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The transmembrane death receptor Fas transduces apoptotic signals upon binding its ligand, FasL. Although Fas is highly expressed in cancer cells, insufficient cell surface Fas expression desensitizes cancer cells to Fas-induced apoptosis. Here, we show that the increase in Fas microaggregate formation on the plasma membrane in response to the inhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis. We used a clinically accessible Rho-kinase inhibitor, fasudil, that reduces endocytosis dynamics by increasing plasma membrane tension. In combination with exogenous soluble FasL (sFasL), fasudil promoted cancer cell apoptosis, but this collaborative effect was substantially weaker in nonmalignant cells. The combination of sFasL and fasudil prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft mouse model. Our results demonstrate that sFasL has strong potential for apoptosis-directed cancer therapy when Fas microaggregate formation is augmented by mechano-inhibition of endocytosis.

Cerebral organoids offer significant potential for neuroscience research as complex in vitro models that mimic human brain development. However, challenges related to their quality and reproducibility hinder their reliability. Discrepancies in morphology, size, cellular composition, and cytoarchitectural organization limit their applications, particularly in disease modeling, drug screening, and neurotoxicity testing. Critically, current methods for organoid characterization often lack standardization, restricting their broader applicability. To address the need for standardized quality assessment of cerebral organoids, we developed a Quality Control (QC) methodology for 60-day cortical organoids, evaluating five key criteria using a scoring system: morphology, size and growth profile, cellular composition, cytoarchitectural organization, and cytotoxicity. We implemented a hierarchical approach, beginning with non-invasive assessments to exclude low-quality organoids, while reserving in-depth analyses for those that passed the initial evaluation. To validate this framework, we exposed 60-day cortical organoids to graded doses of hydrogen peroxide (H2O2), inducing a range of quality outcomes. The QC system demonstrated its robustness by accurately discriminating organoid qualities. Our proposed QC framework is designed to be user-friendly, flexible, and broadly applicable, making it suitable for routine assessment of cerebral organoid quality. Additionally, its scalability enables industrial applications, offering a valuable tool for advancing both fundamental and pre-clinical research.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-14425-x.

Nuclear exclusion of the RNA- and DNA-binding protein TDP-43 can induce neurodegeneration in different diseases. Diverse processes have been implicated to influence TDP-43 mislocalization, including disrupted nucleocytoplasmic transport (NCT); however, the physiological pathways that normally ensure TDP-43 nuclear localization are unclear. The six-transmembrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) cleaves the glycosylphosphatidylinositol (GPI) anchor that tethers some proteins to the membrane. Here we show that GDE2 maintains TDP-43 nuclear localization by regulating the dynamics of canonical Wnt signaling. Ablation of GDE2 causes aberrantly sustained Wnt activation in adult neurons, which is sufficient to cause NCT deficits, nuclear pore abnormalities, and TDP-43 nuclear exclusion. Disruption of GDE2 coincides with TDP-43 abnormalities in postmortem tissue from patients with amyotrophic lateral sclerosis (ALS). Further, GDE2 deficits are evident in human neural cell models of ALS, which display erroneous Wnt activation that, when inhibited, increases mRNA levels of genes regulated by TDP-43. Our study identifies GDE2 as a critical physiological regulator of Wnt signaling in adult neurons and highlights Wnt pathway activation as an unappreciated mechanism contributing to nucleocytoplasmic transport and TDP-43 abnormalities in disease. Synopsis Nuclear exclusion of TDP-43 is observed in various pathologies, but the physiological mechanisms that ensure its nuclear localization are not well-known. This work shows that inhibition of persistent Wnt activation in neurons by GDE2 prevents TDP-43 nuclear exclusion. GDE2 inhibits canonical Wnt signaling in adult postmitotic neurons.Sustained activation of canonical Wnt signaling in neurons disrupts the nuclear pore complex, impairs nucleocytoplasmic transport, and results in TDP-43 nuclear exclusion.iPS neurons from patients with C9orf72 ALS show decreased GDE2 expression and increased activation of canonical Wnt signaling.Inhibition of Wnt activation mitigates TDP-43 dysfunction in C9orf72 iPS neurons. GDE2 maintains TDP-43 nuclear localization by inhibiting Wnt activation in neurons.

AbstractBackground and purposeIn this study, we applied an induced pluripotent stem cell (iPSC)?based model of inherited erythromelalgia (IEM) to screen a library of 281 small molecules, aiming to identify candidate pain?modulating compounds.Experimental approachHuman iPSC?derived sensory neuron?like cells, which exhibit action potentials in response to noxious stimulation, were evaluated using whole?cell patch?clamp and microelectrode array (MEA) techniques.Key resultsSensory neuron?like cells derived from individuals with IEM showed spontaneous electrical activity characteristic of genetic pain disorders. The drug screen identified four compounds (AZ106, AZ129, AZ037 and AZ237) that significantly decreased spontaneous firing with minimal toxicity. The calculated IC50 values indicate the potential efficacy of these compounds. Electrophysiological analysis confirmed the compounds’ ability to reduce action potential generation in IEM patient?specific iPSC?derived sensory neuron?like cells.Conclusions and implicationsOur screening approach demonstrates the reproducibility and effectiveness of human neuronal disease modelling offering a promising avenue for discovering new analgesics. These findings address a critical gap in current therapeutic strategies for both general and neuropathic pain, warranting further investigation. This study highlights the innovative use of patient?derived iPSC sensory neuronal models in pain research and emphasises the potential for personalised medicine in developing targeted analgesics.Key points Utilisation of human iPSCs for efficient differentiation into sensory neuron?like cells offers a novel strategy for studying pain mechanisms.IEM sensory neuron?like cells exhibit key biomarkers and generate action potentials in response to noxious stimulation.IEM sensory neuron?like cells display spontaneous electrical activity, providing a relevant nociceptive model.Screening of 281 compounds identified four candidates that significantly reduced spontaneous firing with low cytotoxicity.Electrophysiological profiling of selected compounds revealed promising insights into their mechanisms of action, specifically modulating the NaV 1.7 channel for targeted analgesia. In this study, Thornton and colleagues utilised an induced pluripotent stem cell (iPSC)?based model of inherited erythromelalgia (IEM) to screen a library of 295 small molecules in search of potential pain?modulating compounds. Their screening identified four compounds that significantly reduced spontaneous firing in iPSC?derived nociceptor?like cells, with minimal associated toxicity .