forms of this rare cancer. Notably, PHOX2B is highly expressed in most neuroblastoma cells. Despite its significance, little is known about the regulation of PHOX2B gene expression, and limited attention has been given to the genomic features of the antisense strand at the PHOX2B locus, although the presence of an antisense transcript is suggested by bioinformatics analyses. In this study, we characterize the recently annotated human antisense transcript PHOX2B-AS1 and the previously unidentified mouse antisense Phox2b transcript. Our findings reveal that PHOX2B positively regulates PHOX2B-AS1 expression and that inhibiting the antisense transcript reduces PHOX2B protein levels. Together, these results provide strong evidence for the existence of a gene antisense to PHOX2B and highlight a strict correlation and reciprocal regulation between PHOX2B and PHOX2B-AS1.

Aging is associated with an attrition of cell subcellular components of the endomembrane system due to long-term exposure to environmental stresses or physical/chemical insults. Tissue morphology physiology and function are strongly dependent on the linked dynamic activities of the endomembrane of organelles of the endomembrane system (ES). This is especially true for nervous tissue and the brain where ES components must interact over long (neurite) distances for proper synapse functions. As the endoplasmic reticulum (ER) is the primary organelle responsible for generating plasma membrane and subcellular membrane components, dysregulation of the ER has a significant role in nervous tissue physiology and diseases in which membrane function is critical, such as in age associated Alzheimer's Disease (AD). Understanding why the ER fails to respond effectively to stress may provide a promising platform for developing antiaging treatments. In this review we characterizegene pathways induced by the transmembrane (TMEM) protein, TMEM230 in the ER in Alzheimer's disease (AD). TMEM230 upregulates oxidative phosphorylation and mitochondria pathways associated cell metabolism. High levels of expression of TMEM230 associated with AD, due chronic inflammation drives hyperoxidation leading to aberrant structural changes in the tethering of the mitochondria and ER membrane and consequently, intra-organelle calcium balance. Sustained elevated levels of expression of TMEM230 leads to catastrophic oxidative stress and irreversible mitochondria damage as seen in some AD patients. Our studies support that Parkinson's Disease and Huntington's Disease may be similarly driven by chronic high levels TMEM230 which results in decoupling of ER-mitochondrial regulation.

Psychiatric disorders often arise from the interaction between genetic predisposition and chronic psychosocial stress, yet the molecular programs determining resilience versus susceptibility remain incompletely understood. Building on evidence that the transcriptional corepressor LSD1 links environmental stress to neuronal gene regulation, we investigated whether isoform-specific regulation of LSD1 splicing contributes to stress adaptation. Using a mouse model of chronic social defeat stress, we analyzed LSD1 microexon E8a splicing in the hippocampus of resilient and susceptible animals. RNA-seq was performed after the last stress session to capture genome-wide transcriptional responses during the window of LSD1 splicing regulation. Comparative analyses with published LSD1 knockdown, LSD1 ChIP-seq and chronic stress datasets were conducted. Hippocampal samples from suicide victims were analyzed to assess translational relevance. Analysis of LSD1 splicing dynamics revealed that resilient mice, but not susceptible animals, retained the ability to reiterate acute stress-induced exon E8a skipping after repeated stress exposure, preserving the capacity to upregulate the enzymatically active ubLSD1 isoform in the hippocampus. In susceptible mice this inducible splicing response was absent. Mechanistically, splicing regulation involved the long non-coding RNA MALAT1, which controls the neurospecific splicing factor nSR100, a regulator of LSD1 exon E8a inclusion. Reduced MALAT1 expression in susceptible mice coincided with marked overactivation of stress-responsive genes revealed by RNA-seq. Approximately 15% (86 of 595) of genes deregulated in susceptible versus resilient hippocampi overlapped with transcripts modulated by LSD1 knockdown in an independent neuronal system. Of these, 25 were direct LSD1 ChIP-seq targets. ESR1 emerged as a regionally divergent upstream regulator associated with susceptibility. The MALAT1-nSR100-LSD1 axis represents a regulatory pathway modulating stress adaptation. Downregulation of ubLSD1 and MALAT1 in the hippocampus of suicide victims recapitulates the molecular phenotype observed in stress-susceptible mice, linking disruption of this pathway to pathological behavioral outcomes.

Lamin A/C is a nuclear type V intermediate filament protein part of the meshwork structure underlying the inner nuclear membrane (nuclear lamina), which plays numerous roles, including maintenance of nuclear shape, heterochromatin organization, and transcriptional regulation. Our group has demonstrated the role of Lamin A/C in different pathophysiological conditions. Here, we investigated for the first time how Lamin A/C affects neuronal maturation in rat cerebellar granule cells (GCs). Primary rat cerebellar GCs where we silenced the Lmna gene constituted our key model; this provided a rather homogeneous cellular system showing a neuronal population in vitro. We then validated our findings in another in vivo murine model with knock-out of the Lmna gene and in an in vitro human neuronal model with silencing of the LMNA gene. We observed across three different models that Lamin A/C down-regulation affects neurons maturation by protecting the cells from glutamate-evoked excitotoxicity and correlates with an inhibition of calcium influxes and a down-regulation of pro-inflammatory cytokine pathways. Consistent with previous findings from our group, this study corroborates that Lamin A/C plays a key role in neural development and opens new significant implications for a better comprehension of the mechanisms involved in neurodegenerative diseases, where changes in the nuclear envelope are linked to neuroinflammatory processes and damage.

Clonally established tumor cell lines often do not recapitulate the behavior of cells in tumors. The sequencing of a whole tumor tissue may not uncover transcriptome profiles induced by the interactions of all different cell types within a tumor. Interferons for instance have a vast number of binding sites in their target genes. Access to the DNA binding sites is determined by the epigenomic state of each different cell type within a tumor mass. To understand how genes such as interferons appear to have both tumor-promoting and tumor-inhibiting functions, single-cell transcript analysis was performed in the breast cancer tissue of HER2+ (epidermal growth factor receptor 2) patients. We identified that potential antagonistic oncogenic activities of cells can be due to diverse expression patterns of genes with pleiotropic functions. Molecular pathways both known and novel were identified and were similar with those previously identified for patients with rheumatoid arthritis. Our study demonstrates the efficacy in using single-cell transcript analysis to gain insight into genes with apparent contradictory or paradoxical roles in oncogenesis.

Aging is often a choice between developing cancer or autoimmune disorders, often due in part to loss of self-tolerance or loss of immunological recognition of rogue-acting tumor cells. Self-tolerance and cell recognition by the immune system are processes very much dependent on the specific signatures of glycans and glycosylated factors present on the cell plasma membrane or in the stromal components of tissue. Glycosylated factors are generated in nearly innumerable variations in nature, allowing for the immensely diverse role of these factors in aging and flexibility necessary for cellular interactions in tissue functionality. In previous studies, we showed that differential expression of TMEM230, an endoplasmic reticulum (ER) protein was associated with specific signatures of enzymes regulating glycan synthesis and processing and glycosylation in rheumatoid arthritis synovial tissue using single-cell transcript sequencing. In this current study, we characterize the genes and pathways co-modulated in all cell types of the synovial tissue with the enzymes regulating glycan synthesis and processing, as well as glycosylation. Genes and biological and molecular pathways associated with hallmarks of aging were in mitochondria-dependent oxidative phosphorylation and reactive oxygen species synthesis, ER-dependent stress and unfolded protein response, DNA repair (UV response and P53 signaling pathways), and senescence, glycolysis and apoptosis regulation through PI3K-AKT-mTOR signaling have been shown to play important roles in aging or neurodegeneration (such as Parkinson’s and Alzheimer’s disease). We propose that the downregulation of TMEM230 and RNASET2 may represent a paradigm for the study of age-dependent autoimmune disorders due to their role in regulating glycosylation, unfolded protein response, and PI3K-AKT-mTOR signaling.

ABSTRACT Alterations in glycoconjugate profiles are thought to promote changes in cell‐to‐cell and cell‐to‐intracellular and extracellular scaffold interactions in human disease. The nearly unlimited number of “glycoforms” that may exist in nature are difficult to study due to glycosylation and glycoconjugate modifications being associated with non‐genome coded posttranscription and post‐translation processes. Specific products generated by glycosylation are dependent on concentration and sub‐cellular locations of glycan synthesis and processing enzymes. An indirect “high‐throughput” approach to study glycosylation is to characterize glycan processing enzymes (hydrolases and transferases) by single cell sequencing of all cell types in tissue of human diseases. We previously identified TMEM230 as an endoplasmic reticulum (ER) associated protein that regulates NOTCH glycoprotein receptor and ligand signaling in zebrafish blood vessel formation and destructive remodeling capacities of diverse cell types including fibroblast, phagocytic and immune system cells in patients with cancer or granulomatous systemic vasculitis autoimmune disorder. NOTCH signaling represents a paradigm in glycan mediated signal transduction and supports the role of TMEM230 in glycan modifications. The ER initiates the earliest steps of glycoconjugate synthesis, sorting, and trafficking. As blood vessel and tissue remodeling, and Notch signaling are hallmarks of autoimmune disorders, we investigated whether aberrant TMEM230 expression was also associated with changes in expression of glycan processing enzymes in patients with rheumatoid arthritis (RA). In this current study, single cell sequencing analysis supported that TMEM230 expression was downregulated in all cell types associated with synovial tissue of RA patients while glycan processing enzymes were predominantly upregulated. In contrast, TMEM230 was upregulated in patients with high‐grade compared to low‐grade gliomas as it was N‐linked glycosylation (GlcNAc), and glycoprotein and glycosaminoglycan expression. Our collective results support that TMEM230 regulates glycan/glycoconjugate processing enzymes in RA and the expression of protein glycoconjugate in aggressive gliomas. TMEM230 may therefore be a therapeutic target and marker for clinical treatment for glycosylation induced human autoimmunity disorders or cancer.

Disease phenotypes can be described as the consequence of interactions among molecular processes that are altered beyond resilience. Here, we address the challenge of assessing the possible alteration of intra- and inter-cellular molecular interactions among processes or cells. We present an approach, designated as "Ulisse", which complements the existing methods in the domains of enrichment analysis, pathway crosstalk analysis and cell-cell communication analysis. It applies to gene lists that contain quantitative information about gene-related alterations, typically derived in the context of omics or multi-omics studies. Ulisse highlights the presence of alterations in those components that control the interactions between processes or cells. Considering the complexity of statistical assessment of network-based analyses, crosstalk quantification is supported by two distinct null models, which systematically sample alternative configurations of gene-related changes and gene-gene interactions. Further, the approach provides an additional way of identifying the genes associated with the phenotype. As a proof-of-concept, we applied Ulisse to study the alteration of pathway crosstalks and cell-cell communications in triple negative breast cancer samples, based on single-cell RNA sequencing. In conclusion, our work supports the usefulness of crosstalk analysis as an additional instrument in the "toolkit" of biomedical research for translating complex biological data into actionable insights.

Congenital Central Hypoventilation Syndrome (CCHS) is a rare, life-threatening genetic disorder of the autonomic nervous system characterized by alveolar hypoventilation and generalized dysautonomia. CCHS is caused by heterozygous PHOX2B mutations, predominantly polyalanine repeat expansion (95% of cases) and, less frequently, frameshift mutations (5%). To address the lack of disease models, we generated five human induced pluripotent stem cell (hiPSC) lines derived from patients carrying +5Ala, +6Ala and +11Ala expansion mutations. These hiPSC lines exhibited undifferentiated hPSC phenotype, pluripotency, normal karyotype, and retention of the pathogenic genotype, providing a reliable in vitro platform for elucidating CCHS molecular mechanisms and disease pathogenesis.

High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors.

BackgroundA reliable preclinical model of patient-derived organoids (PDOs) was developed in a case study of a 69-year-old woman diagnosed with breast cancer (BC) to investigate the tumour evolution before and after neoadjuvant chemotherapy and surgery. The results were achieved due to the development of PDOs from tissues collected before (O-PRE) and after (O-POST) treatment.MethodsPDO cultures were characterized by histology, immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), confocal microscopy, flow cytometry, real-time PCR, bulk RNA-seq, single-cell RNA sequencing (scRNA-seq) and drug screening.ResultsBoth PDO cultures recapitulated the histological and molecular profiles of the original tissues, and they showed typical mammary gland organization, confirming their reliability as a personalized in vitro model. Compared with O-PRE, O-POST had a greater proliferation rate with a significant increase in the Ki67 proliferation index. Moreover O-POST exhibited a more stem-like and aggressive phenotype, with increases in the CD24low/CD44low and EPCAMlow/CD49fhigh cell populations characterized by increased tumour initiation potential and multipotency and metastatic potential in invasive lobular carcinoma. Analysis of ErbB receptor expression indicated a decrease in HER-2 expression coupled with an increase in EGFR expression in O-POST. In this context, deregulation of the PI3K/Akt signalling pathway was assessed by transcriptomic analysis, confirming the altered transcriptional profile. Finally, transcriptomic single-cell analysis identified 11 cell type clusters, highlighting the selection of the luminal component and the decrease in the number of Epithelial-mesenchymal transition cell types in O-POST.ConclusionNeoadjuvant treatment contributed to the enrichment of cell populations with luminal phenotypes that were more resistant to chemotherapy in O-POST. PDOs represent an excellent 3D cell model for assessing disease evolution.

Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretion which contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis.

TMEM230 promotes antigen processing, trafficking, and presentation by regulating the endomembrane system of membrane bound organelles (lysosomes, proteosomes and mitochondria) and phagosomes. Activation of the immune system requires trafficking of various cargos between the endomembrane system and cell plasma membrane. The Golgi apparatus is the hub of the endomembrane system and essential for the generation, maintenance, recycling, and trafficking of the components of the endomembrane system itself and immune system. Intracellular trafficking and secretion of immune system components depend on mitochondrial metalloproteins for ATP synthesis that powers motor protein transport of endomembrane cargo. Glycan modifying enzyme genes and motor proteins are essential for the activation of the immune system and trafficking of antigens between the endomembrane system and the plasma membrane. Recently, TMEM230 was identified as co-regulated with RNASET2 in lysosomes and with metalloproteins in various cell types and organelles, including mitochondria in autoimmune diseases. Aberrant metalloproteinase secretion by motor proteins is a major contributor to tissue remodeling of synovial membrane and joint tissue destruction in rheumatoid arthritis (RA) by promoting infiltration of blood vessels, bone erosion, and loss of cartilage by phagocytes. In this study, we identified that specific glycan processing enzymes are upregulated in certain cell types (fibroblast or endothelial cells) that function in destructive tissue remodeling in rheumatoid arthritis compared to osteoarthritis (OA). TMEM230 was identified as a regulator in the secretion of metaloproteinases and heparanase necessary tissue remodeling in OA and RA. In dendritic (DC), natural killer and T cells, TMEM230 was expressed at low or no levels in RA compared to OA. TMEM230 expression in DC likely is necessary for regulatory or helper T cells to maintain tolerance to self-antigens and prevent susceptibility to autoimmune disease. To identify how TMEM230 and the endomembrane system contribute to autoimmunity we investigated, glycan modifying enzymes, metalloproteinases and motor protein genes co-regulated with or regulated by TMEM230 in synovial tissue by analyzing published single cell transcriptomic datasets from RA patient derived synovial tissue.

We recently identified TMEM230 as a master regulator of the endomembrane system of cells. TMEM230 expression is necessary for promoting motor protein dependent intracellular trafficking of metalloproteins for cellular energy production in mitochondria. TMEM230 is also required for transport and secretion of metalloproteinases for autophagy and phagosome dependent clearance of misfolded proteins, defective RNAs and damaged cells, activities that decline with aging. This suggests that aberrant levels of TMEM230 may contribute to aging and regain of proper levels may have therapeutic applications. The components of the endomembrane system include the Golgi complex, other membrane bound organelles, and secreted vesicles and factors. Secreted cellular components modulate immune response and tissue regeneration in aging. Upregulation of intracellular packaging, trafficking and secretion of endosome components while necessary for tissue homeostasis and normal wound healing, also promote secretion of pro-inflammatory and pro-senescence factors. We recently determined that TMEM230 is co-regulated with trafficked cargo of the endomembrane system, including lysosome factors such as RNASET2. Normal tissue regeneration (in aging), repair (following injury) and aberrant destructive tissue remodeling (in cancer or autoimmunity) likely are regulated by TMEM230 activities of the endomembrane system, mitochondria and autophagosomes. The role of TMEM230 in aging is supported by its ability to regulate the pro-inflammatory secretome and senescence-associated secretory phenotype in tissue cells of patients with advanced age and chronic disease. Identifying secreted factors regulated by TMEM230 in young patients and patients of advanced age will facilitate identification of aging associated targets that aberrantly promote, inhibit or reverse aging. Ex situ culture of patient derived cells for identifying secreted factors in tissue regeneration and aging provides opportunities in developing therapeutic and personalized medicine strategies. Identification and validation of human secreted factors in tissue regeneration requires long-term stabile scaffold culture conditions that are different from those previously reported for cell lines used as cell models for aging. We describe a 3 dimensional (3D) platform utilizing non-biogenic and non-labile poly e-caprolactone scaffolds that supports maintenance of long-term continuous cultures of human stem cells, in vitro generated 3D organoids and patient derived tissue. Combined with animal component free culture media, non-biogenic scaffolds are suitable for proteomic and glycobiological analyses to identify human factors in aging. Applications of electrospun nanofiber technologies in 3D cell culture allow for ex situ screening and the development of patient personalized therapeutic strategies and predicting their effectiveness in mitigating or promoting aging.

Glial cells provide physical and chemical support and protection for neurons. Secreted neurotrophic factors, scaffolds and vesicles regulate in addition to normal homeostasis, repair following neural tissue injury or degeneration. We previously demonstrated that human CNS glia have revascularization capacity by inducing endothelial cell sprouting and microchanneling driven by glial cell secreted components. The clinical potential of using secreted factors and vesicles generated by glial cells in conditioned media was shown by their re-normalization of pathological blood vessels and 3D tissue wound healing and remodeling capacity. To identify secreted components of glial cells and how CNS and PNS glial cells differentially regulate neural tissue homeostasis, disease and injury response, we developed a novel culture method for various types of glial cells that allow for isolating and characterizing secreted glial components, free of animal factors and contaminants normally associated with serum-based culture conditions. Our culture method will enable to identify secreted components using ultrasensitive RNA, glycoconjugate and protein analysis technologies. The characterization of glial secreted factors involved microchanneling may help in development of new clinical therapies for glial and neural cells or axons to migrate and reform connections.