Biology subjects

Piscopo, V. E. C.

Publications and source records attributed to Piscopo, V. E. C..

9 recordsLinked to original sources

bioRxiv2026

Microbiota-derived extracellular vesicles link intestinal dysbiosis to neuroimmune activation in long COVID

Post COVID-19 condition (Long COVID, LC) is frequently accompanied by persistent neurological symptoms, but the mechanisms linking intestinal dysbiosis to neuroinflammation remain unclear. Here we identify gut microbiota-derived extracellular vesicles (GMEVs) as functional mediators linking LC-associated dysbiosis to systemic and neuroimmune inflammation. In a longitudinally characterized cohort, individuals with LC and neurological symptoms exhibit a persistent intestinal microbiome signature. Transplantation of LC-associated microbiota into germ-free mice induces intestinal barrier disruption and neuroinflammatory phenotypes. GMEVs from individuals with LC activate inflammasome-associated programs and impair epithelial barrier function, promote inflammatory responses in macrophages, and induce coordinated pro-inflammatory transcriptional programs in human induced pluripotent stem cell (iPSC)-derived microglia. Chronic oral administration of LC-derived GMEVs remodels the microbiota and induces intestinal and systemic inflammation with glial activation in vivo. Together, these findings support a vesicle-centered framework in which microbiota-derived extracellular vesicles translate dysbiosis into sustained immune and neuroimmune activation in a post-viral inflammatory state.

microbiology↗

bioRxiv2025

Single nuclear RNA sequencing shows altered microglial and astrocytic functions in post-mortem Parkinson's disease tissue

BackgroundParkinsons disease (PD) is a neurodegenerative disease marked by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and formation of misfolded protein aggregates. A growing body of research has implicated glial cell dysfunction in PD etiology, including the concentration of activated glial cells around protein aggregates in post-mortem tissue. Disruptions in the balance of pro- and anti-inflammatory immune response functions of the microglia and astrocytes is believed to contribute towards neurons being lost as the disease progresses. However, the molecular mechanisms remain unclear. To shed light on the role of inflammation in PD, this study analyses two public single nuclear RNA sequencing datasets of the SNpc from patient and control postmortem brain to identify altered molecular pathways in PD-associated microglia and astrocytes. ResultsThe results show that both cell types have a significant upregulation in heat shock binding and misfolded protein response pathways, likely in response to the accumulation of protein aggregates. Microglia annotated with the MKI67 marker gene show a decreased expression in PD patient derived tissue. Markers associated with activated/reactive states in astrocytes and microglia are upregulated in PD samples. Notably, expression of genes associated with resting state microglia and non-inflammatory reactive state microglia are downregulated in PD microglia, including P2RY12, CSF1R, CSF2RA, CSF3R, and TGFBR1. Concurrently, genes associated with activated microglial states such as HSP90AB1 and GPNMB are upregulated. Among the top downregulated functions, genes associated with ion channel functions are downregulated in both astrocytes and microglia. ConclusionsTaken together, the findings imply that astrocytes and microglia respond to protein misfolding pathology in PD by upregulating chaperone protein folding functions. Additionally, the profile of upregulated genes implies that pathways responding to oxidative stress are also activated. The downregulation of inflammation-associated genes in PD microglia paired with the upregulation of misfolding protein response pathways, suggests a switch from immune receptor functions to protein aggregate clearance by the end of disease stages. Finally, GPNMB emerged as a potential target for therapeutic intervention, as the one primary non-HSP gene that is significantly increased in PD-associated microglia. AbbreviationsParkinsons disease (PD); neurodegenerative diseases (NDD); dopaminergic neurons (DNs); substantia nigra pars compacta (SNpc); microglia (Mg); Lewy body dementia (LBD); single nuclear RNA sequencing (snRNA-seq), single cell RNA sequencing (scRNA-seq); Gene Ontology (GO)

neuroscience↗

bioRxiv2025

Cell type specific analysis of ALS associated proteins reveals immune-regulated targets

A substantial fraction of amyotrophic lateral sclerosis (ALS)-associated proteins remain poorly characterized, in part because of the limited availability of validated research antibodies. We established knockout (KO)-based antibody characterization workflows and demonstrated that widely used antibodies against the major ALS-associated protein C9orf72 lacked specificity (Laflamme et al., 2019). We subsequently scaled this framework to systematically benchmark research antibodies, revealing that up to 61% fail to perform as recommended by manufacturers (Ayoubi et al., 2023). Here, we extend this approach by establishing the ALS-Reproducible Antibody Platform (ALS-RAP), a comprehensive effort to generate a publicly available dataset of KO-validated antibodies targeting proteins encoded by ALS risk genes. In total, we characterized 303 antibodies against 33 ALS-associated proteins to identify high-quality reagents for use in western blot, immunoprecipitation, and immunofluorescence. Using these antibodies, we profiled protein levels across human induced pluripotent stem cell (iPSC)-derived and primary neurological cell types. These analyses revealed diverse cellular distributions and higher levels of several ALS-associated proteins in glial populations, consistent with emerging evidence for immune contributions to ALS. Together, ALS-RAP provides a validated antibody toolbox and protein expression resource to support the study of ALS-associated proteins.

neuroscience↗

bioRxiv2025

The microtubule-binding protein EML3 is required for mammalian embryonic growth and cerebral cortical development; Eml3 null mice are a model of cobblestone brain malformation

The cerebral cortex is a multi-layered structure generated through the migration of neural precursors from their birthplace in the ventricular zone to their destination within the cortical plate. Neuronal migration defects are responsible for many human pathologies collectively called neuronal migration disorders, which include subcortical band heterotopia and cobblestone brain (COB) malformation. One example of a protein involved in a neuronal migration disorder is the echinoderm microtubule-associated protein-like 1 (EML1) protein, one of six members of the mammalian EML family. Absence of EML1 protein results in subcortical band heterotopia in mice and humans. Here, we report that absence of the paralogous protein EML3 leads to delayed embryonic development and small size, and a COB-like phenotype with neuronal ectopias in the dorsal telencephalon. We found that EML3 is expressed in the neuroepithelium and meningeal mesenchyme when those tissues participate in pial basement membrane (PBM) formation. Transmission electron microscopy demonstrated that the extracellular matrix of the PBM is structurally abnormal in Eml3 null mice when the first radially migrating neurons arrive. The reduced structural integrity of the PBM leads to focal over-migration of neurons into the subarachnoid space. These findings strengthen the link between the EML protein family and cortical neuronal migration defects by identifying Eml3 as the first EML family member whose absence leads to over-migration of neuroblasts. Moreover, we report the first COB-like phenotype with PBM structural defects when a single microtubule-associated protein is deleted.

developmental biology↗

bioRxiv2024

Single Cell RNAseq to identify subpopulations of glial progenitors in iPSC-derived oligodendroglial lineage cultures

Cellular heterogeneity is a common issue in differentiation protocols of oligodendrocytes (OLs) from human induced pluripotent stem cells. Our previous work described a novel method to generate OLs and highlighted the presence of glial progenitors. Here, we unravel the glial heterogeneity and characterize the response of isolated subpopulations to differentiation. This study provides a novel tool for studying the dynamics of glial development in vitro and on a transcriptomic level.

neuroscience↗

bioRxiv2024

Pro-Inflammatory Molecules Implicated in Multiple Sclerosis Divert the Development of Human Oligodendrocyte Lineage Cells

Background and ObjectivesOligodendrocytes (OL) and their myelin-forming processes are targeted and lost during the disease course of Multiple Sclerosis (MS), targeted by infiltrating leukocytes and their effector cytokines. Myelin repair is considered to be dependent on recruitment and differentiation of oligodendrocyte precursor cells (OPCs). The basis of failure of re-myelination during the disease course of MS remains to be defined. The aim of this study is to determine the impact of pro-inflammatory molecules tumor necrosis factor (TNF) and interferon gamma (IFN{gamma}) on the differentiation of human OPCs. MethodsWe generated human OPCs from induced pluripotent stem cells with a reporter gene under the OL-specific transcription factor SOX10. We treated the cells in vitro with TNF or IFN{gamma} and evaluated effects in terms of cell viability, expression of OL-lineage markers, and co- expression of astrocyte markers. To relate our findings to the molecular properties of OPCs as found in the MS brain we re-analyzed publicly available single nuclear RNAseq datasets. ResultsOur analysis indicated that both TNF and IFN{gamma} decreased the proportion of cells differentiating into the OL-lineage; consistent with previous reports. We now observe the TNF effect is linked to aberrant OPC differentiation. A subset of O4+, reporter-positive cells co- expressed the astrocytic marker Aquaporin-4 (AQP4). On the transcriptomic level, the cells acquire an astrocyte-like signature alongside a conserved reactive phenotype. Analysis of single- nuclear RNAseq datasets from human MS brain revealed a subset of OPCs expressing an astrocytic signature. DiscussionIn the context of MS, these results imply that OPCs are present but inhibited from differentiating along the OL-lineage, with a subset acquiring a reactive and stem-cell like phenotype, reducing their capacity to contribute towards repair. These findings help define a potential basis for the impaired myelin repair in MS and provide a prospective route for regenerative treatment.

neuroscience↗

bioRxiv2024

The functional and pathogenic consequences of fibrinogen on human oligodendroglia

Fibrinogen is a blood-derived protein involved in coagulation, and can make its way into the central nervous system (CNS) following breakdown of the blood-brain barrier. This molecule has been implicated in multiple sclerosis (MS), a disease marked by inflammation and demyelination in the CNS, as well as other neurological disorders. However, the effect of this molecule has not been studied on human myelinating cells. This study examines how fibrinogen influences human oligodendrocyte (OL) lineage cells at various stages of development. Using induced pluripotent stem cell-derived (iPSC) OL precursors and human primary OLs, we examined the effects of fibrinogen on cell differentiation, viability and myelination-related function. Here we show that fibrinogen induces an aberrant differentiation of early lineage OLs, by inhibiting their maturation and inducing an astrocytic phenotype, as seen in previous studies. On mature OLs, fibrinogen was found to promote myelination capacity as shown by ensheathment assays as well as on the RNA level. These effects were associated with the activation of BMP signalling, both in early and mature OLs. Transcriptomic analysis of human MS brain tissue shows similar pro-myelination changes in a subset of OLs, suggesting in vivo relevance. These findings indicate that fibrinogen has a lineage-dependent effect, where it may be inhibitory earlier in the lineage while promoting OL function in later stages. Understanding this dual role will provide insight into remyelination failure in MS and highlights the importance of timing and target in future therapeutic strategies. Significance StatementIn multiple sclerosis (MS), the blood protein fibrinogen leaks into the brain and has been shown to interfere with myelin repair. This study demonstrates that fibrinogen has opposite effects on human oligodendrocyte-lineage cells depending on their stage of maturation. While it blocks the differentiation of early-stage cells, it enhances the functional capacity of mature oligodendrocytes. These findings help explain why remyelination may fail in MS and suggest that fibrinogen could both hinder and support repair, depending on the cell context. This dual role has important implications for developing stage-specific therapies for MS.

neuroscience↗

bioRxiv2023

The Use of a SOX10 Reporter Towards Ameliorating Oligodendrocyte Lineage Differentiation from Human Induced Pluripotent Stem Cells

Oligodendrocytes (OLs) are key players in the central nervous system, critical for the formation and maintenance of the myelin sheaths insulating axons, ensuring efficient neuronal communication. In the last decade, the use of human induced pluripotent stem cells (iPSCs) has become essential for recapitulating and understanding the differentiation and role of OLs in vitro. Current methods include overexpression of transcription factors for rapid OL generation, neglecting the complexity of OL lineage development. Alternatively, growth factor-based protocols offer physiological relevance but struggle with efficiency and cell heterogeneity. To address these issues, we created a novel SOX10-P2A-mOrange iPSC reporter line to track and purify oligodendrocyte precursor cells (OPCs). Using this reporter cell line, we analyzed an existing differentiation protocol and shed light on the origin of glial cell heterogeneity. Additionally, we have modified the differentiation protocol, towards enhancing reproducibility, efficiency, and terminal maturity. Our approach not only advances OL biology but holds promise to accelerate research and translational work with iPSC-derived OLs. Main PointsO_LIThe differentiation of iPSCs in Oligodendrocyte Precursor Cells (OPCs) and Oligodendrocytes (OLs) is a notoriously difficult technique and often displays variable efficiency and cellular heterogeneity. C_LIO_LIWe engineered a novel reporter line carrying the fluorescent protein mOrange under the control of the OL-specific transcription factor SOX10 to track, purify and characterize OLs. C_LIO_LIBy experimenting with diverse differentiation media, we improved the generation of SOX10-positive cells. Consequently, these cells exhibited increased consistency and effectiveness in evolving into myelinating OLs. C_LI

cell biology↗

bioRxiv2023

An adapted stem cell-derived microglia protocol for the study of microgliopathies and other neurological disorders

BackgroundAdult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a primary microgliopathy caused by pathogenic variants in the colony-stimulating factor 1 receptor (CSF1R) gene. Since CSF1R signaling is crucial for microglia development, survival and function, induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial defects caused by ALSP patient-specific CSF1R variants. MethodsSerial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an ALSP patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. ResultsThe newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to primary human microglia and with higher phagocytic and inflammatory competence at [~]3-fold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression. Finally, ALSP patient-derived cells showed surprisingly high phagocytic capacity, which was associated with higher lysosomal content. ConclusionsWe optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory and phagocytic activities.

neuroscience↗