Biology subjects

Bertucci, T.

Publications and source records attributed to Bertucci, T..

8 recordsLinked to original sources

bioRxiv2026

Multiomic Profiling of 85 iPSC Lines from Familial Dementia Reveals Cellular Diversity and Regulators of Organoid Development

Patient-derived induced pluripotent stem cells (iPSCs) are used for disease modeling and therapeutic development, yet their systematic molecular and genetic characterization is uncommon. Here, we performed whole genome sequencing (WGS), bulk/single-cell RNA-sequencing (RNA-seq) and DNA methylation analysis on 85 iPSC lines harboring MAPT mutations and corresponding isogenic controls. Single-cell RNA-seq revealed reproducible inter- and intra-line heterogeneity related to quality/pluripotency and nominated surface markers to quantify iPSC subclusters. WGS detected unintended editing events and structural variants, including the 20q21.31 duplication, missed by standard assays. We identified pathways correlated with neural organoid formation efficiency and with genome-editing and clonal-selection effects, underscoring the need to use unedited lines as isogenic controls. This comprehensive dataset improves the utility of the MAPT iPSC collection and provides proof of principle supporting in-depth genomic characterization to improve iPSC utility in biological research.

genomics↗

bioRxiv2026

Single Cell Transcriptomics and Surface Protein Expression Reveal Distinct Cellular and Molecular Phenotypes in Human RPESC-RPE and PSC-RPE

Current retinal pigment epithelium (RPE) cell replacement strategies in trials for age-related macular degeneration (AMD) are based on either pluripotent stem cell (PSC) or adult RPE stem cell (RPESC) sources. We used Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) to simultaneously assess single-cell transcriptomic and surface protein information, comparing these two RPE sources. Both RPESC-RPE and PSC-RPE expressed key RPE markers and exhibited cellular heterogeneity. However, RPESC-RPE had higher expression of genes related to mature retinal functions, whereas PSC-RPE had greater expression of genes involved in stem cell development and differentiation. We identified two surface proteins that distinguished the cell types. The "dont eat me" signal, CD24, was detected robustly on adult RPESC-RPE cells, while CD57 was detected on most PSC-RPE cells. The differences in gene and surface protein expression suggest that the two RPE sources differ in functional, adhesion, and immunomodulatory properties, which may impact transplantation outcomes.

cell biology↗

bioRxiv2026

Inosine misincorporation into mRNA triggers the integrated stress response and activates an innate immune gene expression signature

The inosine triphosphate pyrophosphatase (ITPase) enzyme restricts levels of the non-canonical nucleotides (deoxy)inosine triphosphate (dITP/ITP) and prevents their aberrant misincorporation into nucleic acids. ITPase deficiency is associated with dilated cardiomyopathy and epileptic encephalopathy in humans and is usually fatal in infancy. It leads to pronounced inosine misincorporation into RNA but the cellular consequences of this misincorporation are not well understood and the pathogenic basis of ITPase deficiency remains unknown. Here we show that cellular transfection of mRNA with inosine misincorporation activates the integrated stress response (ISR) with an innate immune gene expression signature. This stress response triggers stress granule formation and is modulated by the double stranded RNA sensor protein Kinase R (PKR). Inosine nucleoside treatment of ITPase-deficient cells leads to endogenous inosine misincorporation into mRNA and activation of the ISR. Further, differentiation of human ITPase-deficient induced pluripotent stem cells into neurons results in a low-level stress response. Thus, our study establishes inosine misincorporation into mRNA as an unappreciated form of cellular stress. This is normally prevented by the ITPase enzyme, with implications for the pathogenesis of ITPase deficiency.

molecular biology↗

bioRxiv2025

MAPT Splicing Modulators as a Therapeutic Strategy for Tauopathies

Tauopathies are neurodegenerative diseases characterized by the abnormal accumulation of microtubule-associated protein tau (MAPT) in the brain. These disorders, like frontotemporal dementia (FTD-Tau), currently lack effective therapies and can occur sporadically or be inherited when associated with MAPT gene mutations. The MAPT gene region encompassing exon 10 and adjacent introns is a hotspot for pathogenic variants, including splicing mutations that enhance exon 10 inclusion and increase 4R tau expression, and gain-of-function mutations that generate aggregation-prone mutant 4R tau protein. For these 4R-specific tauopathies, a targeted mRNA splicing approach that promotes exon 10 exclusion may offer therapeutic benefit. In this study, we discovered novel splicing modulator compounds (SMCs) that promote MAPT exon 10 exclusion, and demonstrated their efficacy in FTD patient-derived neuronal models carrying the tau-P301L gain-of-function mutation or the tau-S305N splicing mutation. Treatment with SMC reduced 4R tau expression and decreased the accumulation of hyperphosphorylated tau (pTau), oligomeric and insoluble tau, thereby rescuing tau-associated neuronal toxicity. Importantly, our lead SMC corrected the 3R/4R splice ratio in vivo and significantly reduced pTau in the brain of a gene- replacement (GR) mouse model expressing the human tau-N279K splicing mutation. These findings support the therapeutic potential of this class of small molecules and establish MAPT pre- mRNA splicing modulation as a promising strategy for the treatment of 4R tauopathies. One Sentence SummaryDiscovery of SMCs that correct MAPT splicing, reduce 4R tau, and rescue pathology in patient- derived neuronal and in vivo models of 4R tauopathies.

neuroscience↗

bioRxiv2025

APOE-ϵ4-induced Fibronectin at the blood-brain barrier is a conserved pathological mediator of disrupted astrocyte-endothelia interaction in Alzheimer's disease

Blood-brain barrier (BBB) dysfunction is a key feature of Alzheimers disease (AD), particularly in individuals carrying the APOE-{varepsilon}4 allele. This dysfunction worsens neuroinflammation and hinders the removal of toxic proteins, such as amyloid-beta (A{beta}42), from the brain. In post-mortem brain tissues and in animal models, we previously reported that fibronectin accumulates at the BBB predominantly in APOE-{varepsilon}4 carriers. Furthermore, we found a loss-of-function variant in the fibronectin 1 (FN1) gene significantly reduces aggregated fibronectin levels and decreases AD risk among APOE-{varepsilon}4 carriers. Yet, the molecular mechanisms downstream of fibronectin at the BBB remain unclear. The extracellular matrix (ECM) plays a crucial role in maintaining BBB homeostasis and orchestrating the interactions between BBB cell types, including endothelia and astrocytes. Understanding the mechanisms affecting the ECM and BBB cell types will be critical for developing effective therapies against AD, especially among APOE-{varepsilon}4 carriers. Here, we demonstrate that APOE-{varepsilon}4, A{beta}42, and inflammation drive the induction of FN1 expression in several models including zebrafish, mice, iPSC-derived human 3D astrocyte and 3D cerebrovascular cell cultures, and in human brains. Fibronectin accumulation disrupts astroglial-endothelial interactions and the signalling cascade between vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor (HBEGF) and Insulin-like growth factor 1 (IGF1). This accumulation of fibronectin in APOE-{varepsilon}4- associated AD potentiates BBB dysfunction, which strongly implicates reducing fibronectin deposition as a potential therapeutic target for AD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/634732v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1efff03org.highwire.dtl.DTLVardef@14f40cdorg.highwire.dtl.DTLVardef@27743aorg.highwire.dtl.DTLVardef@3fa9a9_HPS_FORMAT_FIGEXP M_FIG C_FIG Accessibility textThis image illustrates the effects of different APOE isoforms (ApoE-{varepsilon}3 and ApoE-{varepsilon}4) on blood-brain barrier (BBB) integrity, focusing on the molecular interactions between astrocytes and endothelial cells. This figure emphasizes the detrimental effects of ApoE-{varepsilon}4 on BBB integrity via fibronectin accumulation and altered signaling pathways. The top section provides a schematic overview of the blood-brain barrier, highlighting astrocytes, endothelial cells, and their interface. The left panel represents the ApoE-{varepsilon}3 condition: Normal fibronectin (FN1) levels support healthy interactions between astrocytes and endothelial cells. Growth factors, including VEGFA, HBEGF, and IGF1, maintain BBB integrity through their respective receptors (VEGFR and EGFR). Green arrows indicate activation of these signaling pathways. The right panel depicts the ApoE-{varepsilon}4 condition: Elevated fibronectin (FN1) disrupts astrocyte-endothelium interactions. FN1 binds integrins and activates focal adhesion kinase (FAK), inhibiting VEGFA, which is required for endothelial HBEGF that in turn activates IGF1 signaling. Red symbols indicate inhibition of HBEGF, VEGFA, and IGF1 pathways, leading to BBB dysfunction. HighlightsAPOE-{varepsilon}4 drives fibronectin deposition in Alzheimers, disrupting astrocyte-endothelia interactions. APOE-{varepsilon}4 and fibronectin co-localize, forming aggregates at blood-brain barrier (BBB). Fibronectin alters the signaling between VEGF, IGF1, and HBEGF impairing BBB function. Reducing fibronectin restores BBB integrity and offsets APOE-{varepsilon}4 pathology.

cell biology↗

bioRxiv2024

Fully Human Bifunctional Intrabodies Achieve Graded Reduction of Intracellular Tau and Rescue Survival of MAPT Mutation iPSC-derived Neurons

Tau protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimers disease, frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP), spurring development of tau-lowering therapeutic strategies. Here, we report fully human bifunctional anti-tau-PEST intrabodies that bind the mid-domain of tau to block aggregation and degrade tau via the proteasome using the ornithine decarboxylase (ODC) PEST degron. They effectively reduced tau protein in human iPSC-derived cortical neurons in 2D cultures and 3D organoids, including those with the disease-associated tau mutations R5L, N279K, R406W, and V337M. Anti-tau-hPEST intrabodies facilitated efficient ubiquitin-independent proteolysis, in contrast to tau-lowering approaches that rely on the cells ubiquitination system. Importantly, they counteracted the proteasome impairment observed in V337M patient-derived cortical neurons and significantly improved neuronal survival. By serial mutagenesis, we created variants of the PEST degron that achieved graded levels of tau reduction. Moderate reduction was as effective as high reduction against tau V337M-induced neural cell death.

neuroscience↗

bioRxiv2023

Improved Protocol for Reproducible Human Cortical Organoids Reveals Early Alterations in Metabolism with MAPT Mutations

Human pluripotent stem cell (hPSC)-derived cortical organoids are powerful models but are often limited by low efficiency, variability, and stress-related artifacts. To address these challenges, we developed a scalable organoid platform with end-to-end quality control (QC) metrics spanning manufacturing and single-cell RNA-sequencing (scRNA-seq), developed using eight MAPT mutation isogenic line sets relevant to frontotemporal dementia (FTD-tau). Using a 96 slit-well format, we achieved [~]100% production efficiency across 64 lines. Controlled-release FGF2 enhanced iPSC pluripotency and reduced mesendodermal contaminants, while optimized SB431542 dosing enhanced cortical patterning across lines with variable TGFBR1/ALK5 expression. The resulting organoids displayed transcriptomic profiles and low-stress signatures closely aligned with the developing human cortex. Applying a cortical organoid scRNA-seq index (CortiCOSI), we identified early dysregulation of phosphatase regulators (PPP2CA, ANP32A) and the prefoldin subunit PFDN6 in MAPT V337M excitatory neurons before tau hyperphosphorylation and oligomerization. This platform improves scalability, reproducibility, and mechanistic insight in cortical organoid studies.

neuroscience↗

bioRxiv2021

Glutamatergic dysfunction precedes neuron loss in cerebral organoids with MAPT mutation.

Frontotemporal dementia (FTD) due to MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations due to the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, and glutamatergic signaling pathways and regulators including the RNA-binding protein ELAVL4. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function and build-up of tau and P-tau S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons of layers affected in patients. Mutant neurons are susceptible to glutamate toxicity which was rescued pharmacologically by treatment with the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede cell death, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.

neuroscience↗